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[ "<title>Introduction</title>", "<p id=\"Par6\">Childhood anxiety disorders represent a significant public health challenge and are associated with poor social, academic and health outcomes [##REF##22581270##1##], family dysfunction [##UREF##0##2##], and significant economic burden [##REF##23884863##3##]. They are the most common mental health problem experienced by children—with a worldwide prevalence rate of 6.5% [##REF##25649325##4##]. Untreated, childhood anxiety disorders have a poor prognosis [##REF##24219155##5##], predict a range of other mental health problems including depressive and substance use disorders in adolescence and adulthood [##REF##25496295##6##], and are associated with poor occupational outcomes [##REF##24456837##7##].</p>", "<p id=\"Par7\">More than three decades of research have demonstrated that psychological interventions are efficacious in the treatment of childhood anxiety [##UREF##1##8##]. The “gold-standard” intervention, child-focused CBT (typically 10–16 sessions in duration), has consistently been shown to be an effective treatment when compared to waitlist and placebo controls across meta-analyses [##UREF##2##9##–##REF##33196111##11##]. However, the meta-analyses highlight two concerning facts: (1) a significant proportion of anxious youth do not respond to their initial course of treatment, and (2) in spite of some promising indications of sustained improvements [##REF##28181040##12##], relapse in the long-term appears to be common [##REF##29960692##13##]. The assumption that child-focused CBT does indeed represent the gold-standard intervention has been further challenged by the most recent Cochrane review examining child-focused CBT in the treatment of childhood anxiety [##REF##33196111##11##]. The review concludes that, while CBT is—in the short-term—an effective treatment compared to wait list or no treatment control conditions, there is little to no evidence that CBT is superior to treatment as usual or alternative treatments (though the authors note that the amount and quality of evidence for this latter finding limits the confidence that should be placed in it). The authors also note that, “we still know little about how best to efficiently improve outcomes” ([##REF##33196111##11##], p. 2).</p>", "<p id=\"Par8\">The role of parents in the etiology of childhood anxiety (e.g., [##REF##28867938##14##, ##REF##30472334##15##]) would appear to offer one avenue for efficiently improving outcomes for children with anxiety disorders and has resulted in a body of research evaluating parent + child focused interventions. However, meta-analyses do not support the intuitive hypothesis that these interventions will produce superior outcomes compared to child-focused CBT [##REF##22459788##10##, ##REF##33196111##11##]. Most recently, the role of parents in treatment has been approached differently, with parent-only interventions receiving increased attention. Across a wide range of emotional and behavioural difficulties in children, parent-only interventions have been found to be both valued by and experienced as acceptable by parents [##REF##31820298##16##]. Randomized controlled trials (RCTs) have provided encouraging results for studies evaluating various parent-only interventions in the treatment of childhood anxiety. This includes therapist-supported bibliotherapy [##REF##22545740##17##, ##REF##16313883##18##], as well as clinic-based, parent-only interventions ranging from six [##REF##28641122##19##] to 12 weekly sessions [##REF##25617254##20##]. However, many of these studies are limited by small sample sizes (the studies cited here had sample sizes ranging from 49 to 194) and a lack of long-term follow-up (of the eight studies cited here, three reported only post-treatment follow-up, two reported 6-month follow-up and three reported 12-month follow-up data). That being noted, studies have shown that parent-only interventions produce: (1) superior outcomes compared to a waitlist control [##REF##28641122##19##, ##REF##23969483##21##, ##REF##19457471##22##]; (2) equivalent results to a parent + child intervention [##REF##19457471##22##, ##REF##10517054##23##]; and (3) noninferior results to child-focused CBT [##REF##30851397##24##]. A recent meta-analysis of 29 studies concluded that parent-only interventions have a significant treatment effect when compared to waitlist controls; however, no differences were found when compared to active interventions [##REF##35460744##25##]. Thus, parent-only interventions appear to be a promising way to treat childhood anxiety disorders, offering several potential benefits including the capacity to directly target hypothesised anxiety-maintaining parent behaviours.</p>", "<p id=\"Par9\">Interestingly, regardless of the nature of the intervention, treatment outcomes have tended to focus solely on the “identified” anxious child, as opposed to family-level outcomes. Parental anxiety is the traditional exception to this trend, and some recent studies have examined family functioning as an outcome. One outcome that, to the best of our knowledge, has not been reported upon is sibling anxiety—despite the fact that 12% of siblings of children being treated for an anxiety disorder meet criteria for a previously undiagnosed anxiety disorder themselves [##UREF##3##26##]. Interest in the effects of treatment on siblings is justified because siblings are among the most important developmental influences on child development. Siblings impact each other in many ways, which include but are not limited to, influencing each other’s acquisition of interpersonal skills [##UREF##4##27##], emotional development and adjustment [##UREF##5##28##], development of mental health and behavioural problems [##REF##23159327##29##], and providing protection against the adverse effects of marital discord [##REF##2295580##30##]. Hence, in this study, we examined the effects of the parent focused intervention on the siblings of target children as well.</p>", "<p id=\"Par10\">The current study evaluated two different versions of a parent-only intervention (Fear-Less Triple P; FLTP; [##UREF##6##31##]): a standard 6-week group format and a one-day intensive workshop format. In a previous study, it has been shown that the 6-week group format produced diagnostic outcomes that were superior to a waitlist control comparison; and that were comparable to outcomes reported in other studies examining child-focused CBT, although it must be noted that FLTP has not yet been compared with either treatment as usual or an active control [##REF##28641122##19##]. At six sessions (~ 9 h clinical contact), FLTP is already briefer than the majority of parent-only interventions (typically 8–12 sessions).</p>", "<p id=\"Par11\">The one-day workshop format (~ 6 h clinical contact) was developed to address the most significant issue facing the field of child anxiety treatment outome research. Namely, efficacious psychological interventions do not have a meaningful public health impact [##REF##33196111##11##]. Community studies suggest that, while the proportion of youth seeking treatment for anxiety has increased [##UREF##7##32##] in recent years, only a very small proportion are able to access specialist mental health support, with an even smaller proportion (~ 2%) receiving an evidence-based intervention such as child-focused CBT [##REF##31965309##33##]. Common barriers include parents’ perceptions of the social stigma for their children, cost (money and time), and access to care. Recent trends in psychotherapy research have witnessed the development and use of brief and more intensive approaches (brief, intensive and concentrated [BIC] protocols [##REF##28772195##34##]) that are designed to reach more children and their families, to be more efficient, and to be more cost-effective. CBT, given its evidentiary support and its sound underlying therapeutic principles, is uniquely positioned to be in the foreground of this movement. In general, these approaches have modified more traditional or conventional CBT approaches by reducing either the number of sessions or the time period over which the sessions are delivered. Such programs have been developed for youth with specific phobia, obsessive–compulsive disorder, and separation anxiety disorder [##REF##28772195##34##]. However, at this point, a comparable transdiagnostic intervention for anxiety disorders more broadly does not exist. This is especially important in as much as current evidence suggests that clinical contact time is not related to children’s outcomes [##REF##33196111##11##]; and indeed, that the number of treatment sessions/weeks is significantly and negatively related to youth’s post-treatment outcomes [##REF##28221063##35##].</p>", "<p id=\"Par12\">In this study, we evaluated the efficacy of the one-day Fear-Less workshop (a BIC protocol), with the 6-week group program as the control. Primary outcomes related to anxiety diagnostic status. Secondary outcomes included: parent and child ratings of anxiety, independent evaluator (IE)-rated improvement, parent ratings of anxiety-enhancing parenting, parent-rated treatment satisfaction, parent ratings of family functioning, parent anxiety and stress, and anxiety for the sibling closest in age to the identified child. It was hypothesised that:<list list-type=\"order\"><list-item><p id=\"Par13\">The workshop and the 6-week program would each result in comparable reductions in child anxiety symptoms (as assessed by questionnaire, diagnostic interview and IE ratings of improvement), which would be maintained at follow-up.</p></list-item><list-item><p id=\"Par14\">The workshop and the 6-week program would be highly acceptable to parents. The question of whether they would be equally acceptable to parents was viewed as exploratory in nature.</p></list-item></list></p>", "<p id=\"Par15\">Finally, in the absence of sufficient research in the area of parent-only interventions—we aimed to explore the impact of the workshop and the 6-week program on sibling anxiety, parental anxiety and stress, and family dysfunction.</p>" ]
[ "<title>Methods</title>", "<title>Transparency and openness</title>", "<p id=\"Par16\">This research meets Level 1 (Disclosure) for all eight aspects of research planning and reporting of the TOP Guidelines as well as Level 2 (Requirement) for data citation, design and analysis transparency, and study and analysis plan preregistration. We report how we determined our sample size, all data exclusions (if any), all manipulations, and all measures in the study, and we follow Journal Article Reporting Standards (JARS) [##UREF##8##36##]). All data, analysis code, and research materials are available upon request from the authors. Data were analyzed using IBM Statistics SPSS Version 25 [##UREF##9##37##]. The trial—which was begun in 2015—was listed with the Australian and New Zealand Clinical Trials Registry (ACTRN 210 12615001284550), and included a basic analysis plan.</p>", "<title>Participants</title>", "<p id=\"Par17\">Participant families were recruited through the media and local schools in metropolitan Brisbane between September 2015 and July 2017. Interestingly, 12 participant families came from suburbs designated as ‘rural and regional’ by the Australian Department of Home Affairs. To be included in the study, children were required to be 7–14 years of age and to meet diagnostic criteria for a primary diagnosis of a DSM-5 [##UREF##10##38##] anxiety disorder. The exclusion criteria were (a) parent is unable to understand and participate in the treatment; (b) child is concurrently receiving ongoing treatment for anxiety; and (c) child has a significant physical or intellectual impairment. Primary anxiety disorder diagnoses included separation anxiety disorder (<italic>n</italic> = 7, 9.6%), social anxiety disorder (<italic>n</italic> = 19, 26.0%), specific phobia (<italic>n</italic> = 20, 27.4%), and generalized anxiety disorder (<italic>n</italic> = 27, 37.0%); there were no statistical differences in the number of children with each primary anxiety disorder diagnosis across the two treatment conditions. Participants were not excluded from the study if the child met criteria for additional anxiety disorders or for a co-morbid non-anxiety diagnosis. In fact, 84% (<italic>n</italic> = 61) of children met criteria for one or more comorbid anxiety disorder diagnoses and about 43% (<italic>n</italic> = 31) of participants met criteria for a secondary, non-anxiety diagnosis (e.g., ADHD, major depressive disorder, oppositional defiant disorder). The final sample included 73 children and adolescents (Mean age = 8.40 years, 74% male sex<xref ref-type=\"fn\" rid=\"Fn1\">1</xref>) and their parents. Participant, parent, and family characteristics are presented in Table ##TAB##0##1##.</p>", "<p id=\"Par19\">Additionally, as noted below, parents also completed a measure of anxiety symptomology for the participating child’s closest-in-age sibling. Of the 73 participating children, 64 had one or more siblings. Siblings ranged in age from 2 to 26-years-old (M = 8.09, SD = 4.14); 52% were male sex. Of the siblings, 42 (66%) were younger than the target child, 21 (33%) were older, and 1 was a twin. Furthermore, 33 (52%) of the sibling pairs were the same sex and 31 (48%) were the opposite sex. Whether the target child had a sibling or not, the age and sex of the sibling closest in age, and whether the sibling was older or younger or of the same or different sex than the targeted child did not differ significantly between the group and workshop conditions.</p>", "<p id=\"Par20\">Families were given a full description of the study before giving written informed assent/consent. A total of 77 families were tested for eligibility: three families did not meet the inclusion criteria and one family declined to participate. Thus, a total of 73 families were randomized to either the six-week group condition (<italic>n</italic> = 34) or the one-day workshop (<italic>n</italic> = 39). Participant flow through the study is summarized in Fig. ##FIG##0##1##.</p>", "<title>Procedure</title>", "<p id=\"Par21\">Ethical approval was obtained through the University of Queensland (#2014001727). Families attended an initial (pre) assessment interview at the university’s Psychology Clinic, where informed consent was obtained from all participants. Pre-treatment assessments were conducted by authors VC, IG, MJ and NRA (the first is an experienced clinical psychologist and researcher in the field of child anxiety; the other assessors were postgraduate clinical psychology trainees at the time). Assessments of child anxiety disorder and severity, children and siblings’ anxiety symptoms, parents’ own emotional symptoms, and family-level functioning were completed at four time-points: before treatment, 1-week post-treatment, and at 6- and 12-month follow-up. The assessment battery was completed by the target child’s mother in 79% of the families, by the father in 1% of the families, and by two parents (depending on timepoint) in 20% of the families. Families were compensated with a $20 gift card for each completed follow-up assessment.</p>", "<p id=\"Par22\">Parents were randomly allocated to either the six-week group or one-day workshop condition via a computerized random generator with a 1:1 ratio. Post-treatment and follow-up interviews were completed either face to face or via telephone. Four additional postgraduate clinical psychology trainees served as independent evaluators (IEs) and conducted all follow-up assessments. All IEs were blind to participants’ condition and the design of the study. All interviewers had previous experience in administering the ADIS-IV-C/P, with training having involved a mix of videotaped and live diagnostic interviews. All interviewers met a reliability criterion of 85% in terms of the inter-rater reliability rating for both diagnoses and clinical severity ratings between the trainee and an expert diagnostician (the first author). All interviews were recorded and another trained IE (not involved in conducting any of the interviews) viewed a random twenty percent of interviews over the course of the study in order to ensure there was no interviewer drift. The inter-rater reliability was excellent for the primary diagnosis assigned (K = 0.98).</p>", "<p id=\"Par23\">Of those randomized to treatment, retention of participants at the post-treatment, 6-month and 12-month time-points for the 6-week group was 85%, 82% and 71%. For those assigned to the one-day workshop, the rates were 77%, 72%, and 59%.</p>", "<title>Measures</title>", "<title>Clinician measures</title>", "<title>Structured diagnostic interviews with parents</title>", "<p id=\"Par24\">Caseness was determined based on outcomes of The Anxiety Disorders Interview Schedule for DSM-IV for Children—Parent Version [##UREF##11##39##]. The interview was modified to be consistent with the DSM-5 criteria [##UREF##10##38##]. Based on parent report, overall child anxiety diagnoses and clinical severity ratings (CSR) were assigned, where a CSR of the primary anxiety diagnosis of 4 or greater on a 9-point scale (moderate to severe) was considered to meet diagnostic criteria. Wherever possible, follow-up diagnostic interviews were conducted face to face at the Psychology Clinic, with a small proportion conducted over the telephone to accommodate parents. Research indicates that administration of the interview via telephone has good inter-rater reliability [##REF##9874902##40##] and good comparability to the face-to face version of the interview [##REF##15725972##41##].</p>", "<title>Clinical global impressions: improvement scale (CGI-I)</title>", "<p id=\"Par25\">The Clinical Global Impression—Improvement Scale (CGI-I), a seven-point scale ranging from 1 = very much improved to 7 = very much worse [##UREF##12##42##] was used to determine overall improvements in child anxiety. Scores of 1 and 2 indicate intervention success. Overall mean inter-rater reliability for the team of IEs was excellent (ICC = 0.917).</p>", "<title>Questionnaire measures</title>", "<title>Children’s anxiety symptoms</title>", "<p id=\"Par26\">Children completed the Spence Children Anxiety Scale (SCAS [##REF##9648330##43##]); a 45-item self-report measure of anxiety symptomology. It consists of six anxiety subscales that comprise a total score which is reported in the current study. Cronbach’s alpha in this study for the total score was 0.923. Parents also completed the parent version of the Spence Children Anxiety Scale (SCAS-P; [##REF##9648330##43##, ##REF##15149901##44##]) for the child in question (Cronbach's alpha in this study was 0.885).</p>", "<title>Family-level outcomes</title>", "<p id=\"Par27\">Sibling anxiety, parent anxiety and stress symptoms, and overall family functioning were assessed as secondary outcomes using the SCAS-P, the Depression Anxiety and Stress Scale (DASS-21 [##UREF##13##45##]), and the Family Assessment Device—General Functioning Subscale (FAD-GF [##UREF##14##46##]) respectively. Parents were asked to complete the SCAS-P about the sibling closest in age to the ‘identified child’. They also completed the Depression Anxiety and Stress Scale, a 21-item self-report adult measure designed to measure the symptoms of depression, anxiety and stress; the anxiety and stress scores are reported in the current study (Cronbach's alpha in this study was 0.738 [DASS Anxiety], and 0.825 [DASS Stress]). The FAD-GF is a 12-item self-report measure that utilizes a four-point Likert scale (1 = strongly agree and 4 = strongly disagree) to indicate problematic functioning in the family. Lower scores indicate better functioning. Internal consistency in this study was good (α = 0.858).</p>", "<title>Intervention</title>", "<p id=\"Par28\">Triple P—Positive Parenting Program [##REF##18729665##47##] is a public health approach designed to strengthen parenting and support families. It is a multilevel parenting intervention of varying intensities. The intervention is offered at five levels, ranging from a universal public communication campaign on positive parenting (Level 1) to intensive parenting interventions for severe and complex presentations within families (Level 5). Considerable evidence has been found for the efficacy of Triple P [##REF##18509758##48##, ##REF##24842549##49##].</p>", "<p id=\"Par29\">Fear-Less Triple P (FLTP [##UREF##6##31##, ##UREF##15##50##]) is a Level 4, Triple P intervention for childhood anxiety. The parent-only CBT intervention consists of a suite of programs allowing for flexibility of delivery. This study investigated the outcomes of delivery modes of the six-weekly group sessions and the one-day workshop format. FLTP is designed to empower parents to take on and enhance their role as the most powerful agent of change for their children. Based on principles of transfer of control and parental modeling, the program teaches parents about effective cognitive-behavioural strategies for managing anxiety, and targets parenting behaviours and family accommodations implicated in the etiology of childhood anxiety (e.g., overprotectiveness, encouragement of avoidance). Thus, it equips parents to ‘coach’ their children in learning cognitive-behavioural strategies for managing anxiety while also focusing on parent–child relationship dynamics in the context of responding to children’s anxiety. Content covered in FLTP includes: psychoeducation about anxiety and parents’ potential role in the maintenance of children’s anxiety; promoting emotional resilience in children; modelling; the role of thoughts in anxiety and mental flexibility; avoidance and exposure; parental strategies for responding to children’s anxiety; and problem solving. Key concepts are incorporated in homework tasks. For example, cognitive restructuring is practiced at home where children are asked by their parents to generate as many interpretations as possible of ambiguous hypothetical child-focused situations.</p>", "<title>Fear-less triple P group program</title>", "<p id=\"Par30\">The standard FLTP group program consists of six, 90-min weekly sessions (approximately 9 h). Seven groups were run (4–8 families per group) and delivered at the Psychology Clinic (on weekday evenings) by two postgraduate clinical trainee psychologists, trained in the intervention. Supervision was provided by the first author, a licensed clinical psychologist and the lead author of the program. Each weekly session included in-session activities and homework tasks to apply the core concepts and strategies. Families attended an average of 5.4 of the 6 sessions (SD = 0.77). Of the 34 families randomized to the group condition, 29 had only the mother attend each session, two had only the father attend, two had both mother and father attend each session, and one had the mother attend all six sessions while the father also attended two sessions.</p>", "<title>Fear-less triple P workshop</title>", "<p id=\"Par31\">The FLTP workshop consists of a 1-day program (6 h). It was delivered at the Psychology Clinic by a licensed clinical psychologist and co-facilitated with one or two postgraduate clinical psychology trainees who received training in the intervention. Three workshops were delivered (on weekend days) with each one attended by between 7 and 17 families. Of the 39 families assigned to the workshop condition, 26 had only the mother attend the workshop, three had only fathers attend, and 10 had two parents in attendance.</p>", "<title>Comparison of the two treatment modes</title>", "<p id=\"Par32\">Both group and workshop formats provided the same core therapeutic concepts, examples and activities; with both incorporating didactic content delivery (via PPT slide presentation and participants’ workbooks). Following treatment, all families were contacted for a brief (15–20 min) phone call one-week post-intervention to give caregivers a chance to review the strategies and problem-solve any concerns arising since program completion. In both the group and workshop modalities, all sessions were recorded. Independent research assistants, who were blind to the study design, reviewed 20% of randomly chosen therapy session recordings for treatment adherence. Of the planned intervention content, 100% was covered as intended in both formats.</p>", "<p id=\"Par33\">The delivery mode of intervention content was the main factor that distinguished the two modes. While both formats used the same PPT slide presentation and the parent workbook as their basis, families in the workshop condition had fewer opportunities to complete activities within the session (with only the most important activities and exercises done in-session) and instead were encouraged to work through these at home. Thus, while families in the workshop condition were engaged in some active learning activities, there was a heavier emphasis on didactic content presentation, with regular check-ins for questions. The conditions also differed in terms of the time allowed for in-group informal discussions. The six-week group program allowed opportunities for parents to not only network but also to discuss knowledge gained during previous weeks and share experiences, whereas the one-day workshop provided relatively little time for this to occur.</p>", "<title>Data analyses</title>", "<p id=\"Par34\">All analyses were conducted in IBM Statistics SPSS Version 25 [##UREF##9##37##]. Standard significance testing was used to explore all primary and secondary outcomes. Longitudinal, multi-level mixed models were used to explore whether SCAS-P total scores, SCAS-C total scores, FAD scores, sibling SCAS-P,<xref ref-type=\"fn\" rid=\"Fn2\">2</xref> and parental DASS stress and anxiety scores significantly improved over time and whether there were differences in the changes over time between the treatment conditions. In each model, assessment time points (Level 1) were nested within participants (Level 2) and treatment condition was a Level 2 predictor.</p>", "<p id=\"Par36\">Additionally, Pearson chi-square tests were used to determine whether the number of children who no longer met diagnostic criteria for their primary anxiety disorder or any anxiety disorder differed between the two conditions. Lastly, differences in parent satisfaction ratings at the post-treatment assessment and CGI scores at each of the follow-up assessments were assessed using independent samples <italic>t</italic>-tests.</p>", "<p id=\"Par37\">Missing data at the follow-up assessment time points was accounted for using the multiple imputation procedure in SPSS for all analyses except for the longitudinal, multi-level models. In those analyses, missing data was accounted for by using restricted maximum likelihood estimation which allowed all data at each time point to be utilized without excluding participants who did not complete all measures or attend every time point.</p>", "<p id=\"Par38\">Power analyses indicated that with α = 0.025 and power = 0.80, our acquired sample size was sufficient to detect moderate to large effect sizes, but not small effect sizes.</p>" ]
[ "<title>Results</title>", "<p id=\"Par39\">See Table ##TAB##1##2## for all variable means and standard deviations at each assessment session.</p>", "<title>Child level outcomes</title>", "<title>Anxiety diagnoses</title>", "<p id=\"Par40\">Chi-square analyses indicated that there were no significant differences in the number of children who no longer met criteria for their primary anxiety disorder at post-treatment, χ<sup>2</sup>(1, <italic>N</italic> = 73) = 0.06, <italic>p</italic> = 0.808, 6-month follow-up, χ<sup>2</sup>(1, <italic>N</italic> = 73) = 1.94, <italic>p</italic> = 0.164, or 12-month follow-up, χ<sup>2</sup>(1, <italic>N</italic> = 73) = 0.30, <italic>p</italic> = 0.586, assessments. Of the 39 participants in the workshop condition, 23 (59.0%), 31 (79.5%), and 34 (87.2%) were diagnosis free of their primary anxiety disorder at the post-treatment, 6-month follow-up, and 12-month follow-up assessments, respectively. Of the 34 participants in the group condition, 21 (61.8%), 31 (91.1%), and 31 (91.1%) were free of their primary anxiety disorder at the post-treatment, 6-month follow-up, and 12-month follow-up assessments, respectively (Fig. ##FIG##1##2##). Mean clinical severity ratings (CSRs) of the primary diagnosis reflect the diagnostic status data and are presented in Table ##TAB##1##2##.</p>", "<p id=\"Par41\">Furthermore, chi-square analyses indicated that there were no significant differences in the number of children who no longer met criteria for <italic>any</italic> anxiety disorder at the post-treatment, χ<sup>2</sup> (1, <italic>N</italic> = 73) = 0.21, <italic>p</italic> = 0.644, 6-month follow-up, χ<sup>2</sup>(1, <italic>N</italic> = 73) = 0.00, <italic>p</italic> = 0.964, or 12-month follow-up, χ<sup>2</sup> (1, <italic>N</italic> = 73) = 3.06, <italic>p</italic> = 0.080, assessments. Of the 39 participants in the workshop condition, 14 (35.9%), 30 (76.9%), and 34 (87.2%) were free of all anxiety disorders at the post-treatment, 6-month follow-up, and 12-month follow-up assessments, respectively. Of the 34 participants in the group condition, 14 (41.2%), 26 (76.5%), and 24 (70.6%) were free of all anxiety disorders at the post-treatment, 6-month follow-up, and 12-month follow-up assessments, respectively (Fig. ##FIG##1##2##).</p>", "<title>Anxiety symptoms</title>", "<p id=\"Par42\">SCAS-P scores significantly reduced over time,<italic> F</italic>(3, 71) = 6.94, <italic>p</italic> &lt; 0.001, and the interaction between assessment session and treatment condition was not significant using the p &lt; 0.025 criterion,<italic> F</italic>(3, 71) = 3.93, <italic>p</italic> = 0.034, indicating that improvement in SCAS-P scores across the four assessment sessions did not differ between the treatment conditions (Fig. ##FIG##2##3##).</p>", "<p id=\"Par43\">Significance testing indicated that, although on average SCAS-C total scores reduced across time, this reduction was not statistically significant,<italic> F</italic>(3, 71) = 2.33, <italic>p</italic> = 0.088, and did not vary by treatment condition,<italic> F</italic>(3, 71) = 1.04, <italic>p</italic> = 0.386 (Fig. ##FIG##2##3##).</p>", "<title>Global improvement</title>", "<p id=\"Par44\">At the post-treatment assessment there was a significant difference in IE rated improvement between the two conditions, <italic>t</italic>(71) = 2.90, <italic>p</italic> = 0.004. Mean CGI scores indicated that participants in the workshop condition were “minimally improved” while participants in the group condition were “much improved.” However, at the 6-month, <italic>t</italic>(71) = 0.75, <italic>p</italic> = 0.455, and 12-month, <italic>t</italic>(71) = -0.99, <italic>p</italic> = 0.323, follow-up assessments the difference between the conditions was nonsignificant and, on average, participants in both conditions were rated as being “much improved.”</p>", "<title>Family level outcomes</title>", "<title>Sibling anxiety</title>", "<p id=\"Par45\">Mixed-models analysis demonstrated that across the two interventions Sibling SCAS-P scores significantly reduced over time, <italic>F</italic>(3, 71) = 5.49, <italic>p</italic> = 0.009. However, the time by group analysis was not significant,<italic> F</italic>(3, 71) = 1.01, <italic>p</italic> = 0.345, indicating that improvement in SCAS-P scores did not differ between the treatment conditions (Fig. ##FIG##2##3##).</p>", "<title>Parental stress and anxiety</title>", "<p id=\"Par46\">Results for parental scores on the stress and anxiety subscales of the DASS were similar; across the two interventions parental stress,<italic> F</italic>(3, 71) = 0.78, <italic>p</italic> = 0.512, and anxiety,<italic> F</italic>(3, 71) = 1.12, <italic>p</italic> = 0.355, did not significantly change over time, nor was there a significant interaction between time and treatment condition for stress, <italic>F</italic>(3, 71) = 0.17, <italic>p</italic> = 0.917, or anxiety, <italic>F</italic>(3, 71) = 0.70, <italic>p</italic> = 0.558. Importantly, parental stress and anxiety scores fell in the “normal” range at the pre-treatment assessment and remained in that range at each of the follow-up sessions.</p>", "<title>Family functioning</title>", "<p id=\"Par47\">Mixed-models analysis demonstrated that across the two interventions FAD-GF family functioning scores did not significantly change over time,<italic> F</italic>(3, 71) = 2.95, <italic>p</italic> = 0.046, nor was there a significant interaction between time and treatment condition, <italic>F</italic>(3, 71) = 1.02, <italic>p</italic> = 0.395.</p>", "<title>Treatment satisfaction</title>", "<p id=\"Par48\">Parent satisfaction ratings were high at the post-treatment assessment. The difference in parent satisfaction, <italic>t</italic>(71) = 0.183, <italic>p</italic> = 0.855 between the two interventions was nonsignificant.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par49\">The present pilot study provides preliminary confirmation for the primary hypothesis that FLTP, a parent-only intervention for childhood anxiety would produce comparable positive diagnostic outcomes when delivered in a one-day workshop format compared to a multi-session group format. Thus, 76.9% and 87.2% of children whose parents were assigned to the workshop were free of any anxiety disorder at 6- and 12-month follow-up respectively, compared to 76.5% and 70.6% of children whose parents were assigned to the 6-week group. Moreover, at post-treatment, 59% and 61.8% of children whose parents were assigned to the one-day workshop and 6-week group conditions respectively were free of their primary diagnosis. These results are consistent with post-treatment results for other parent-focused programs such as the 12-session SPACE program (68.8% diagnosis free [##REF##30851397##24##]) and guided parent-delivered CBT (50% diagnosis free for the full program—two face to face and four telephone sessions—and 39% for the brief program—two face to face and two telephone sessions [##REF##23969483##21##]). Of these two parent-focused interventions, outcomes beyond post-treatment were reported only for the guided parent-delivered CBT intervention (53% free of any anxiety diagnosis for the full guided CBT group and 55% anxiety diagnosis free for the brief guided CBT group at 6-month follow-up [##REF##23969483##21##]). Together, these findings appear to provide additional support for the effectiveness of FLTP [##REF##28641122##19##] both in the 6-week and one-day workshop formats. To the best of our knowledge, the one-day workshop format of FLTP is the first BIC for transdiagnostic anxiety disorders in children to be evaluated. The diagnostic outcomes produced by this protocol represent exciting—if preliminary—new directions.</p>", "<p id=\"Par50\">Data from the questionnaire measures of anxiety symptomatology and the IE-rated improvement scale provide varying levels of support for the effectiveness of FLTP and the lack of difference between the workshop and 6-week group formats. Parental report on the SCAS-P indicated a significant reduction over time, with no difference found between treatment conditions. However, while child reports on the SCAS-C reduced over time, these reductions were not statistically significant. No difference was found between the treatment conditions. It is worth noting that there was a very large range in children’s scores on the SCAS-C (in both treatment conditions and at all time points), as reflected in the large standard deviations. This may have contributed to the failure to find a significant reduction over time.</p>", "<p id=\"Par51\">IE ratings of improvement on the CGI-I indicated that, by the 6- and 12-month follow-up assessments, there was no difference in improvement ratings across the conditions—with all participants, on average, being rated as ‘much improved’. As hypothesized, both formats of FLTP were highly acceptable and satisfactory to families, with no differences found between conditions.</p>", "<p id=\"Par52\">Finally, we explored the impact of FLTP on family-level outcomes. The present study extends the existing literature by showing that both FLTP formats produced comparable positive effects at follow up for the siblings of children with anxiety problems. This finding is important as a significant number of children with diagnosed anxiety problems have siblings with similar problems. If an intervention targeting one child can produce changes in parenting practices that can then be applied to other children in the family, the family level benefits can be considerable. The precise mechanisms through which treatment affected siblings are not clear. Parents may have simply applied the same techniques to different children in the family, siblings may have learned from their siblings’ experiences through observational learning, or reductions in sibling avoidance may have reduced the opportunity for siblings to become anxious in similar situations. Although mean-level family functioning improved from pre-treatment to the 12-month follow-up assessment, there were no significant time or time by condition effects obtained. This was also the case for maternal stress and anxiety scores on the DASS. However, these scores were in the normal range at pre-treatment, with little room for change. Future research could examine the mechanisms through which parenting interventions influence siblings and potentially counter family stress levels when they are observed.</p>", "<p id=\"Par53\">It is important to note that, all findings must be considered in light of the fact that this study was under-powered to detect small between-group differences. Given this limitation, findings in relation to the lack of between-group differences must be interpreted with caution.</p>", "<p id=\"Par54\">Collectively these preliminary findings add to the growing evidence that the systematic targeting of family interactional processes hypothesized to maintain childhood anxiety can be effective treatments in their own right, producing outcomes comparable to “gold standard” CBT interventions for childhood anxiety. The findings are consistent with the wider parenting literature that demonstrates that, for parents of children with conduct problems, low intensity parent-only interventions delivered in a brief, intensive group format or as self-directed online can be as effective as the same content delivered in person in more sessions over a longer time period [##REF##24842549##49##, ##REF##28167330##51##, ##REF##32220142##52##].</p>", "<p id=\"Par55\">Parenting programs that can be delivered in a one-day workshop format are likely to be much more cost effective and accessible for parents as they involve less total time for the parent, lower transportation expenses (less time, fewer trips, reduced parking costs), and reduced likelihood of parents dropping out or missing sessions. However, new funding mechanisms are needed to enable practitioners to be reimbursed for delivering these intensive programs rather than the more customary individual consultation sessions on an hourly basis.</p>", "<p id=\"Par56\">Interestingly, in this study, despite the metropolitan-focused recruitment, over 16% of participant families lived in areas of the state classified as rural and regional—a higher proportion of non-metropolitan families than we have seen in our previous trials. During the assessment process, numerous parents (particularly rural and regional families) anecdotally expressed a preference for the one-day workshop; with many families explicitly requesting assignment to the workshop condition (both before and after assignment) and requiring an explanation of random assignment. Many participant parents reflected on the attraction of a ‘one-off’ intervention, with reduced time, travel and need for childcare the most cited advantages. Relatedly, of two-parent families, a greater percentage had both parents in attendance for the one-day workshop condition compared to the 6-week group condition—reflecting (according to participant feedback) the fact that it is easier to arrange childcare for a single event. Based on these anecdotal observations, it is proposed that a one-day workshop parenting program offers considerable advantages for parents—especially rural and regional parents—compared to a weekly group program (even one as brief as the 6-session FLTP program). The significance of this study lies not in the finding that a 6-h treatment produces similar outcomes to a 9-h treatment; but rather in the finding that a one-day workshop format of FLTP appears to produce similar outcomes to a multi-session group format of the same intervention.</p>", "<p id=\"Par57\">The present findings need to be interpreted in light of the strengths and limitations of our study. Strengths include use of a randomized design, comprehensive outcome assessment including clinical diagnostic measures to establish caseness, multi-informant assessment, a sample with significant non-anxiety comorbidities, high fidelity delivery of both intervention conditions, and inclusion of measures (e.g., family functioning) to study putative mechanism of change. Limitations include the small sample and subsequent lack of power to detect small effect sizes; and recruitment of a sample through community outreach rather than case ascertainment through clinical referral. This latter limitation is mitigated somewhat by the requirement that all participating children met diagnostic criteria and all children experienced significant and interfering levels of anxiety. The study is also constrained by limited data from fathers and the fact that the diagnostic interview was completed by parents only. Participating parents were a relatively homogeneous sample in terms of ethnicity, income, education level and marital status, with an under-representation of more social disadvantaged or minority families. This clearly limits the generalizability of findings. Another important limitation is the attrition rate of participants over follow-up—in particular the workshop participants at the 12-month follow-up point. Data are not available on whether any participating families accessed additional therapeutic services during the follow up period. Although measures of mechanisms of change were included, these were not explored statistically in this paper. This represents an important future direction. Relatedly, it would have been useful to have included a measure of family accommodation in this study—the lack of measurement of this construct is a limitation. It is also noteworthy that our observations about the cost effectiveness of the intervention will require further validation through assessment of the actual costs and benefits incurred by both parents and practitioners. Finally, we do not know whether parent preferences interact with the observed outcomes. It is possible that reception of the preferred delivery format could influence parent satisfaction with treatment or child outcomes.</p>", "<p id=\"Par58\">In a world where childhood anxiety is on the rise; “in person” attendance at health services has become increasingly difficult; and access to mental health services has never been more challenging, two observations in relation to FLTP are worth making. First, an obvious area for future program innovation and evaluation is the development and evaluation of both telehealth delivery and an online format of FLTP. Second, and finally, the advantages and need for an efficacious and brief parent-only program in treating childhood anxiety may never have been more relevant.</p>" ]
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[ "<title>Objective</title>", "<p id=\"Par1\">Parent-only cognitive-behavioural therapy (CBT) interventions have promise for youth with anxiety disorders. Fear-Less Triple P (FLTP) is one such intervention that has been found comparable to child-focused CBT. Although traditionally administered in six sessions, a one-day workshop format of FLTP was developed to improve accessibility. The current study compared the effectiveness of the six-session and one-day workshop formats.</p>", "<title>Method</title>", "<p id=\"Par2\">Seventy-three youth (mean age, 8.4 years; 74% male) were randomized to traditional FLTP (6-week group) or the one-day workshop format. Anxiety diagnostic status, self- and parent-reported anxiety symptoms scores, independent evaluator-rated improvement, treatment satisfaction, and measures of family functioning were included to assess treatment outcome. Data were collected prior to treatment, and 1-week, 6-months, and 12-months following treatment.</p>", "<title>Results</title>", "<p id=\"Par3\">Both conditions resulted in significant improvement in child anxiety symptom scores per parent report (on both questionnaire and diagnostic interview measures). Furthermore, significant decreases in sibling anxiety were observed in both treatment conditions. There were no statistically significant differences between conditions on any outcome measure.</p>", "<title>Conclusions</title>", "<p id=\"Par4\">Results of this study add to the growing evidence that brief, low-intensity, parent-only interventions can effectively target child psychopathology. These brief interventions are ideal for families for whom the resources and time required to commit to a standard multi-week intervention are prohibitive.</p>", "<p id=\"Par5\"><italic>Registration of Clinical Trials</italic>: This trial was registered with the Australian and New Zealand Clinical Trials Registry (ACTRN 12615001284550).</p>", "<title>Keywords</title>" ]
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[ "<title>Acknowledgements</title>", "<p>The authors wish to thank the parents and children who took part in this trial; as well as the many postgraduate clinical psychology students from the University of Queensland who assisted. In the public, commercial, or not-for-profit sectors.</p>", "<title>Author contributions</title>", "<p>VC: conceptualization, methodology, investigation, project administration, supervision, writing—original draft preparation. SR: data curation, formal analysis, writing—original draft preparation. IH: investigation, project administration, writing—review and editing. MJ<italic>:</italic> investigation, project administration, writing—review and editing. NRA: investigation, project administration, writing—review and editing. TO: conceptualization, formal analysis, writing- reviewing and editing. MS: conceptualization, writing- reviewing and editing.</p>", "<title>Funding</title>", "<p>This research did not receive any specific grant from funding agencies.</p>", "<title>Availability of data and materials</title>", "<p>This research meets Level 1 (Disclosure) for all eight aspects of research planning and reporting of the TOP Guidelines as well as Level 2 (Requirement) for data citation, design and analysis transparency, and study and analysis plan preregistration. We report how we determined our sample size, all data exclusions (if any), all manipulations, and all measures in the study, and we follow Journal Article Reporting Standards (JARS) [##UREF##8##36##]). All data, analysis code, and research materials are available upon request from the authors. Data were analyzed using IBM Statistics SPSS Version 25 [##UREF##9##37##]. The trial – which was begun in 2015 – was listed with the Australia and New Zealand Clinical Trials Registry (ACTRN 12615001284550), included a basic analysis plan.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par59\">Ethical approval was obtained through the University of Queensland, Australia. HREC reference #2014001727.</p>", "<title>Competing interests</title>", "<p id=\"Par60\">The Parenting and Family Support Centre is partly funded by royalties stemming from published resources of the Triple P—Positive Parenting Program, which is developed and owned by The University of Queensland (UQ). Royalties are also distributed to the Faculty of Health and Behavioural Sciences at UQ and contributory authors of published Triple P resources. Triple P International (TPI) Pty Ltd is a private company licensed by Uniquest Pty Ltd on behalf of UQ, to publish and disseminate Triple P worldwide. The authors of this report have no share or ownership of TPI. Dr. Cobham and Dr. Sanders may in future receive royalties and/or consultancy fees from TPI. TPI had no involvement in the study design, collection, analysis or interpretation of data, or writing of this report. Drs. Cobham and Sanders are employees at UQ. The other authors have no competing interests disclosure to make.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Participant flow. Received intervention: participant received the allocated treatment; withdrew: participant voluntarily withdrew from the study and had no further contact with the study; lost to contact: participant was unable to be contacted by the study; did not attend: participant failed to attend scheduled assessment. Remained in study and attended the next assessment point</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Percentage of participants diagnosis free of their primary anxiety disorder or all anxiety disorders across time</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Spence child anxiety scale scores across time</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Participant characteristics</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">M(SD)/<italic>n</italic>(%)</th><th align=\"left\" rowspan=\"2\">t/χ<sup>2</sup></th></tr><tr><th align=\"left\">Group (N = 34)</th><th align=\"left\">Workshop (N = 39)</th></tr></thead><tbody><tr><td align=\"left\">Child age</td><td char=\"(\" align=\"char\">8.65 (1.63)</td><td char=\"(\" align=\"char\">8.18 (1.57)</td><td char=\".\" align=\"char\">−1.25</td></tr><tr><td align=\"left\">Child sex (% male)</td><td char=\"(\" align=\"char\">26 (76.5)</td><td char=\"(\" align=\"char\">28 (71.8)</td><td char=\".\" align=\"char\">0.21</td></tr><tr><td align=\"left\">Number of siblings</td><td char=\"(\" align=\"char\">1.35 (0.76)</td><td char=\"(\" align=\"char\">1.43 (1.24)</td><td char=\".\" align=\"char\">0.31</td></tr><tr><td align=\"left\">Mother education (% completed undergraduate or postgraduate) (n = 28 and 36)</td><td char=\"(\" align=\"char\">23 (82.1)</td><td char=\"(\" align=\"char\">21 (58.3)</td><td char=\".\" align=\"char\">4.27</td></tr><tr><td align=\"left\">Father education (% completed undergraduate or postgraduate) (n = 14 and 16)</td><td char=\"(\" align=\"char\">12 (85.7)</td><td char=\"(\" align=\"char\">8 (50.0)</td><td char=\".\" align=\"char\">4.95</td></tr><tr><td align=\"left\">Mother ethnicity (% white) (n = 26 and 37)</td><td char=\"(\" align=\"char\">26 (100)</td><td char=\"(\" align=\"char\">33 (89.2)</td><td char=\".\" align=\"char\">3.00</td></tr><tr><td align=\"left\">Father ethnicity (% white) (n = 14 and 14)</td><td char=\"(\" align=\"char\">13 (92.9)</td><td char=\"(\" align=\"char\">13 (92.9)</td><td char=\".\" align=\"char\">2.00</td></tr><tr><td align=\"left\">Parental marital status (% parents married) (n = 31 and 37)</td><td char=\"(\" align=\"char\">28 (90.3)</td><td char=\"(\" align=\"char\">28 (75.7)</td><td char=\".\" align=\"char\">4.51</td></tr><tr><td align=\"left\">Parental combined salary (% &gt; $100,000) (n = 30 and 37)</td><td char=\"(\" align=\"char\">25 (83.3)</td><td char=\"(\" align=\"char\">29 (78.4)</td><td char=\".\" align=\"char\">7.82</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Variable means and standard deviations across time</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Pre-treatment</th><th align=\"left\" colspan=\"2\">Post-treatment</th><th align=\"left\" colspan=\"2\">6 month follow-up</th><th align=\"left\" colspan=\"2\">12 month follow-up</th></tr><tr><th align=\"left\">G</th><th align=\"left\">W</th><th align=\"left\">G</th><th align=\"left\">W</th><th align=\"left\">G</th><th align=\"left\">W</th><th align=\"left\">G</th><th align=\"left\">W</th></tr></thead><tbody><tr><td align=\"left\">CSR</td><td align=\"left\">5.50 (1.11)</td><td align=\"left\">5.56 (1.33)</td><td align=\"left\">2.17 (2.56)</td><td align=\"left\">2.52 (2.54)</td><td align=\"left\">0.48 (1.48)</td><td align=\"left\">1.52 (2.55)</td><td align=\"left\">0.92 (2.14)</td><td align=\"left\">1.15 (2.46)</td></tr><tr><td align=\"left\">SCAS-P</td><td align=\"left\">34.28 (12.60)</td><td align=\"left\">32.70 (14.52)</td><td align=\"left\">21.58 (9.83)</td><td align=\"left\">27.92 (12.26)</td><td align=\"left\">21.39 (7.06)</td><td align=\"left\">24.52 (10.78)</td><td align=\"left\">21.11 (10.10)</td><td align=\"left\">23.03 (14.65)</td></tr><tr><td align=\"left\">SCAS-C</td><td align=\"left\">28.44 (19.45)</td><td align=\"left\">32.18 (16.76)</td><td align=\"left\">27.99 (15.02)</td><td align=\"left\">30.69 (19.55)</td><td align=\"left\">23.33 (14.89)</td><td align=\"left\">26.34 (16.51)</td><td align=\"left\">22.68 (22.02)</td><td align=\"left\">22.63 (19.50)</td></tr><tr><td align=\"left\">CGI</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">2.14 (0.94)</td><td align=\"left\">2.89 (1.03)</td><td align=\"left\">1.87 (0.87)</td><td align=\"left\">2.09 (1.20)</td><td align=\"left\">2.18 (1.25)</td><td align=\"left\">1.84 (1.05)</td></tr><tr><td align=\"left\">PSS</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">62.75 (5.55)</td><td align=\"left\">63.01 (6.27)</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">FAD</td><td align=\"left\">1.79 (0.48)</td><td align=\"left\">1.76 (0.41)</td><td align=\"left\">1.81 (0.47)</td><td align=\"left\">1.79 (0.48)</td><td align=\"left\">1.78 (0.46)</td><td align=\"left\">1.77 (0.53)</td><td align=\"left\">1.67 (0.51)</td><td align=\"left\">1.63 (0.48)</td></tr><tr><td align=\"left\">SCAS-P (Sibling)</td><td align=\"left\">17.59 (11.96)</td><td align=\"left\">19.53 (9.42)</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">11.85 (4.13)</td><td align=\"left\">13.60 (5.95)</td><td align=\"left\">9.93 (4.20)</td><td align=\"left\">11.85 (4.61)</td></tr><tr><td align=\"left\">DASS-S</td><td align=\"left\">6.12 (3.79)</td><td align=\"left\">7.29 (4.53)</td><td align=\"left\">5.83 (5.76)</td><td align=\"left\">6.42 (6.33)</td><td align=\"left\">5.84 (7.04)</td><td align=\"left\">6.51 (7.80)</td><td align=\"left\">5.83 (11.67)</td><td align=\"left\">7.35 (13.18)</td></tr><tr><td align=\"left\">DASS-A</td><td align=\"left\">1.73 (2.65)</td><td align=\"left\">2.53 (3.06)</td><td align=\"left\">1.10 (2.22)</td><td align=\"left\">1.56 (2.56)</td><td align=\"left\">0.96 (2.75)</td><td align=\"left\">1.76 (3.48)</td><td align=\"left\">0.84 (8.40)</td><td align=\"left\">0.92 (9.34)</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>All t and χ<sup>2</sup> p &gt; 0.10</p></table-wrap-foot>", "<table-wrap-foot><p><italic>G</italic> group (N = 34), <italic>W</italic> workshop (N = 39), <italic>CSR</italic> clinical severity rating, <italic>SCAS-P</italic> spence child anxiety scale—parent report, <italic>SCAS-C</italic> spence child anxiety scale—child report, <italic>CGI</italic> clinical global impression—improvement, <italic>PSS</italic> parent satisfaction survey, <italic>CSS</italic> client satisfaction survey, <italic>FAD</italic> family assessment device—general functioning scale, <italic>DASS-S</italic> depression, anxiety, and stress scale—stress subscale;—measure not administered at that session</p></table-wrap-foot>", "<fn-group><fn id=\"Fn1\"><label>1</label><p id=\"Par18\">Sex was defined in a binary fashion by the biological attributes at birth that are associated with physical and physiological features of female and male sex.</p></fn><fn id=\"Fn2\"><label>2</label><p id=\"Par35\">Siblings who were under 4-years-old and over 18-years-old (n = 5) were considered outliers and excluded from the analyses presented below. However, the sibling SCAS-P analyses were also run with the outliers included and the results did not differ.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[{"label": ["2."], "surname": ["Jongerden", "B\u00f6gels"], "given-names": ["L", "SM"], "article-title": ["Parenting, family functioning and anxiety-disordered children: comparisons to controls, changes after family versus child CBT"], "source": ["J Child Fam Stud"], "year": ["2015"], "volume": ["24"], "issue": ["7"], "fpage": ["2046"], "lpage": ["2059"], "pub-id": ["10.1007/s10826-014-0005-6"]}, {"label": ["8."], "surname": ["Farrell", "Ollendick", "Muris"], "given-names": ["L", "TH", "P"], "source": ["Innovations in CBT treatment for childhood anxiety, OCD, and PTSD"], "year": ["2019"], "publisher-loc": ["Cambridge"], "publisher-name": ["Cambridge University Press"]}, {"label": ["9."], "surname": ["James", "James", "Cowdrey", "Soler", "Choke"], "given-names": ["AC", "G", "FA", "A", "A"], "source": ["Cognitive behavioural therapy for anxiety disorders in children and adolescents. Cochrane database of systematic reviews"], "year": ["2013"], "publisher-loc": ["Chichester, UK"], "publisher-name": ["John Wiley & Sons Ltd"]}, {"label": ["26."], "surname": ["Dia", "Harrington"], "given-names": ["DA", "D"], "article-title": ["What about me? Siblings of children with an anxiety disorder"], "source": ["Soc Work Res"], "year": ["2006"], "volume": ["30"], "issue": ["3"], "fpage": ["183"], "lpage": ["188"], "pub-id": ["10.1093/swr/30.3.183"]}, {"label": ["27."], "surname": ["Downey", "Condron"], "given-names": ["DB", "DJ"], "article-title": ["Playing well with others in kindergarten: the benefit of siblings at home"], "source": ["J Marriage Fam"], "year": ["2004"], "volume": ["66"], "issue": ["2"], "fpage": ["333"], "lpage": ["350"], "pub-id": ["10.1111/j.1741-3737.2004.00024.x"]}, {"label": ["28."], "surname": ["Kramer"], "given-names": ["L"], "article-title": ["Learning emotional understanding and emotion regulation through sibling interaction"], "source": ["Early Educ Dev"], "year": ["2014"], "volume": ["25"], "issue": ["2"], "fpage": ["160"], "lpage": ["184"], "pub-id": ["10.1080/10409289.2014.838824"]}, {"label": ["31."], "mixed-citation": ["Cobham VE, Sanders MR. Fear-less Triple P. Group workbook for parents of anxious children. Brisbane, Australia: University of Queensland. 2015."]}, {"label": ["32."], "surname": ["Lawrence", "Johnson", "Hafekost", "Zubrick"], "given-names": ["D", "S", "J", "S"], "source": ["The mental health of children and adolescents. Report on the second Australian Child and Adolescent Survey of Mental Health and Wellbeing"], "year": ["2015"], "publisher-loc": ["Canberra"], "publisher-name": ["Springer"]}, {"label": ["36."], "mixed-citation": ["Kazak AE. Journal article reporting standards. 2018."]}, {"label": ["37."], "surname": ["Corp"], "given-names": ["IBM"], "source": ["IBM SPSS statistics for windows, version 25.0"], "year": ["2017"], "publisher-loc": ["Armonk, NY"], "publisher-name": ["IBM Corp"]}, {"label": ["38."], "collab": ["APA"], "source": ["Diagnostic and statistical manual of mental disorders"], "year": ["2013"], "edition": ["5"], "publisher-loc": ["Washington, DC"], "publisher-name": ["APA (DSM-V)"]}, {"label": ["39."], "surname": ["Silverman", "Albano"], "given-names": ["WK", "AM"], "source": ["Anxiety disorders interview schedule for DSM-IV: parent interview schedule (Vol. 1)"], "year": ["1996"], "publisher-loc": ["San Antonio, TX"], "publisher-name": ["Psychological Corporation"]}, {"label": ["42."], "surname": ["Guy"], "given-names": ["W"], "source": ["Clinical global impressions. In: ECDEU assessment manual for psychopharmacology"], "year": ["1976"], "publisher-loc": ["Rockville, MD"], "publisher-name": ["National Institute of Mental Health"], "fpage": ["217"], "lpage": ["221"]}, {"label": ["45."], "surname": ["Lovibond", "Lovibond"], "given-names": ["SH", "PF"], "source": ["Manual for the depression anxiety stress scales"], "year": ["1995"], "edition": ["2"], "publisher-loc": ["Sydney"], "publisher-name": ["Psychology Foundation"]}, {"label": ["46."], "surname": ["Epstein", "Baldwin", "Bishop"], "given-names": ["NB", "LM", "DS"], "article-title": ["The McMaster family assessment device"], "source": ["J Marital Fam Ther"], "year": ["1983"], "volume": ["9"], "issue": ["2"], "fpage": ["171"], "lpage": ["180"], "pub-id": ["10.1111/j.1752-0606.1983.tb01497.x"]}, {"label": ["50."], "mixed-citation": ["Cobham VE, Sanders MR. Fear-less Triple P. Group workbook for parentsof anxious children. Brisbane, Australia: University of Queensland. 2009."]}]
{ "acronym": [], "definition": [] }
52
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2024-01-14 23:43:47
Child Adolesc Psychiatry Ment Health. 2024 Jan 13; 18:8
oa_package/6c/58/PMC10787495.tar.gz
PMC10787497
38216873
[ "<title>Background</title>", "<p id=\"Par4\">\n<italic>Riemerella anatipestifer</italic> (<italic>R. anatipestifer</italic>, RA) is an important veterinary pathogen belonging to the family <italic>Weeksellaceae</italic> [##REF##31608019##1##]. RA poses a threat to domestic ducks, geese, and turkeys, causing acute or chronic septicemia [##UREF##0##2##]. Since 1982, when Bisgaard established the <italic>R. anatipestifer</italic> serotyping scheme [##REF##18770200##3##], at least 21 serovars have been reported around the world [##REF##12396349##4##]. And there was no cross-reactivity between different serovars [##REF##26017672##5##]. There are several sets of reference strains that have been used for serovar designation by different groups [##REF##18770200##3##, ##REF##18680018##6##, ##REF##23391177##7##]. Recently, Omaleki et al. reconfirmed the serovar reference strains and identified 17 different serovars, which is the latest validated serotyping scheme [##REF##32990455##8##]. Nonetheless, the serotyping of <italic>R. anatipestifer</italic> still relies heavily on reference strains and antisera, and no molecular serotyping methods have been proposed.</p>", "<p id=\"Par5\">In most bacteria, the surface polysaccharide structures exhibit intraspecies diversity, which is usually associated with serological phenotype [##REF##29885806##9##, ##REF##33505361##10##]. The capsular polysaccharide (CPS) synthesis gene cluster was frequently utilized as a molecular serotyping target due to its genetic diversity [##REF##29885806##9##, ##REF##19521509##11##, ##REF##18508935##12##]. Notably, due to the high correlation between the genetic signature of CPS gene cluster and the serovar phenotype, CPS related genes have been widely used as targets for molecular serotyping of many bacteria [##REF##18508935##12##–##REF##28551457##14##]. The most commonly targeted genes are <italic>wzx</italic> and <italic>wzy</italic>, which encode the oligosaccharide unit flippase (Wzx) and polymerase (Wzy), respectively. This gene pair plays a crucial role in CPS synthesis, which is directly linked to the specificity of CPS [##REF##19521509##11##, ##REF##35404221##13##]. Several studies have focused on the genes that are related to lipopolysaccharide (LPS) and CPS in <italic>R. anatipestifer</italic> [##REF##26266750##15##–##REF##30223890##17##], but the information they provide regarding serovar characteristics is limited. Previous studies have speculated that the serological characteristics of <italic>R. anatipestifer</italic> are associated with surface polysaccharides [##UREF##1##18##]. Our recent study indicates that the CPS of <italic>R. anatipestifer</italic> determines the serological specificity of serovar 2, rather than the LPS [##UREF##2##19##], while the genetic loci determining the serovars of <italic>R. anatipestifer</italic> remain unclear.</p>", "<p id=\"Par6\">With the development of whole-genome sequencing technology and the accumulation of genomic data, genome-wide association studies (GWAS) have become a powerful tool in bacterial research to reveal the genetic basis of important phenotypes. Recently, several methods have been introduced to assess the correlation between bacterial phenotypes and genotypes [##REF##27887642##20##, ##UREF##3##21##]. Among these, the pan-genome-wide association study (Pan-GWAS), based on the typical gene presence/absence features of bacterial accessory genomes, has proven effective in studies of bacterial resistance and pathogenicity [##UREF##4##22##, ##REF##33097713##23##]. There have been several pan-genomic studies of <italic>R. anatipestifer</italic> [##UREF##5##24##, ##REF##31004458##25##]. However, to the best of our knowledge, no study has used the pan-GWAS approach to determine the association between genotypes and phenotypes in <italic>R. anatipestifer</italic>.</p>", "<p id=\"Par7\">In the current study, we used pan-GWAS to identify the genetic loci associated with serovars. And we further analyzed the biological functions of the serologically associated genetic loci and preliminarily characterized our findings using gene knockout methods. These findings will provide a basis for further exploration of the molecular mechanism of <italic>R. anatipestifer</italic> serological phenotypes and provide direction for the establishment of molecular serotyping methods.</p>" ]
[ "<title>Materials and methods</title>", "<title>Bacterial strain and whole-genome sequencing</title>", "<p id=\"Par8\">The <italic>R. anatipestifer</italic> strains and the published genome data employed in this study are listed in Supplementary Table ##SUPPL##0##1##. One representative strain of each serovar was chosen for further presentation. Serovar representative strains and serotyping information were shown in Table ##TAB##0##1##. And all representative strains were obtained from the Culture Collection University of Gothenburg (CCUG). Other strains of <italic>R. anatipestifer</italic> used in this study were identified, characterized and archived by the Research Center of Avian Diseases, Sichuan Agricultural University (Chengdu, Sichuan, China).\n</p>", "<p id=\"Par9\">The <italic>R. anatipestifer</italic> strains were grown in tryptic soy broth (TSB) and tryptic soy agar (TSA), at 37 °C for 12 h under microaerophilic conditions. <italic>R. anatipestifer</italic> genomic DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN BIOTECH, China). Whole-genome sequencing was performed using the Illumina HiSeq 2500 platform at the Beijing Genomics Institute (BGI, Shenzhen, China). Short-reads were filtered by Fastp (v0.19.4, default settings) [##REF##30423086##26##] and draft genomes were assembled using SPAdes (v 3.11.0, default parameters with --careful flag) [##REF##22506599##27##].</p>", "<p id=\"Par10\">All <italic>R. anatipestifer</italic> strains were confirmed by RA-specific, 16S rRNA PCR assays (universal primer pairs 27F and 1410R), and genomic average nucleotide identity, as described previously [##REF##23391177##7##, ##REF##25690020##28##, ##REF##30504855##29##]. and non-redundant isolates (&gt; 2000 SNPs/INDELs) were identified using custom scripts based on the NUCmer program (version 4.0.0beta2, <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/youngDouble/MUMmerSNP2VCF_script\">https://github.com/youngDouble/MUMmerSNP2VCF_script</ext-link>) [##UREF##6##30##].</p>", "<title>Agglutination test using the antisera</title>", "<p id=\"Par11\">The serovars of <italic>R. anatipestifer</italic> involved in this study were determined by slide agglutination according to Bisgaard [##REF##18770200##3##]. Standard serotyping antisera were obtained from RIPAC-LABOR GmbH (Potsdam, Germany), and those antisera have been extensively tested to indicate no cross-reactivity [##REF##18680018##6##, ##REF##23391177##7##]. As our representative strains are mainly from Bisgaard’s scheme [##REF##18770200##3##], we have labelled the serovar types according to the recent study by Omaleki et al. [##REF##32990455##8##]. For non-serovar reference strains, we have labelled them with temporary serovar types. The serovar represented by CCUG 18373 is labeled as undefined type 1 (U1) and the serovar represented by CCUG 25013 is labeled as undefined type 2 (U2) (Table ##TAB##0##1##).</p>", "<title>Genome wide association study of <italic>R anatipestifer</italic> serovar phenotypes</title>", "<p id=\"Par12\">To explore the association between <italic>R. anatipestifer</italic> serovars and genetic characteristics, a pan-genome-wide association study (Pan-GWAS) was performed. To ensure statistical power, GWAS was performed for the three most prevalent serovars in China. Specifically, the <italic>R. anatipestifer</italic> genome was annotated using Prokka (version 1.14.6, default parameters) [##REF##24642063##31##], and the pan-genome containing 45 strains of <italic>R. anatipestifer</italic> was reconstructed with Roary (version 3.12.0, with identity threshold of protein = 90) [##REF##26198102##32##]. Furthermore, Scoary (v1.6.16) [##REF##27887642##20##] was used to perform the Pan-GWAS with the <italic>gene_presence_absence</italic> file generated by Roary (only serovars containing more than 10 strains were considered). Scoary’s <italic>P</italic>-value and <italic>Q</italic>-value (adjusted <italic>P</italic>-value, adjust algorithm: Benjamini-Hochberg method) cut-offs were set to 0.05, the sensitivity cut-off was set to 95% and specificity to 85%. Next, we mapped the genes that were significantly associated with the serovar to the corresponding genome to obtain the distribution characteristics. Contig comparisons were generated with Easyfig (v2.2) [##REF##21278367##33##].</p>", "<title>Functional speculation of the gene cluster</title>", "<p id=\"Par13\">To explore the function of serovar-related genetic loci, genome-wide biosynthetic gene clusters (BGCs) of <italic>R. anatipestifer</italic> was predicted with antiSMASH (version 4.2.0, parameter setting: --clusterblast --subclusterblast --knownclusterblast --smcogs --inclusive --borderpredict) [##REF##31032519##34##]. BGCs analysis was performed again by DeepBGC [##REF##31400112##35##], which uses deep learning strategies to mine biosynthetic gene clusters in the microbial genome. The results of the above two methods will be considered comprehensive.</p>", "<title>Gene boundary determination of <italic>R. anatipestifer</italic> CPS gene cluster</title>", "<p id=\"Par14\">Based on the results of biosynthetic gene cluster mining, we further determined the boundaries of the <italic>R. anatipestifer</italic> CPS gene cluster. Specifically, we retrieved 450 known CPS gene clusters from the NCBI Nucleotide database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/nuccore\">https://www.ncbi.nlm.nih.gov/nuccore</ext-link>) (Supplementary Table ##SUPPL##0##2##). We downloaded the protein sequence of these gene clusters, used CD-HIT (version 4.8.1, parameter setting: -c 1 -aS 0.95) [##REF##16731699##36##] to remove redundancies and constructed the database. TblastN (version 2.6+) was used to map these proteins to the <italic>R. anatipestifer</italic> genome, and the resulting filtering thresholds were as follows: coverage ≥50% (−qcov_hsp_perc 50), e-value≤1e-5 (−evalue 1e-5). Subsequently, the densely mapped regions in the genome are considered as candidates for the CPS gene cluster. Finally, combined with the prediction results of BGCs, the boundary of the gene cluster was determined by manual inspection.</p>", "<title>Annotation of the CPS synthesis gene cluster</title>", "<p id=\"Par15\">Protein-encoding genes were predicted using Prokka [##REF##24642063##31##] and NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [##REF##33270901##37##] with default parameters. To assign functions to the predicted genes, the Conserved Domains Database (CDD) [##REF##25414356##38##] was used to search for conserved domains with an E-value threshold of 0.01. Meanwhile characteristic gene annotation of genes was performed using Blastp (v2.6+) against Non-Redundant (NR, <ext-link ext-link-type=\"uri\" xlink:href=\"https://ftp.ncbi.nlm.nih.gov/blast/db/\">https://ftp.ncbi.nlm.nih.gov/blast/db/</ext-link>) database. The E-value and query coverage were set at 1e-5 and 50% respectively. Wzx and Wzy are key proteins in CPS synthesis and possess a typical multi-transmembrane structure [##REF##32680453##39##]. Therefore, for the identification of Wzx and Wzy, TMHMM2.0 [##UREF##7##40##] was used to predict the transmembrane regions of proteins.</p>", "<title>Inter- and intra-serovars comparison of CPS gene cluster</title>", "<p id=\"Par16\">Gene cluster nucleotide sequence alignment was performed using MAFFT [##REF##12136088##41##] in automatic mode, and then Mega X [##REF##27004904##42##] with default parameters and 1000 bootstrap replicates were used to reconstruct the Neighbor-joining (NJ) [##REF##3447015##43##] phylogenetic tree.</p>", "<p id=\"Par17\">Blast (v2.6+) and Easyfig (v2.2) [##REF##21278367##33##] were used for inter- and intra-serovar CPS gene cluster comparisons. Clustal Omega Web services (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\">https://www.ebi.ac.uk/Tools/msa/clustalo/</ext-link>, default settings) was used to calculate the percentage identity of all Wzx and Wzy protein sequences.</p>", "<title>Conservation analysis of CPS locus in family <italic>Weeksellaceae</italic></title>", "<p id=\"Par18\">To investigate the conservation of the CPS locus of <italic>R. anatipestifer</italic> in closely related species within the family <italic>Weeksellaceae</italic>, the multi-gene search method was implemented against representative genome database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://ftp.ncbi.nlm.nih.gov/genomes/refseq/\">https://ftp.ncbi.nlm.nih.gov/genomes/refseq/</ext-link>, data as of November 12, 2022) [##REF##24316578##44##]. Specifically, Multigeneblast [##REF##23412913##45##] was used to find homologues of <italic>R. anatipestifer</italic> CPS gene cluster from the representative genomes of <italic>Weeksellaceae</italic> species (based on published data). In addition, we used Easyfig to compare the collinearity of the best homologues.</p>", "<title>Construction of <italic>R. anatipestifer wzy</italic> mutant strain CH-2Δ<italic>wzy</italic></title>", "<p id=\"Par19\">To further characterize the relationship between the predicted CPS gene cluster and capsule synthesis, we performed a deletion mutation in the predicted key gene. Briefly, the <italic>wzy</italic> gene (<italic>G148_RS04365</italic>) was deleted by allelic exchange using the recombinant suicide vector pYA4278 (Supplementary Fig. ##SUPPL##1##1##a, Kong et al. [##REF##1929280##46##]; donated by Professor Kong). Briefly, upstream (L) and downstream (R) fragments of the <italic>R. anatipestifer</italic> CH-2 <italic>wzy</italic> gene were amplified by PCR from the genome using <italic>wzy</italic>-Left F and <italic>wzy</italic>-Left R, and <italic>wzy</italic>-Right F and <italic>wzy</italic>-Right R primers, respectively. A 1145-bp Spec<sup>R</sup> cassette was PCR-amplified from the pYES1 new plasmid using the Spc F and Spc R primers. The three fragments were then spliced together in vitro by overlap extension using the <italic>wzy</italic>-Left F and <italic>wzy</italic>-Right R primers, producing the LSR fragment. Adenosine nucleotides were added to both ends of the PCR product, which was then ligated to the AhdI-digested T-cloning suicide vector pYA4278 to generate pYA4278-LSR, which carries a deletion of the entire <italic>wzy</italic> gene. Subsequently, pYA4278-LSR was successively transformed into <italic>E. coli</italic> X7213λpir [##REF##9511756##47##]. <italic>E. coli</italic> X7213λpir (Donor) and <italic>R. anatipestifer</italic> CH-2 (Recipient) were mixed in a 10 mM MgSO<sub>4</sub> solution and incubated on TSB agar with diaminopimelic acid at 37 °C for 24 h. Spec<sup>R</sup> transconjugants were further selected in media containing spectinomycin (40 μg/ml). The detailed steps of this study refer to the methods of Luo et al. [##REF##25690020##28##]. To confirm the <italic>R. anatipestifer</italic> mutant CH-2Δ<italic>wzy</italic>, we performed PCR targeting the transconjugants (see Supplementary Fig. ##SUPPL##1##1##b for details). The above strains and plasmids are preserved at the Research Center of Avian Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, China. The primers used for construction of the above strains and plasmids are listed in Table ##TAB##1##2##.\n</p>", "<title>Microscopic imaging of capsules by India ink staining and transmission electron microscopy</title>", "<p id=\"Par20\">Capsule staining of <italic>R. anatipestifer</italic> was carried out using an improved Indian ink staining method as previously described [##UREF##8##48##]. Briefly, one drop of Indian ink and one drop of bacterial suspension were mixed on a glass slide, spread thinly, and air-dried. The slide was then counterstained with 1% crystal violet for 1 minute, gently rinsed with distilled water, air-dried, and observed by optical microscope.</p>", "<p id=\"Par21\">The wild-type (CH-2) and mutant (CH-2Δ<italic>wzy</italic>) strains were washed twice with phosphate-buffered saline at 5000 r/min for 10 minutes after overnight culture. The precipitate was treated with 2.5% glutaraldehyde (pH 7.2) for 2 hours. After washing 20 times with ultrapure water, the cells were adsorbed onto copper grids and then stained with the phosphotungstic acid solution for 5 minutes. The capsule was observed by field-emission transmission electron microscope (TEM, FEI Tecnai G2 F20, 200 kV).</p>" ]
[ "<title>Results</title>", "<title>The serovars of <italic>R. anatipestifer</italic></title>", "<p id=\"Par22\">In this study, the genome data of <italic>R. anatipestifer</italic> involved a total of eleven serovars, including Serovar U1 (<italic>n</italic> = 10), Serovar 2 (<italic>n</italic> = 11), Serovar 3 (<italic>n</italic> = 1), Serovar 4 (<italic>n</italic> = 1), Serovar 5 (<italic>n</italic> = 1), Serovar 6 (<italic>n</italic> = 3), Serovar 9 (<italic>n</italic> = 1), Serovar 11 (<italic>n</italic> = 1), Serovar 8 (<italic>n</italic> = 5), Serovar U2 (<italic>n</italic> = 10), and Serovar 12 (<italic>n</italic> = 1), which were determined by slide agglutination or from references. All strains and their information are shown in Supplementary Table ##SUPPL##0##1##.</p>", "<title>The gene cluster associated with serovar phenotype of <italic>R. anatipestifer</italic></title>", "<p id=\"Par23\">To screen for loci associated with serovars, the GWAS was performed with Scoary on the serovars containing more than 10 strains (serovars U1, 2, U2). According to the Pan-GWAS filtering threshold, we obtained a total of 27 target genes, and each serovar harbors 9 associated genes (Fig. ##FIG##0##1##a, Supplementary Table ##SUPPL##0##3##). Next, we mapped these genes to the corresponding genome and found that these genes were close to each other and formed a gene cluster. Interestingly, according to the BGCs results predicted by antiSMASH, the gene clusters mentioned above were labelled as polysaccharide biosynthetic gene clusters. Furthermore, the presence of <italic>wza</italic> and <italic>wzc</italic> gene indicates that this gene cluster is responsible for the biosynthesis of CPS. (Supplementary Table ##SUPPL##0##4##). Based on these results, we speculate that the serovar-specific gene cluster was CPS biosynthesis gene cluster of <italic>R. anatipestifer</italic>.</p>", "<p id=\"Par24\">We further compared the distribution of the gene cluster among different serovars, and the results showed that the position of the gene cluster was relatively conserved in the genome of <italic>R. anatipestifer</italic> (Fig. ##FIG##1##2##). In short, the gene region has conserved fragments of 4 and 5 genes at the beginning and end, respectively (Fig. ##FIG##1##2##).</p>", "<p id=\"Par25\">To determine the boundaries of the CPS gene cluster, we focused on those locations with a high density of genes associated with CPS synthesis. The results show a distinct boundary in the region of the putative CPS gene cluster (Fig. ##FIG##0##1##b, Supplementary Fig. ##SUPPL##1##2##). Therefore, we speculate that that the CPS gene cluster of <italic>R. anatipestifer</italic> is located between the regulatory protein coding gene <italic>recX</italic> and the ribosomal protein S12 methylthiotransferase coding gene <italic>rimO</italic>, both of which are highly conserved in the genome of <italic>R. anatipestifer</italic> (Fig. ##FIG##1##2##).</p>", "<title>Inter- and intra-serovars comparison of CPS gene cluster</title>", "<p id=\"Par26\">Based on the positional conservation of the CPS gene cluster, we extracted the CPS gene cluster sequences from serovar representative strains (Supplementary Table ##SUPPL##0##5##). The length of the gene clusters from 22.76 kb (serovar 8) to 30.18 kb (serovar U1), GC content between 32.55% (serovar 10) and 34.05% (serovar 2), which was significantly different from the genomic GC content (upper quartile: 34.98%, lower quartile: 34.82%, mean: 34.95%; paired t-test: <italic>p</italic>-value &lt; 0.0001). We annotated the CPS gene clusters of the serovar representative strains, the results are shown in Supplementary Table ##SUPPL##0##4##. These gene clusters contain an average of 23 CDSs (ranging from 19 to 27). It is worth noting that all serovar CPS gene clusters contain <italic>wza</italic>, <italic>wzc, wzx, wzy, rfbA, rfbB, and rfbC</italic> genes. The presence of the set of <italic>wzx</italic> and <italic>wzy</italic> genes indicates that CPS synthesis in <italic>R. anatipestifer</italic> may be a Wzx/Wzy-related processing pathways (Supplementary Table ##SUPPL##0##4##, Supplementary Fig. ##SUPPL##1##3##).</p>", "<p id=\"Par27\">Furthermore, a NJ phylogenetic tree based on the complete sequence of the CPS gene cluster and a synteny analysis of the CPS gene cluster (DNA sequence identity cut-off: 69%) are shown in Fig. ##FIG##1##2##. As expected, strains of the same serogroup have more similar CPS gene cluster structures to each other and clearly cluster together in the same phylogenetic clade. Except for a gene insertion event in CCUG25001, the genetic structure of the CPS gene cluster of all serovar 2 strains were highly similar (Fig. ##FIG##1##2##). According to the annotation results of Prokka and PGAP, the predicted function of the inserted gene is O-acetylase involved in peptidoglycan or LPS synthesis. Additionally, the cluster of serovar 5 and those of serovar 2 differed by only two genes (<italic>wzx</italic> and a glycosyltransferase coding gene). The gene clusters of serovar U1, 6, U2 strains have considerable similarity within the serogroup. Despite the relative diversity of the gene clusters of serovar 8 strains, their <italic>wzx</italic> and <italic>wzy</italic> are also identical. Comparative phylogenetic analysis revealed that the evolutionary trends of the core genome is inconsistent with CPS gene clusters (Fig. ##FIG##1##2##).</p>", "<p id=\"Par28\">Next, we analysed the identity of the Wzx and Wzy sequences of all the strains by and constructed the NJ phylogenetic tree. Overall, Wzx and Wzy are serovar-specific, and much greater differences exist among the different serovars (Fig. ##FIG##2##3##a). Phylogenetic analysis of Wzx and Wzy, especially Wzx, clearly distinguishes the clades of different serovars. The minimum identity for the same serovars was 92.15% for Wzx and 95.14% for Wzy; the maximum identity for Wzx was 28.02% for different serovars and 26.50% for Wzy, except for serovars 5 with 2, which were 99.75% for Wzy (Fig. ##FIG##2##3##b).</p>", "<title>Conserved loci in other <italic>Weeksellaceae</italic> species</title>", "<p id=\"Par29\">The synteny analysis of homologous gene clusters in <italic>Weeksellaceae</italic> indicated that the locus of the CPS gene cluster was conserved among closely related species (Supplementary Fig. ##SUPPL##1##4## and ##SUPPL##1##5##). Specifically, the upstream gene arrangement (<italic>recx</italic>-<italic>gdr</italic>-<italic>wza</italic>-<italic>wzc</italic>) of <italic>R. anatipestifer</italic> CPS gene cluster was highly conserved. <italic>Chryseobacterium</italic> and <italic>R. anatipestifer</italic> were the same (<italic>recx</italic> and <italic>rimO</italic>) at the beginning and end of the region.</p>", "<p id=\"Par30\">As expected, this locus is also conserved in <italic>Elizabethkingia sp.</italic> and <italic>Chryseobacterium sp.</italic> (Supplementary Fig. ##SUPPL##1##4##b and c). Furthermore, many glycosyltransferases related to polysaccharide synthesis are distributed in this region in both genera. It is worth mentioning that <italic>rfbA, rfbB, and rfbC</italic> (<italic>Elizabethkingia sp.</italic>), LPS export system ATP-binding protein gene (<italic>lptB, Elizabethkingia sp.</italic>), ligase gene (<italic>Chryseobacterium sp.</italic>), and oligosaccharide flippase gene (<italic>Chryseobacterium sp.</italic>) were also present in the conserved region, and they are usually involved in the synthesis of CPS and LPS.</p>", "<p id=\"Par31\">Regarding the other two species of <italic>Riemerella</italic>: <italic>Riemerella columbina</italic> and <italic>Riemerella columbipharyngis</italic>, a similar gene cluster was found in <italic>Riemerella columbina</italic> DSM 16469 (Supplementary Fig. ##SUPPL##1##4##d). Furthermore, the genes encoding oligosaccharide repeat unit polymerase (Wzy) and oligosaccharide flippase (Wzx) were annotated in the cluster. However, compared with <italic>R. anatipestifer</italic>, the cluster region is significantly rearranged in <italic>Riemerella columbina</italic>. However, we could not detect similar genetic regions in <italic>Riemerella columbipharyngis</italic>.</p>", "<title>Identification and characterization of <italic>R. anatipestifer</italic> CH-2Δ<italic>wzy</italic></title>", "<p id=\"Par32\">The <italic>wzy</italic> of <italic>R. anatipestifer</italic> CH-2 was knocked out by allelic exchange, and the mutant CH-2Δ<italic>wzy</italic> was identified by PCR (Supplementary Fig. ##SUPPL##1##1##b). CH-2Δ<italic>wzy</italic> amplified the 16S rRNA fragment, Spec<sup>R</sup> cassette fragment, and LSR fragment, but did not amplify the <italic>wzy</italic> fragment. All amplicons were confirmed by Sanger sequencing. After continuous culture for 30 generations, the genetic stability of the CH-2Δ<italic>wzy</italic> mutant was confirmed by the same PCR test (Supplementary Fig. ##SUPPL##1##1##c).</p>", "<p id=\"Par33\">The results of India ink staining showed that there was a white ring-like capsule structure around the wild-type strain CH-2 (Fig. ##FIG##3##4##a), while there was none around the mutant strain CH-2Δ<italic>wzy</italic> (Fig. ##FIG##3##4##b). The capsule structure observed by transmission electron microscopy indicated that there was a layer of furry substance on the surface of the wild-type strain (Fig. ##FIG##3##4##c), and the capsule of the mutant strain (Fig. ##FIG##3##4##d) was thinner than the wild-type strain. Furthermore, the antisera slide agglutination test indicated that CH-2Δ<italic>wzy</italic> could agglutinate with the antisera from multiple serovars (Supplementary Fig. ##SUPPL##1##6##).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par34\">Serotyping is an important method for characterizing <italic>R. anatipestifer</italic>, but corresponding molecular typing studies are still lacking. A recent study using time-of-flight mass spectrometry combined with machine learning algorithms has achieved recognition of serovars 1 and 2 [##UREF##9##49##]. Although the study was limited to a non-representative test set and did not indicate its corresponding molecular basis, its results suggest the feasibility of molecular serotyping of <italic>R. anatipestifer</italic>.</p>", "<p id=\"Par35\">Pan-GWAS have been applied in various bacteria to explore the genetic basis of various phenotypes [##UREF##4##22##, ##REF##33097713##23##, ##REF##33536414##50##]. In the present study, we used Pan-GWAS to identify the genetic loci significantly associated with three prevalent serovars of <italic>R. anatipestifer</italic>. Further functional analysis of the loci suggested that these loci are responsible for the synthesis of CPS. The result is in agreement with our recent finding that the CPS of <italic>R. anatipestifer</italic> determines the serological specificity of the serovar 2 strain [##UREF##2##19##]. This is also consistent with previous studies in other species, suggesting that each serovar corresponds to a specific CPS synthesis gene cluster [##REF##29885806##9##, ##REF##12517878##51##, ##REF##23628376##52##].</p>", "<p id=\"Par36\">Based on the results of the association study between serovar and genome, we predicted and analysed the CPS gene cluster of <italic>R. anatipestifer</italic>. We observed that the CPS gene cluster exhibits a genetic structure with highly conserved regions at both ends and a diversified middle region. The structure is similar to that of <italic>Klebsiella pneumoniae</italic>, <italic>E. coli</italic>, and <italic>Acinetobacter baumannii</italic>, and <italic>Vibrio parahaemolyticus</italic>, with typical genes (i.e., <italic>wza</italic> and <italic>wzc</italic>) located in the start region of the gene cluster [##REF##33505361##10##, ##REF##18508935##12##, ##REF##16756484##53##, ##REF##32118530##54##]. Despite some minor differences, the phylogenetic relationships between gene clusters of the same serovar are closer together. There are non-essential gene differences in the CPS gene cluster of serovar 9 strains of <italic>Streptococcus suis</italic>, but these differences did not cause phenotype change [##REF##23416996##55##]. Considering the significant differences in isolation time and geographical location between recent <italic>R. anatipestifer</italic> isolates and the serovar reference strains, these differences in the CPS gene cluster appear to be explainable. The CPS gene clusters of serovars 2 and 5 differ by only three genes, of which <italic>wzx</italic> is one of the key genes determining serological specificity. This phenomenon is also observed in other bacteria [##REF##33505361##10##, ##REF##32118530##54##, ##REF##26493302##56##]. Notably, we observed phylogenetic inconsistency between the CPS gene clusters and the core genomes, which may be due to stronger selection pressure on capsule antigens [##REF##30037568##57##]. The presence of <italic>wzx</italic> and <italic>wzy</italic> implies that the CPS of <italic>R. anatipestifer</italic> are processed via the Wzx/Wzy-dependent pathway [##REF##32680453##39##]. <italic>wzx</italic> and <italic>wzy</italic> are widely used for capsule serotyping due to their excellent serovar specificity [##REF##18508935##12##, ##REF##16390959##58##], which has also been confirmed in our study. In <italic>R. anatipestifer</italic>, <italic>wzx</italic> can perfectly distinguish strains of different serovars, while <italic>wzy</italic> was slightly less effective as it cannot differentiate between serovar 2 and 5. Similar reports of two capsule serovars sharing the same <italic>wzy</italic> gene have been described in <italic>Klebsiella spp</italic> [##REF##26493302##56##].</p>", "<p id=\"Par37\">In this study, we also performed a conservative analysis of the CPS synthesis gene cluster of other <italic>Weeksellaceae</italic> species. It is noteworthy that a similar genetic locus is harbored in some species of <italic>Chryseobacterium</italic> and <italic>Elizabethkingia</italic>. To the best of our knowledge, there are no evidence-based reports of CPS synthetic gene clusters in <italic>Chryseobacterium</italic> and <italic>Elizabethkingia</italic>. Despite this limitation, we found several genes related to polysaccharide synthesis in these regions, such as <italic>wbpA</italic>, <italic>wbpD</italic>, <italic>wbpE</italic>, <italic>lptB</italic> and the ABC transporter ATP-binding protein gene [##REF##28394325##59##, ##REF##24852504##60##]. This is consistent with the previously putative capsular polysaccharide synthesis gene cluster of <italic>Elizabethkingia</italic> species [##REF##27461509##61##]. Therefore, for some species of <italic>Chryseobacterium</italic> and <italic>Elizabethkingia</italic>, the above mentioned genomic region may also be the locus of the CPS synthesis gene cluster.</p>", "<p id=\"Par38\">Furthermore, <italic>wzy</italic> gene (<italic>G148_RS04365</italic>) was deleted from <italic>R. anatipestifer</italic> CH-2. The absence of the capsule suggests that the <italic>wzy</italic> gene plays a crucial role in capsule synthesis, and previous study have shown that inactivation of <italic>wza</italic> in the CPS gene cluster also leads to the same phenotype [##REF##28442426##16##]. Another study showed that knocking out the <italic>AS87_04050</italic> gene (coding Vi polysaccharide biosynthesis protein) from the CPS locus of <italic>R. anatipestifer</italic> Yb2 can alter serological characteristics, and the mutant strains exhibit a rough morphology [##REF##25303276##62##], which is a typical feature of capsule loss [##REF##19871689##63##]. And in the present study, the mutant strain CH-2Δ<italic>wzy</italic> could agglutinate with antisera from multiple serovar. Similarly, deletion of the <italic>M949_1603</italic> gene (coding glycosyltransferase family 2 protein) in <italic>R. anatipestifer</italic> CH-1 results in cross-reactivity [##REF##26266750##15##]. And we have confirmed that <italic>M949_1603</italic> gene is also located in the CPS gene cluster region. One possible explanation for the phenomenon of cross-reactivity is that the absence of capsule leads to the exposure of highly conserved epitopes. These results may indicate that the CPS of <italic>R. anatipestifer</italic> is the major antigenic component related to serological characteristics.</p>" ]
[ "<title>Conclusion</title>", "<p id=\"Par39\">In this work, we revealed that association between the putative CPS gene cluster and the serovar types of <italic>R. anatipestifer</italic> through a genome-wide association studies. The CPS synthesis gene cluster of <italic>R. anatipestifer</italic> is serovar-specific. Moreover, the inactivation of the <italic>wzy</italic> gene results in defective capsule phenotype and cross-agglutination. This study provides new insights into the molecular basis of serotyping in <italic>R. anatipestifer</italic> and provides ideas for the development of molecular serotyping methods.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">The disease caused by <italic>Riemerella anatipestifer</italic> (<italic>R. anatipestifer</italic>, RA) results in large economic losses to the global duck industry every year. Serovar-related genomic variation, such as the O-antigen and capsular polysaccharide (CPS) gene clusters, has been widely used for serotyping in many gram-negative bacteria. RA has been classified into at least 21 serovars based on slide agglutination, but the molecular basis of serotyping is unknown. In this study, we performed a pan-genome-wide association study (Pan-GWAS) to identify the genetic loci associated with RA serovars.</p>", "<title>Results</title>", "<p id=\"Par2\">The results revealed a significant association between the putative CPS synthesis gene locus and the serological phenotype. Further characterization of the CPS gene clusters in 11 representative serovar strains indicated that they were highly diverse and serovar-specific. The CPS gene cluster contained the key genes <italic>wzx</italic> and <italic>wzy</italic>, which are involved in the Wzx/Wzy-dependent pathway of CPS synthesis. Similar CPS loci have been found in some other species within the family <italic>Weeksellaceae</italic>. We have also shown that deletion of the <italic>wzy</italic> gene in RA results in capsular defects and cross-agglutination.</p>", "<title>Conclusions</title>", "<p id=\"Par3\">This study indicates that the CPS synthesis gene cluster of <italic>R. anatipestifer</italic> is a serotype-specific genetic locus. Importantly, our finding provides a new perspective for the systematic analysis of the genetic basis of the <italic>R anatipestifer</italic> serovars and a potential target for establishing a complete molecular serotyping scheme.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s12864-024-09988-4.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>Not applicable.</p>", "<title>Authors’ contributions</title>", "<p>ZY designed the work, performed data analysis, and wrote the manuscript. XY assisted with molecular experiments. MW , RJ, SC, and ML managed and supervised the project. XZ, QY, YW, SZ, JH, XO, SM, QG, DS, and BT reviewed and revised the manuscript. DZ and AC conceptualized and designed the work, provided funding support, and revised the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This work was supported by Sichuan Science and Technology Program (2020YJ0330); Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System (SCCXTD-2021-18); Earmarked Fund for China Agriculture Research System (CARS-42-17).</p>", "<title>Availability of data and materials</title>", "<p>All genomic data generated or analysed in this study are archived in the NCBI Genome Database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/assembly/\">https://www.ncbi.nlm.nih.gov/assembly/</ext-link>) and accession numbers can be found in Supplementary Table ##SUPPL##0##1##.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par40\">Not applicable.</p>", "<title>Consent for publication</title>", "<p id=\"Par41\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par42\">The authors declare no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Location of genes significantly associated with serovars. <bold>a</bold> And the specificity and the sensitivity of genes significantly associated with serovars U1, 2, and U2. The size of the shape indicates sensitivity; colour indicates negative log of adjusted <italic>P</italic>-value. <bold>b</bold> Gene cluster location and boundary determination. The dot plot represents the hits of genes related to CPS on the genome, and the size of the dot indicates the coverage length. Interval markers on gene clusters indicate the BGC regions predicted by DeepBGC and antiSMASH</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Comparison between CPS gene cluster phylogeny and core genome phylogeny. NJ phylogenetic tree and genetics structure of the CPS gene cluster are shown on the left, and the core genome phylogenetic tree is shown on the right. The same strain IDs are linked using dashed lines. Bootstrap values (greater than 50%) are indicated in red</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p><bold>a</bold> Phylogenetic tree constructed by the neighbor joining method based on the Wzx (left) and Wzy (right) protein sequences. <bold>b</bold> Identity of Wzx (upper triangle) and Wzy (lower triangle) sequences</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Capsule staining (<bold>a</bold>, <bold>b</bold>) and transmission electron microscope (<bold>c</bold>, <bold>d</bold>). The capsule of the wild-type strain CH-2 (<bold>a</bold>) and mutant strain CH-2Δ<italic>wzy</italic> (<bold>b</bold>) was stained and observed at 1000× magnification. The red arrows indicate the transparent capsule structures. The microstructure of wild-type strain CH-2 (<bold>c</bold>) and mutant strain CH-2Δ<italic>wzy</italic> (<bold>d</bold>) at 130000× magnification</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Serovar representative strains in this study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>CCUG ID of representative strains</th><th>Strains name</th><th>Serovar</th><th>Bisgaard (1982) Serovar Reference Strains [##REF##18770200##3##]</th><th>Bisgaard (1982) Serovar [##REF##18770200##3##]</th><th>Rubbenstroth et al. (2013) Reference strains [##REF##23391177##7##]</th><th>Rubbenstroth et al. (2013) Serovar [##REF##23391177##7##]</th><th>Omaleki et al. (2021) Bisgaard Serovar Reference Strains [##REF##32990455##8##]</th><th>Omaleki et al. (2021) Bisgaard Serovar [##REF##32990455##8##]</th></tr></thead><tbody><tr><td>CCUG18373</td><td>P-892</td><td>U1</td><td/><td/><td>DRL-24105</td><td>1</td><td/><td/></tr><tr><td>CCUG25001</td><td>HPRS 2591</td><td>2</td><td>HPRS 2591</td><td>2</td><td>DRL-24046</td><td>2</td><td>HPRS 2591</td><td>2</td></tr><tr><td>CCUG25002</td><td>HPRS 2212</td><td>3</td><td>HPRS 2212</td><td>3</td><td>DRL-26338</td><td>3</td><td>HPRS 2212</td><td>3</td></tr><tr><td>CCUG25004</td><td>HPRS 2514</td><td>5</td><td>HPRS 2514</td><td>5</td><td>DRL-24123</td><td>5</td><td>HPRS 2514</td><td>5</td></tr><tr><td>CCUG25005</td><td>HPRS 2336</td><td>6</td><td>HPRS 2336</td><td>6</td><td>P-2123</td><td>6</td><td>HPRS 2336</td><td>6</td></tr><tr><td>CCUG25008</td><td>HPRS 2174</td><td>8</td><td>HPRS 2174</td><td>8</td><td>HPRS-2199</td><td>10</td><td>HPRS 2174</td><td>8</td></tr><tr><td>CCUG25010</td><td>HPRS 2528</td><td>9</td><td>HPRS 2528</td><td>9</td><td>DRL-27179</td><td>7</td><td>HPRS 2528</td><td>9</td></tr><tr><td>CCUG25011</td><td>HPRS 2564</td><td>10</td><td>HPRS 2564</td><td>10</td><td>HPRS-2565</td><td>4</td><td>HPRS 2564</td><td>10</td></tr><tr><td>CCUG25013</td><td>Singapore 8</td><td>U2</td><td/><td/><td>DRL-28020</td><td>11</td><td/><td/></tr><tr><td>CCUG25054</td><td>HPRS 2560</td><td>11</td><td>HPRS 2560</td><td>11</td><td>DRL-26220</td><td>8</td><td>HPRS 2560</td><td>11</td></tr><tr><td>CCUG25055</td><td>8755/9</td><td>12</td><td>8755/9</td><td>12</td><td>8755/9</td><td>12</td><td>8755/9</td><td>12</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Primers for identification of <italic>wzy</italic> deletion</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Name of pimer</th><th>Targeted gene;Description</th><th>Product Length(bp)</th><th/><th>Sequence</th><th>Source or reference</th></tr></thead><tbody><tr><td rowspan=\"2\">wzy-Left</td><td rowspan=\"2\">G148_RS04360; Amplification of the <italic>wzy</italic> upstream fragment</td><td rowspan=\"2\">609</td><td>F</td><td>AAGAACATTACCCATATCCTATCGTTTCGACGGTA</td><td>This study</td></tr><tr><td>R</td><td>TTCTGTCCTGGCTGGTTTTACGAATATTTGTAAGATA</td><td>This study</td></tr><tr><td rowspan=\"2\">wzy-Right</td><td rowspan=\"2\">G148_RS04370; Amplification of the <italic>wzy</italic> downstream fragment</td><td rowspan=\"2\">602</td><td>F</td><td>CCAAGGTAGTCGGCAAATAATTTTATGAAAAAAGTAC</td><td>This study</td></tr><tr><td>R</td><td>TACATGAGAAACCACAAAAGCCTCTTTGGGAATA</td><td>This study</td></tr><tr><td rowspan=\"2\">wzy</td><td rowspan=\"2\">RA-CH-2 (G148_RS04365); Amplification of the wzy</td><td rowspan=\"2\">886</td><td>F</td><td>TCCAATGGGTTTACTTTCTTGTAACTTTGTCT</td><td>This study</td></tr><tr><td>R</td><td>CGTAATGGTTGGTTGAGATTCATTGGAG</td><td>This study</td></tr><tr><td rowspan=\"2\">LSR</td><td rowspan=\"2\">G148_RS04360 + spec; Identification of transconjugants</td><td rowspan=\"2\">1199</td><td>F</td><td>AGGTAGATAGGGCAAGTATGGCTTTTTCG</td><td>This study</td></tr><tr><td>R</td><td>ACCGTAACCAGCAAATCAATATCACTGTG</td><td>This study</td></tr><tr><td rowspan=\"2\">16S rRNA_RA</td><td rowspan=\"2\">16S rRNA; Identification of species</td><td rowspan=\"2\">960</td><td>F</td><td>CTTCGGATACTTGAGAGCG</td><td>[##REF##25690020##28##]</td></tr><tr><td>R</td><td>GCAGCACCTTGAAAATTGT</td><td>[##REF##25690020##28##]</td></tr><tr><td rowspan=\"2\">Spec</td><td rowspan=\"2\"><italic>spec;</italic> Amplification of the <italic>spec</italic></td><td rowspan=\"2\">1145</td><td>F</td><td>TCTTACAAATATTCGTAAAACCAGCCAGGACAGAAAT</td><td>This study</td></tr><tr><td>R</td><td>ACTTTTTTCATAAAATTATTTGCCGACTACCTTGGTG</td><td>This study</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>" ]
[ "<table-wrap-foot><p><italic>CCUG </italic>Culture Collection University of Gothenburg, <italic>U1 </italic>Undefined type 1, <italic>U2 </italic>Undefined type 2</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"12864_2024_9988_Fig1_HTML\" id=\"MO1\"/>", "<graphic xlink:href=\"12864_2024_9988_Fig2_HTML\" id=\"MO2\"/>", "<graphic xlink:href=\"12864_2024_9988_Fig3_HTML\" id=\"MO3\"/>", "<graphic xlink:href=\"12864_2024_9988_Fig4_HTML\" id=\"MO4\"/>" ]
[ "<media xlink:href=\"12864_2024_9988_MOESM1_ESM.xlsx\"><caption><p><bold>Additional file 1: Supplementary Table 1. </bold>The strains and the published genome data used in this study. <bold>Supplementary Table 2.</bold> Information of 450 known capsular polysaccharide synthesis gene cluster from the NCBI. <bold>Supplementary Table 3.</bold> The specificity and the sensitivity of genes significantly associated with serovar U1, 2, and U2. <bold>Supplementary Table 4.</bold> Functional prediction of the gene in capsular polysaccharide synthesis gene cluster of serovar representative strain. <bold>Supplementary Table 5.</bold> Location, length and GC content of capsular polysaccharide synthesis gene cluster of serovar representative strain.</p></caption></media>", "<media xlink:href=\"12864_2024_9988_MOESM2_ESM.docx\"><caption><p><bold>Additional file 2: Supplementary Figure 1. </bold>a) Construction of R. anatipestifer wzy mutant strain CH-2Δwzy. b) Identification of R. anatipestifer CH-2Δwzy. Lane M: DL2000 DNA Marker; Lanes 1-3: 16S rRNA F and 16S rRNA R, which amplify a 960 bp fragment from R. anatipestifer 16S rRNA. Order: Wild-type(CH-2), mutant(CH-2Δwzy), and negative control (distilled water); Lanes 4-6: Spec F and Spec R, which amplify a 1180 bp fragment from the SpecR cassette. Order: Positive control (pYES1 new), mutant (CH-2Δwzy), and negative control (distilled water); Lanes 7-9: wzy F and wzy R, which amplify an 886 bp fragment from the wzy gene. Order: Wild-type(CH-2), mutant (CH-2Δwzy), and negative control (distilled water); Lanes 10-11: LSR F and LSR R, which amplify a 1199 bp fragment from the SpecR cassette, indicating that it was inserted in the correct position in the R. anatipestifer CH-2 genome. Order: Mutant (CH-2Δwzy), Negative control (distilled water). c) Identification of R. anatipestifer CH-2Δwzy after continuous culture for 30 generations. Lane M1: DL15000 DNA Marker; Lane M2: DL2000 DNA Marker. The rest of the lanes are identical to (b). <bold>Supplementary Figure 2.</bold> Gene cluster location and boundary determination of serovar U1 and serovar U2. The dot plot represents the hits of genes related to CPS on the genome, and the size of the dot indicates the coverage length. Interval markers on gene clusters indicate the BGC regions predicted by DeepBGC and antiSMASH. <bold>Supplementary Figure 3.</bold> The prediction of transmembrane helices in amino acid sequences encoded by wzx and wzy. <bold>Supplementary Figure 4.</bold> Conserved loci in other Weeksellaceae species. a) The genetic locus of the CPS biosynthesis gene cluster in R. anatipestifer is conserved among the closest species. b) Conserved structure in multiple Elizabethkingia species. c) Conserved structure in multiple Chryseobacterium species. d) Comparison of the CPS gene cluster between R. anatipestifer (CH-2) and R. columbina (DSM 16469). <bold>Supplementary Figure 5.</bold> 16S rDNA NJ phylogenetic tree of closely related species (representative genome from NCBI) for R. anatipestifer. 16S rDNA nucleotide sequence alignment was performed using MAFFT and tree was reconstructed by MEGA X with default parameters and 1000 bootstrap replicates. <bold>Supplementary Figure 6.</bold> Agglutination test of R. anatipestifer mutant CH-2Δwzy. The Mutants are capable of reacting to multiple antisera. And the details of the reaction differed from the wild type.</p></caption></media>" ]
[{"label": ["2."], "surname": ["Boulianne", "Blackall", "Hofacre", "Ruiz", "Sandhu", "Hafez", "Chin", "Register", "Jackwood"], "given-names": ["M", "PJ", "CL", "JA", "TS", "HM", "RP", "KB", "MW"], "italic": ["Pasteurellosis"], "source": ["Diseases of poultry"], "year": ["2020"], "edition": ["14"], "publisher-name": ["Wiley"], "fpage": ["831"], "lpage": ["889"]}, {"label": ["18."], "mixed-citation": ["Brogden KA, Rhoades KR, Rimler RB. Serologic types and physiologic characteristics of 46 avian Pasteurella anatipestifer cultures. Avian Dis. 1982;7"]}, {"label": ["19."], "mixed-citation": ["Liu Y, Luo S, Yang Z, Wang M, Jia R, Chen S, et al. Capsular polysaccharide determines the serotyping of "], "italic": ["Riemerella anatipestifer"]}, {"label": ["21."], "mixed-citation": ["Lees JA, Galardini M, Bentley SD, Weiser JN, Corander J. Pyseer: a comprehensive tool for microbial pangenome-wide association studies. Bioinformatics. 2018;3"]}, {"label": ["22."], "surname": ["Yuan", "Li", "Xu", "Sun", "Shao", "Zhang", "Li", "Fan", "Xue", "Chen"], "given-names": ["J", "Y-Y", "Y", "B-J", "J", "D", "K", "D-D", "Z-B", "W-H"], "article-title": ["Molecular signatures related to the virulence of "], "italic": ["Bacillus cereus"], "source": ["Msystems."], "year": ["2019"], "volume": ["4"], "issue": ["6"], "fpage": ["12"], "pub-id": ["10.1128/mSystems.00745-19"]}, {"label": ["24."], "surname": ["Zhu", "Yang", "Xu", "Wang", "Jia", "Chen", "Liu", "Zhao", "Yang", "Wu"], "given-names": ["D", "Z", "J", "M", "R", "S", "M", "X", "Q", "Y"], "article-title": ["Pan-genome analysis of Riemerella anatipestifer reveals its genomic diversity and acquired antibiotic resistance associated with genomic islands"], "source": ["Function Integr Genomics."], "year": ["2020"], "volume": ["20"], "issue": ["3"], "fpage": ["307"], "lpage": ["320"], "pub-id": ["10.1007/s10142-019-00715-x"]}, {"label": ["30."], "surname": ["Kurtz", "Phillippy", "Delcher", "Smoot", "Shumway", "Antonescu", "Salzberg"], "given-names": ["S", "A", "AL", "M", "M", "C", "SL"], "article-title": ["Versatile and open software for comparing large genomes"], "source": ["Genome Biol."], "year": ["2004"], "volume": ["5"], "issue": ["2"], "fpage": ["1"], "lpage": ["9"], "pub-id": ["10.1186/gb-2004-5-2-r12"]}, {"label": ["40."], "surname": ["Krogh", "Larsson", "von Heijne", "Sonnhammer"], "given-names": ["A", "B", "G", "ELL"], "article-title": ["Predicting transmembrane protein topology with a hidden markov model: application to complete genomes11Edited by F"], "source": ["Cohen J Mol Biol."], "year": ["2001"], "volume": ["305"], "issue": ["3"], "fpage": ["567"], "lpage": ["580"], "pub-id": ["10.1006/jmbi.2000.4315"]}, {"label": ["48."], "mixed-citation": ["Breakwell DP, Moyes RB, Reynolds J. Differential staining of Bacteria: capsule stain. Curr Protoc Microbiol. 2009;15(1)"]}, {"label": ["49."], "mixed-citation": ["Wang Z, Zheng X, Chen J, Xu Z, Dong Y, Xu G, et al. Machine learning combined with MALDI-TOF MS has the potential ability to identify serotypes of the avian pathogen "], "italic": ["Riemerella anatipestifer"]}]
{ "acronym": [], "definition": [] }
63
CC BY
no
2024-01-14 23:43:47
BMC Genomics. 2024 Jan 13; 25:57
oa_package/ae/2c/PMC10787497.tar.gz
PMC10787498
0
[ "<title>Background</title>", "<p id=\"Par12\">An unprecedented number of people are displaced due to conflict or persecution, with United Nations High Commissioner for Refugees (UNHCR) estimating that over 100 million people are forcibly displaced, 41% of whom are children [##UREF##0##1##, ##UREF##1##2##]. The majority of displaced people live in low- and middle-income countries, often in camps and informal settlements [##UREF##1##2##, ##REF##21835460##3##]. Many have been exposed to war but also face ongoing adversities, including limited access to basic resources, housing, education, and health-related services. These cumulative stressors contribute to a significantly elevated risk of associated and often chronic mental health problems and also prevent access to vital treatment [##REF##21835460##3##].</p>", "<p id=\"Par13\">The Syrian civil war led to millions being displaced internally and into surrounding countries. By early 2022, more than five million Syrians were registered as refugees in neighboring countries [##UREF##2##4##]. Nearly a million are registered in Lebanon: a majority live in the Beqaa region with nearly half in tents or other non-permanent shelters [##UREF##3##5##, ##UREF##4##6##]. Few anticipate a return to Syria and most are likely to live in these conditions for the foreseeable future [##UREF##5##7##]. Approximately half of children living in informal settlements in Beqaa are estimated to meet criteria for at least one mental disorder [##UREF##6##8##]; however, mental health service provision is limited and access is challenging. Amongst refugee families in Beqaa, less than 10% who express interest in mental health services for their child end up accessing treatment (Pluess et al., in preparation) and similar problems have been reported for adults in this region [##UREF##7##9##]. Commonly reported barriers include long distances to clinics, lack of transportation, expense, concerns about safety when travelling (risk of violence or being stopped at military checkpoints when many Syrian refugees lack necessary legal documentation), and lack of knowledge about which services to approach and where these services are located [##UREF##7##9##, ##UREF##8##10##]. Furthermore, access to services has been restricted due to the COVID-19 pandemic response; extreme weather events, such as flooding and heavy snow; and civil unrest resulting in widespread road closures. Thus, there is a fundamental need for accessible, effective, and efficient evidence-based mental health treatments for refugees, as well as vulnerable host communities who face similar barriers.</p>", "<p id=\"Par14\">Delivering services via telephone provides one potential solution to increase access when mobility is restricted and resources are sparse. A recent meta-analysis found that remote delivery of therapies, such as via video calls or telephone, can be effective for young people [##REF##34807635##11##]. Access to the technology required for video calls (e.g., smartphones, software, and bandwidth) is often limited in humanitarian settings, so the use of standard telephone calls may have wider applicability. To establish the potential for telephone-delivered therapy to overcome access barriers in humanitarian settings, it is essential to understand whether it is feasible and acceptable both to those providing and those receiving services over the phone. Although there has been a telemedicine expansion during the COVID-19 pandemic [##REF##32605827##12##], there is a dearth of literature on telepsychiatry in low- and middle-income countries or settings such as refugee camps [##REF##30972263##13##] and it is not yet clear whether telephone-delivered therapy is feasible or acceptable in these contexts.</p>", "<p id=\"Par15\">A further challenge in humanitarian and resource-constrained settings is a lack of mental health professionals to deliver interventions [##UREF##9##14##]. However, trained and supervised lay counsellors have been found to be able to effectively deliver face-to-face evidence-based psychological therapies, including transdiagnostic interventions such as Common Elements Treatment Approach (CETA) and Early Adolescents Skills for Emotions (EASE) [##REF##25620867##15##–##REF##34579750##17##]. CETA has been developed specifically for use with lay counsellors in low- and middle-income countries, with evidence of effectiveness in adults [##REF##30532155##18##–##REF##25386945##20##] and preliminary evidence for effectiveness in children [##REF##29868236##21##].</p>" ]
[ "<title>Method</title>", "<title>Aim</title>", "<p id=\"Par16\">In this study we focus on the use of CETA specifically adapted for delivery via telephone – t-CETA – and delivered by trained and supervised lay counsellors, with the aim of exploring whether this is a feasible and acceptable approach to delivering mental health services to children in a humanitarian setting. We do this through thematic content analysis of interviews with counsellors who delivered t-CETA, as well as interviews with children who received t-CETA and their caregivers, asking: (1) <italic>What are the perspectives of counsellors on the delivery of t-CETA to Syrian refugee children over the telephone?</italic> (2) <italic>How did Syrian refugee children and their caregivers experience receiving psychological treatment (t-CETA) over the telephone?</italic> This qualitative approach was chosen to provide an in-depth understanding of potential success factors but also challenges related to the implementation and application of telephone-delivered therapy. To the best of the authors’ knowledge, this is the first study that has systematically studied the feasibility and acceptability of telephone-delivered therapy in a sample of health workers and clients from a humanitarian setting. Data on effectiveness and mechanisms of change of t-CETA are described separately [##UREF##11##22##, ##UREF##12##23##].</p>", "<title>Setting</title>", "<p id=\"Par17\">This paper describes the qualitative component of a mixed-methods randomized controlled trial (RCT) of t-CETA (ClinicalTrials.gov ID: NCT03887312 [##UREF##13##24##]) carried out May-December 2019. Ethical approval was granted by the Institutional Review Board of the American University of Beirut (ref: SBS-2017–0429) and the study was approved by the Ministry of Public Health in Lebanon (ref: 2017/4/49165). Participants in the RCT were Syrian families living in informal tented settlements (ITS) in the Beqaa region of Lebanon.</p>", "<title>Intervention</title>", "<p id=\"Par18\">The therapy delivered via phone was Common Elements Treatment Approach (CETA; [##REF##25620867##15##, ##REF##29868236##21##, ##UREF##14##25##]). CETA is an established transdiagnostic intervention for children with common mental health problems and incorporates evidence-based treatments (mostly using cognitive behavioral therapy [CBT] approaches) into one package, including treatments for depression, anxiety, trauma-related symptoms, externalizing behavior problems, and substance use. CETA can be delivered by lay counsellors after comprehensive training [##REF##22099582##26##]. Most components are delivered to the child, with content repeated with the caregiver to enable them to support their child to complete homework; however, the component for externalizing behavior problems is delivered only to caregivers. For the current study, the original CETA manual underwent linguistic and cultural adaptation for Syrian children, while retaining the essence and content of CETA.</p>", "<p id=\"Par19\">CETA was adapted to be delivered via telephone (t-CETA) jointly by the local team at Médecins du Monde in Lebanon and the CETA team at Johns Hopkins University. Adaptations included shorter, more frequent sessions; strategies for maintaining child engagement over the phone, such as gamifying the content; alternatives to workbooks and written materials; and training counsellors to focus on non-visual cues [##UREF##15##27##]. Safety protocols were developed in consultation with child protection and gender-based violence experts who work with the target community.</p>", "<title>Recruitment and training of lay counsellors</title>", "<p id=\"Par20\">Lay counsellors were recruited to deliver the intervention: requirements included education to bachelor degree level and experience in social work/case management in humanitarian settings with Syrian refugees. The recruitment process included role-plays to evaluate aptitude for counselling, including the ability to deal appropriately and sensitively with issues such as disclosure of maltreatment or self-harm/suicidal ideation. Lay counsellors completed a six-day CETA training course delivered by a CETA expert (SS), followed by an eight-week period of training and practice sessions led by a local psychotherapist (NC). Sessions included roleplay of CETA components and feedback from the supervisor, and education about common mental disorders and how they present in children (two-to-three sessions per week, three-to-four hours per session). Following this, the counsellors delivered CETA under the supervision of the local psychotherapist, who was in turn under the supervision of the CETA expert.</p>", "<title>Participants</title>", "<p id=\"Par21\">Semi-structured interviews were conducted with (1) the two lay counsellors who delivered t-CETA and the supervisor who directly oversaw delivery (all three referred to as counsellors throughout, for simplicity), and (2) 11 children who received t-CETA and their primary caregivers. The counsellors were Lebanese, two females and one male, aged 25–29 years, and had a bachelor’s degree (counsellors) or were a clinical psychologist in training (supervisor). Children and adolescents were eligible for the RCT if aged 8–17 years and had a diagnosis of a common mental disorder (depression, anxiety disorder, PTSD, conduct or oppositional defiant disorder). Full inclusion criteria for the RCT are described in Additional file ##SUPPL##0##1##. Children and caregivers were eligible to take part in the semi-structured interviews if the child had fully or partially (≥ 2 sessions) completed a course of t-CETA. Of N = 12 children who met these criteria, N = 11 participated (n = 8, full course; n = 3, partial course). In all cases, both the caregiver and child took part. The number of sessions via phone ranged from 2–12 (<italic>M</italic>[<italic>SD</italic>] = 7.8 [4.1]). Five children (45%) were female and six (55%) were male, aged 8–17 years (<italic>M</italic>[<italic>SD</italic>] = 10.9 [2.6]). All 11 caregivers were female (10 mothers and one grandmother), aged 29–56 years (<italic>M</italic>[<italic>SD</italic>] = 36.4 [8.4]). All families lived in informal tented settlements.</p>", "<title>Procedure</title>", "<p id=\"Par22\">Counsellors were invited to take part in semi-structured interviews after completion of the RCT. After providing informed consent, semi-structured interviews were conducted online in English by KH between December 2019 and February 2020. Interviews lasted 54–59 min (<italic>SD</italic> = 2.65). Interview questions related to delivery of CETA to refugee children in person and via phone, benefits and challenges of working with a Syrian refugee population, program efficacy, how counsellors worked through challenges they faced, and training and supervision. Interviews were audio recorded, transcribed, and identifying details were removed (see Additional file ##SUPPL##0##1##). No remuneration was provided to counsellors for participation.</p>", "<p id=\"Par23\">Families that received t-CETA were approached by phone after completion of the RCT. Information about the study was read to families over the phone and verbal consent taken from caregivers; verbal assent was taken from children only if their caregiver provided consent. Financial compensation for participants’ time was provided. Semi-structured interviews were conducted over the phone in Arabic by HEK, during November and December 2019. Caregiver interviews lasted 20–57 min<bold>,</bold> and child interviews lasted 20–33 min (total time per family, <italic>M</italic>[<italic>SD</italic>] = 56.64 [13.76]). Children and caregivers were interviewed individually, with the exception of one 11-year-old who was more comfortable being interviewed together with their caregiver. Interview questions related to the experience of receiving telephone-delivered therapy, the extent of improvement of the child’s problems, session content, relationship with the counsellor, and benefits and challenges of receiving therapy over the phone (see Additional file ##SUPPL##0##1##). Interviews were audio-recorded, transcribed, and translated to English, and identifying information removed. Names have been replaced with pseudonyms throughout.</p>", "<title>Analysis</title>", "<p id=\"Par24\">Thematic content analysis was conducted independently for the counsellor interviews and the child and caregiver interviews (Braun &amp; Clarke, 2006). Interviews with counsellors were transcribed, reviewed, and coded by KH. Interviews with children and caregivers were transcribed and translated by HEK, reviewed for accuracy by Syrian and Lebanese researchers, then reviewed and coded by HEK. Data were classified into smaller meaningful categories through a process of open coding [##UREF##16##28##].</p>", "<p id=\"Par25\">Data relevant to the research questions were coded inductively line-by-line by one author (KH, counsellor interviews; HEK, child and caregiver interviews), with codes inductively deriving from the content of the data as opposed to the researchers’ presuppositions. For counsellor interviews, all codes were reviewed by KP, and for child and caregiver interviews, codes were reviewed by KH and/or FM for 55% of interviews. Reviewing authors commented on the classification of text and category labels assigned to them; codes were revised where necessary. Codes were reexamined, related codes grouped together, and these codes sorted into potential themes [##UREF##17##29##]. To promote credibility in the analysis, after theme development, the authors who assisted with coding also assisted with the refinement of themes. Researcher triangulation was employed, with regular meetings to discuss coding and emerging themes, as well as refinement of the final themes together. This was an iterative process, and disagreements in coding and themes were discussed to achieve consensus [##UREF##18##30##].</p>" ]
[ "<title>Results</title>", "<p id=\"Par26\">Three themes emerged from analysis of counsellor interviews. The counsellors believed that (1) counselling over the phone both solves and creates practical and logistical problems, (2) t-CETA is adapted to potential cultural blocks, and (3) t-CETA works and is needed. Four themes emerged from analysis of child and caregiver interviews: (1) counselling over the phone both solves and creates practical and logistical problems, (2) t-CETA works for many families but there are remaining challenges in other families, (3) the relationship between the counsellor and the child and the caregiver is extremely important, and (4) the family’s attitude to mental health influences their understanding of and engagement with counselling. While the analyses were conducted separately, there was significant overlap between themes (Fig. ##FIG##0##1##) and so results are presented by theme, highlighting similarities and differences between the perspectives of the counsellors and families where relevant.</p>", "<title>Counselling over the phone both solves and creates practical and logistical challenges</title>", "<title>Solving logistical issues</title>", "<p id=\"Par27\">The counsellors identified a number of challenges that refugees face in accessing traditional, face-to-face counselling, largely centered on difficulties travelling to a clinic. Because of the pervasive poverty of many Syrian refugees in Lebanon, few own a car and the cost of using taxis or buses is prohibitive. Furthermore, travelling to and from the clinic takes time and may not be possible for those with caring responsibilities. One of the counsellors explained,</p>", "<p id=\"Par29\">Many families echoed these issues. Caregivers described the difficulty of getting to a clinic and the comparative ease of receiving treatment over the phone, describing the expense of transport, time taken to travel, and difficulties when there are other children to take care of or when children attend school. A mother, comparing travelling to the clinic before starting phone sessions, said, “it takes time going and coming. But over the phone, we do the session and that’s it, it does not take a lot of our time.” This point was also made by several children. One mother said that it is not safe to leave other children at home in a camp, and another explained, “I preferred them over the phone, because you know I have young kids … If I went I would have to leave them … on the phone they can be beside me.”</p>", "<p id=\"Par30\">In addition, counsellors explained that parents found it hard to leave the settlement because of fear of “if something happened on the road”, “if a fire happened” in the settlement, or “the danger of being held at a checkpoint” without papers. The risk of sexual violence also impacted the mobility of women and children: “they heard that a woman got harassed by a taxi driver. Everyone stopped going in taxis”. Finally, severe weather conditions were a barrier to accessing in person counselling. A counsellor explained, “Their clothes are not equipped for cold – intense cold – in Beqaa for example. And the roads are not equipped for rain, and for flooding… for snow.” Counsellors pointed out that when a family misses a traditional in-person appointment, typically they would have to wait a week or two until their next scheduled session. If the barriers to attending are unchanged at that point, they will miss the rescheduled appointment too. This leads to many missed in-person appointments. Telephone delivery was seen by counsellors as a way to reach more and harder-to-reach children, to be more logistically flexible, to hold more appointments, and to be able to follow up more easily and rapidly when sessions were missed. This flexibility was also highlighted by mothers who described the counsellor calling to remind them of the appointment the day before and then again in the morning; if it was a bad time when the counsellor called – for example, because the family was out – then the counsellor would call back later to do the session.</p>", "<title>Logistical challenges</title>", "<p id=\"Par31\">Although t-CETA addressed the above problems, counsellors identified that phone-delivery created its own challenges which then had to be addressed. First, many children did not have reliable access to a phone. While some caregivers indicated that this was not a problem because they had two phones or borrowed one from a relative, in many families there was only one phone available due to the “very, very high telephone costs” (counsellor). This phone was typically with the father, potentially leading to issues when the father was at work; “whenever I call, I have to wait for the father until he goes back to the home” (counsellor). To address this, where possible the counsellors would get phone numbers for relatives or neighbours, and try to hold the sessions over this alternative phone. This worked where families felt comfortable with their relative or neighbour knowing that the child was in counselling, but some families wanted to keep this private. A counsellor explained one case where,</p>", "<p id=\"Par33\">Even where access to a phone was consistent, network coverage and phone charging were not always reliable. A counsellor said, “Too many times when I was calling the family, the phone will be off or they lose the battery or we had the bad connection.” Some families also highlighted this. One mother said, “The phone would disconnect a lot. Because … the coverage is very weak … Like now I have to keep my phone always outside, I don’t want to stay sitting outside in the cold so I get coverage.” Counsellors said that this could be a major problem, because when a call was dropped then “we have to call again and call again” and each time the child and counsellor would have to repeat what they were saying, some of which was “very, very sensitive” information which the children would not want to repeat. This slowed some sessions and interrupted their flow.</p>", "<p id=\"Par34\">A second issue that phone delivery raised was in the safety of talking on the phone. A counsellor explained that “the Syrian refugees are very wary of using the phone because their government … spies on people over the phone.” Some families were worried about the government spying on them, or their relatives or neighbours secretly recording their phone calls.</p>", "<p id=\"Par35\">A third issue was lack of privacy. Many refugees live in one or two room tents, often with large families and in close proximity to other tents. One counsellor said, “In a tent, you can hear everything around you. So there’s noise always. Sometimes there’s no doors in the tent, so someone will enter and go during the session.” The tents where the children live are “the least private space ever … if you’re whispering in the room, you’ll hear it in another room” (counsellor). The counsellors would explain to the parents and to the child that the counselling should be done in private, and in most cases, the families tried to respect this despite the logistical difficulties. However, it was not always possible. For example, one mother said it was, “hard for [the counsellor] to hear us and us to hear her … my daughter would go to the other room but you know it’s a tent... as much as you move away you will hear sound.” The counsellors explained that the therapy was less likely to be effective when there is lack of privacy, since the children may not feel comfortable sharing their experiences and feelings in the presence of others.</p>", "<p id=\"Par36\">However, not all families reported these problems and some discussed finding a private space to do the sessions: “I empty the space for him and I give him [the phone] to speak,” (mother); “I sit alone, and we talk, her [counsellor] and I, just me alone … And when she wants my mom, I go out and give her and my mom sits alone” (child).</p>", "<title>Communication over the telephone</title>", "<p id=\"Par37\">Councillors reported some difficulties in comprehension and communication over the phone. In-person, the counsellor “could use a board to explain or the sheets or a material in my hand to explain for the child”, but over the phone they had to explain concepts to the children with no aids. This was challenging because the counsellors would give “examples for shapes or numbers or timeline” in the sessions but these concepts were new to many children because they had had limited access to school. The counsellors had to teach the children these concepts before they could begin the therapy work itself: “after many efforts we could provide the therapy that we were providing. But it took many efforts.” However, the counsellors indicated that it was possible to teach these concepts to the children over the phone and that after this, therapy could begin.</p>", "<p id=\"Par38\">In-person therapy involves both visual and auditory information, but telephone therapy is solely auditory and so some information is lost. Aside from the lack of aids, the impacts of a lack of visual information were viewed to be mixed by the counsellors. The counsellors indicated that this could be a major positive; one felt that children trusted them more because the children could never see any judgement in their expression, and that not being in person allowed the children to “feel free to talk about everything.” It also could be a challenge though, with another counsellor indicating that they could not tell “from his facial expression what he is going through or if he is not understanding” the content. They explained that, “over the phone, you are really reliant on nonverbal cues that are also nonvisual. So it’s double the challenge.” These mixed results may reflect the level of experience of the counsellors: the counsellor who viewed it to be a positive was more experienced, whereas the counsellor who viewed it as a negative had never provided any counselling before. In addition to the above, the counsellors indicated that sometimes the children’s attention drifted, and that being together in person might have reduced this, or that they would have been better able to identify it.</p>", "<p id=\"Par39\">These mixed results were echoed in the interviews with families. Several families said that they would prefer face-to-face therapy because they thought that children would concentrate better, understand more, and find it easier to express themselves. Both caregivers and children emphasized the benefit of being able to see the other person’s reaction, body language and facial expressions. Children would be less likely to get bored, which was more of a problem for phone therapy when children were aged 12 years or younger. However, most caregivers said that they were comfortable using the phone for sessions and one thought that it was more comfortable for children who are shy than seeing a counsellor face-to-face: “She didn’t see her [the counsellor]... so she was able to speak more than if she saw her face-to-face.”</p>", "<p id=\"Par40\">Despite the above challenges, most of the families were very accepting of and positive about telephone delivery. They often indicated to the counsellors that they preferred it to in-person therapy because of the challenges of attending clinics. A counsellor explained that “there are many challenging things, but it’s [because of] their living conditions more than their acceptance for the phone or not.”</p>", "<title>t-CETA is adapted to potential cultural blocks</title>", "<p id=\"Par41\">t-CETA was specifically adapted for use with Syrian refugee children, as well as for lay person delivery, and the counsellors indicated that they felt that it was well-adapted culturally. They felt well trained and prepared to counsel the children. They found the training particularly useful because of the applied components, including extensive role play: “we apply all the components. We apply it. We do role plays for everything. We cover all the manual. We’ve covered many challenges that may appear.” By having lay counsellors with prior experience of case management/social work with Syrian refugees, t-CETA was delivered by people who knew the context and challenges well. Having telephone delivery and lay counsellors also enabled the counsellors to be more informal with the children than is typical in therapy: “I feel like I’m at ease when I’m working with them because the therapy context in this situation is more human than the clinical private setting … with refugees … it’s friendlier, it’s more accessible for patients” (counsellor).</p>", "<p id=\"Par42\">By adapting t-CETA to the context and having it delivered by local counsellors, the counsellors discussed mental health problems in a locally appropriate way,</p>", "<p id=\"Par44\">The counsellors were also careful because of potential stigma around having a mental health problem or seeking counselling. Phone therapy was useful in obviating this stigma and therefore in having higher levels of adherence to therapy, “because it’s basically done in the total privacy of the house. So no one would know that this person is going to a therapist or seeking mental health care” (counsellor). Reducing the “risk of being stigmatized and judged” was viewed as particularly important in reaching families with female children because accessing mental health services could impact girls’ later marital opportunities. To ensure that they did not inadvertently let others know that a child was having therapy, the counsellors had a protocol for how to talk to whoever picked up the phone, and for how to contact neighbours or relatives; “We try to get to know who is in the household, their full names to know if someone responded other than the child, that they know everything or not to see how to respond.” If the counsellor did not know the person who picked up the phone, they explained that they were calling from Médecins du Monde but did not make any reference to mental health services. Overall, having local lay counsellors receive in-depth training and extensive supervision was described by the counsellors as being both effective and appropriate, and as good strategy for identifying and addressing potential cultural issues.</p>", "<title>The relationship between the counsellor and the child and caregiver was extremely important</title>", "<title>Building rapport and trust</title>", "<p id=\"Par45\">The adaptation to cultural blocks identified by counsellors was reflected in children’s and caregivers’ views about the relationship and rapport between them and the counsellors. This influenced children’s willingness to engage with counselling. Both children and caregivers said that they trusted the counsellor and were made to feel comfortable and relaxed in the sessions. One mother said, “Every week I used to wait for the interview. I’d like talking to her. I feel relaxed and he [child] relaxes.” They highlighted the character and skills of the counsellors, saying that they were knowledgeable, respectful, accommodating, and treated them with attentiveness and care. This had a positive impact on the sessions. The mother of a 14-year-old said that the counsellor:</p>", "<p id=\"Par47\">It was clear that the counsellors could build rapport and this provided a strong foundation for the counselling, enabling children to talk honestly. Several caregivers said that their child was able to be open with the counsellor, sometimes sharing things that they had not shared with family members including details about traumatic experiences. Children were more likely to accept advice from counsellors: the counsellor “would wake him up, like give him advice… he would hear it from her and not hear it from me” (mother).</p>", "<p id=\"Par48\">Caregivers highlighted that children felt able to talk honestly because the sessions were conducted in private with the counsellor, without the caregiver being present. The counsellor independently spoke to caregivers about the general content of sessions (without giving details), meaning that caregivers were aware of the types of problems their child had raised and the skills they were being taught to address them. This built trust, making it easier for children to speak honestly and for caregivers to trust in the process. A mother explained,</p>", "<p id=\"Par50\">Caregivers mentioned that they felt comfortable talking openly to the counsellor and trusted the counsellor’s expertise and advice. For some, the feeling that someone was listening to them and helping was important, while children reported feeling more hopeful and positive about life and supported in dealing with specific difficulties.</p>", "<title>Challenges to engagement</title>", "<p id=\"Par51\">In some cases, children found aspects of the sessions difficult and it was challenging to engage with the counsellor at the outset. Some initially refused or felt unsure about talking to the counsellor on the phone and needed reassurance about speaking to the counsellor alone. Caregivers mentioned shyness or holding back because of depression as reasons for this. In other cases, the child would get bored or distracted. Children said that some aspects of the sessions were hard, specifically talking about memories of traumatic events. For others, difficulties related to understanding the counsellor and/or the content. An 11-year-old girl said, “I didn’t used to understand anything from her [the counsellor].” This girl did not like using a phone in general. However, some children who initially found it difficult to engage because of shyness got more comfortable with time. For example, two mothers said that their child found the first two sessions difficult but after that they started to enjoy talking to the counsellor.</p>", "<p id=\"Par52\">Some mothers and children said that there were times when they did not understand session content at first, but the counsellor would explain and repeat content until they understood. One mother commented, “Now if I found something difficult, we tell her about it, she makes me understand it directly… like we never shut the session having not understood what we are doing and talking about.” Both children and caregivers conveyed an impression that the counsellors were good at picking up when something was not understood and worked to ensure that content was understood before they moved on.</p>", "<title>The family’s attitude to mental health influences their understanding and engagement with counselling</title>", "<p id=\"Par53\">Children and caregivers’ attitude to mental health varied and this influenced the extent to which they engaged with counselling.</p>", "<title>Positive attitudes to seeking help</title>", "<p id=\"Par54\">Around half of families reported little stigma or shame: they were keen to seek expert advice, were supportive of therapy, and willing to talk to others outside their family about mental health issues and services. This seemed to contribute to caregivers’ involvement in sessions, children’s willingness to do sessions, as well as families’ overall commitment to therapy. Some drew parallels between mental and physical health and did not see mental health problems as something to be ashamed of, pointing out that “a therapist is exactly like any other doctor” (caregiver). An 8-year-old boy, asked about his reason for doing counselling, drew the link between traumatic memories of events in Syria, his feelings of fear, and his body. One mother emphasized the importance of children talking about psychological difficulties to someone other than their parents, which she said could make them feel safe and listened to, while another said, “it’s not shameful or sinful, something one is learning from.” Some families said that they had shared contact details for the service with others, and several children said that they would share techniques they had learned if they had a friend who was struggling.</p>", "<p id=\"Par55\">Openness to talking about mental health problems related to a greater willingness to seek out help and advice from those perceived to be specialists. The mother of a 9-year-old, said:</p>", "<p id=\"Par57\">Many caregivers encouraged their child to engage with the sessions, with some stating that families have a duty to help children get better. One mother, whose daughter initially found the sessions boring but went on to complete them, explained, “I made her understand that the counsellor is helping her and such, she understood more.” Another highlighted that she and the counsellor both wanted what was best for her son and they worked together to help him. Several caregivers expressed gratitude for the efforts of the counsellors.</p>", "<p id=\"Par58\">Caregivers varied in the way they understood their child’s problems, how much they were involved in sessions, and what they saw as their role in their child’s treatment. One mother showed insight into her daughter’s problems, talking at length about the impact of displacement and ongoing adversity on her daughter’s mental health. Another pointed out that “there are things it’s possible the child doesn’t say [to the counsellor], the mom says it for him,” while a different mother said that she told the counsellor about her daughter’s moods so the counsellor would know that she needs to be patient. One also said that she would also ask the counsellor for advice on dealing with her son’s problems. This suggests that some caregivers saw their role as an ally to the counsellor, engaging with the sessions and working cooperatively to help their child. This included prioritizing the sessions, with one mother saying, “The day she [counsellor] wants to call, if I have something, I postpone it.” Another mother described counselling as “a treatment like any other” that “you have to commit” to. The psychoeducation component of t-CETA emphasizes this, suggesting that at least some families took this on board and committed to ensuring that their child completed the course of counselling.</p>", "<title>Stigma and shame</title>", "<p id=\"Par59\">Other caregivers felt less comfortable disclosing and recommending sessions to others and, in some cases, were less involved in the counselling. For example, a grandmother who largely did not participate in her granddaughter’s sessions said that other people “don’t approve of this thing” and that “it’s secret talk, we can’t say it to anyone.” The child also expressed reluctance to mention counselling to others. One mother said that she would be reluctant to recommend counselling to another family in case they interpreted it as an accusation. She also said that she was initially hesitant about accepting mental health services because of the worry that it meant her son was “crazy”, but accepted when the counsellor reassured her that it was just talking and exercises and did not involve medication.</p>", "<title>Prior experience of mental health services</title>", "<p id=\"Par60\">Several caregivers talked about how the sessions were better than expected. In some cases, this related to previous experience with services for refugees. One mother said, “I thought it was a failing plan… But maybe in my perspective it was a failure because many organizations come and take information and then go.” She seemed surprised that this time there was regular follow up and support. The mother of an 8-year-old boy was initially worried that talking over the phone would not be effective:</p>", "<p id=\"Par62\">Previous experiences of mental health services, including the use of medication, had shaped families’ expectations about counselling, and this was something that was necessary to address through psychoeducation.</p>", "<title>t-CETA works and is needed</title>", "<p id=\"Par63\">The theme derived from interviews with the counsellors, <italic>t-CETA works and is needed</italic>, overlapped substantially with the theme derived from interviews with families, <italic>t-CETA works for many families but there are remaining challenges in other families</italic>, and so they are reported together.</p>", "<title>t-CETA works for many children</title>", "<p id=\"Par64\">All three counsellors indicated the utility of t-CETA at treating psychopathology. They were all hugely positive about this treatment from both an efficacy and logistical perspective. The efficacy was unexpected for counsellors, who were initially wary: “Really I was amazed and surprised how you can afford a full service on the phone.” They described multiple success stories of children whose debilitating mental health problems were alleviated through t-CETA. These changes were observed by the counsellors – who indicated that “you can hear it and feel it” – and were also told to them by the children’s parents: “it’s a really, really amazing feeling when the parents said ‘thank you, thank you very much. There is a big change.’ And you are just doing it on the phone.” Despite some challenges, all of the counsellors recommended the use of telephone-delivered therapy in the future, indicating “I think it’s a successful program. I really like it. I hope it will continue again, or if any INGO adopts this program for delivering it again, because it really works.” They indicated that particularly in a context of yearly funding cycles for NGOs, having a short, targeted telephone delivery of therapy for specific issues “is exactly what works.” The counsellors felt that it clearly addressed many of the logistical and cultural needs of the children and families, enabled more sessions to be held each day, allowed for better and more rapid follow up when appointments were missed, and was useful for reaching children and families who were unable or unwilling to access in-person therapy.</p>", "<p id=\"Par65\">Many families were similarly positive about t-CETA, with children and caregivers reporting that they enjoyed the session activities, learned from the counsellor, were able to apply what they learned, and their problems had improved. Several mothers were aware of coping techniques that their child had learned and applied to real life situations. One mother described her daughter employing cognitive restructuring:</p>", "<p id=\"Par67\">Children and caregivers described that the children employed other coping skills taught to them, such as counting to 10 when mad, writing down things that were bothering them, doing activities like drawing or coloring, walking or playing with friends, or helping their mother at home. Some caregivers noted that their child had learned about feelings, how to manage relationships with family and friends, and coping with nightmares.</p>", "<p id=\"Par68\">Caregivers also talked about techniques that they had learned to manage their child’s problems, including positive reinforcement, allowing their child to go out (e.g., to play with friends), and dealing with bed-wetting. Several talked about positive effects such as developing better understanding of their child’s problems and no longer using corporal punishment. The mother of a 9-year-old boy said,</p>", "<p id=\"Par70\">Most families reported at least some improvement of problems across different settings including home, school, and with friends. Improvements were noted as improved sleep and fewer nightmares; reduced fear and focus on traumatic memories; feeling calmer; reduced fatigue; improved play and relationships with siblings and peers; decreased anger and fighting; and finding studying easier. Improvements were sometimes noted by others: “My neighbor saw him, how he improved, some of them are telling me ‘Give us the number to talk’”. Some caregivers also compared the sessions favorably to other mental health services. One mother said that her son had previously seen a psychiatrist and had been given medication but did not show any improvement; by contrast he “benefitted a lot” from speaking to the t-CETA counsellor.</p>", "<title>Individual and contextual factors pose challenges to treatment</title>", "<p id=\"Par71\">Problems remained in some children, sometimes due to issues like physical illness or situational factors that are not addressed by t-CETA. Some children said that they were not applying what they learned in the sessions or they could not remember all the content. In one case, the grandmother of an 11-year-old girl explained, “She has iron deficiency. Iron deficiency makes someone forget … She forgets what the counsellor Farah told her.” Some caregivers reported continued problems in their child, including fear and difficult memories, anger, tired psychological state/depression, disciplinary issues, and relationship problems.</p>", "<p id=\"Par72\">Some children said that while they felt better during the sessions, this did not last. Often this was related to ongoing adversity, with families reporting stress due to financial and housing problems, family members with physical and mental health problems, the situation in Syria, separation from family and friends, and lack of access to school. A 17-year-old described how her father was sick, in pain and had a “broken psychological state”, and her mother and sister were depressed. She no longer had access to school and had to start working, their tent had flooded, and her brother had moved to Germany. The girl explained, “All the sessions were good with the counsellor. Good, but every time I would be optimistic, I would get a bump to take me down lower … I am talking to you and being hopeful but after I close [the phone] I get sad over my dad and his situation.” This girl completed only two counselling sessions before having to drop out to work.</p>", "<p id=\"Par73\">Counsellors noted that because of the major structural and environmental challenges faced by refugees, mental health treatment delivered in any form (in-person, over the phone, or another way) was likely to be limited in its efficacy. They explained that the challenges faced by refugees in Lebanon may cause further mental health problems or reduce the efficacy of treatment, while noting that these challenges are also part of why there is such an urgent need for treatment. The counsellors described how the mental health problems of the children were often triggered by their living situation:</p>", "<p id=\"Par75\">They were positive about telephone delivery of therapy but cautioned that major structural changes would also need to occur in order to address children’s mental health needs in the most effective and sustainable way.</p>", "<p id=\"Par76\">This was reflected in some families’ expressed need for financial as well as mental health support. One mother explained,</p>", "<p id=\"Par78\">This family talked about multiple problems that they faced and the impact this had on their mental health. This is a stark reminder that refugee families are likely to have multiple unmet needs that will impact on the likelihood of treatment success, regardless of the mode of delivery.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par79\">The aim of this study was to explore the feasibility and acceptability of delivering psychological treatment to children via telephone, by trained and supervised lay counsellors, in the challenging humanitarian context of informal tented settlements in Lebanon. Thematic content analysis of interviews with counsellors who delivered t-CETA and a parallel analysis of interviews with children who received t-CETA and their primary caregivers provided promising evidence suggesting that this approach is feasible and acceptable, while also highlighting challenges that must be addressed if telephone-delivered therapy is to be more widely adopted in humanitarian crisis contexts. This should be considered alongside preliminary evidence from the same sample suggesting that t-CETA is effective in reducing symptoms of psychopathology (with small-medium effect size) and increases the number of children who complete treatment [##UREF##12##23##].</p>", "<title>Telephone delivery solves logistical and cultural challenges</title>", "<p id=\"Par80\">Both counsellors and families highlighted the problems that telephone-delivered therapy helped to solve. Substantial overlap in the issues raised by counsellors and families strengthened the validity of these findings. For example, difficulty with travelling to clinics was frequently reported to be a significant barrier to accessing standard mental health services, echoing other reports from this context [##UREF##7##9##, ##UREF##8##10##]. A telephone-delivered service made access considerably easier as it avoided the cost of taxis, need for childcare, and safety concerns about women and children travelling alone. This is in line with findings from other settings, where high satisfaction with tele-mental health is based on improved access and convenience, and cost savings on travel, childcare, and work commitments [##REF##32594756##31##]. Notably, t-CETA allowed service provision during nationwide protests in Lebanon in 2019 (17 October Revolution) when many services were unable to provide face-to-face appointments. A substantially greater proportion of children in the pilot RCT who received t-CETA accessed treatment (90%) and completed a course of treatment (60%) compared to those who received face-to-face services (60% and 0%, respectively), showing that perceptions of reduced barriers to attendance translated into meaningful differences in retention [##UREF##12##23##]. This has clear implications for other situations when mobility is restricted, such as the COVID-19 pandemic and natural disasters.</p>", "<p id=\"Par81\">Crucially, receiving psychological therapy via phone potentially overcame some cultural blocks; where people do not use mental health services because of stigma, receiving therapy in the home removed the need to visibly attend a mental health service. This benefit has previously been noted for adults, where tele-mental health may remove barriers such as stigma as well as potentially allowing people to talk more freely [##REF##32594756##31##].</p>", "<title>Challenges to address</title>", "<p id=\"Par82\">Various new challenges were created by telephone-delivery, which needed to be addressed. This included a phone not always being available at the right time, poor phone signal, and difficulty for some families in finding a quiet place to do the sessions. These problems are pertinent in this context, where families may only have one phone, live in rural areas with poor network coverage, and live in overcrowded accommodation where it can be difficult to achieve privacy. Developing protocols to manage these difficulties reduced disruption to therapy and risks to confidentiality. This included avoiding disclosing the reason for the call before establishing the identity of the responder, working with the caregiver to find and maintain an appropriate place for the child to take the call, and jointly agreeing a plan for what to do if it was no longer comfortable for the child to talk or if the call was cut off [##UREF##15##27##]. The strategies developed helped to improve the situation over the course of therapy in most cases; however, in some cases there were persistent difficulties, such as background noise. It will be important to determine the suitability of telephone-delivery for individual families, including taking into account their scope to find an appropriate place for the calls. This should be decided on a case-by-case basis rather than assuming that tented settlements are not suitable, as some families were able to maintain a suitable space for the duration of sessions.</p>", "<p id=\"Par83\">Some of the difficulties created by telephone-delivery could be mitigated by the increased flexibility afforded by the approach. For example, it was much easier to rearrange sessions that were missed to later in the same day. Telephone-delivery, including offering some calls during evenings and weekends, resulted in considerably more flexibility than would be available with standard services and so less disruption to treatment flow. Caregivers also talked about feeling more supported when there were regular phone calls to follow up with them, in contrast to clinic-based services they had accessed previously. The ability to flexibly and regularly follow up by phone may act to increase engagement relative to face-to-face services, reflected in the high retention rates seen in the pilot RCT [##UREF##12##23##].</p>", "<title>Adapting communication for telephone-delivery</title>", "<p id=\"Par84\">Both counsellors and families reported that communication with some children was difficult over the phone. Communication was more challenging without visual feedback, making it harder for some children to maintain concentration and for counsellors to judge if children understood or were engaged with session content. For this reason, paying attention to non-visual cues and developing strategies to engage children was a particular focus of training and supervision [##UREF##15##27##]. For example, noticing and responding to hesitant or contradictory answers, prolonged silences, frequent topic changes, or changes in background noise that might indicate that the child was moving around or interacting with other people. Several strategies were employed to help engagement, such as shortening sessions, gamifying sessions by turning steps into activities, involving the child in decision-making and planning, and incorporating short verbal games to boost concentration and build rapport. Counsellors also highlighted that it was difficult to help children understand concepts without printed materials or being able to use pen and paper. This was made more challenging because many children had missed formal schooling and were unfamiliar with concepts (for example, what a triangle is, which was used to convey the links between thoughts, feelings, and behaviors). While the counsellors developed alternative strategies – such as describing a line linking concepts or getting children to use their fingers as a rating scale – this required incorporating a prior step before it was possible to cover the core session content. If possible, providing workbooks prior to commencing telephone-delivered therapy could support the process, taking into account the impact of missed schooling and other challenges on displaced children’s literacy [##UREF##19##32##] to ensure that materials are in an accessible format.</p>", "<p id=\"Par85\">Overall, while both counsellors and families highlighted a range of challenges, in reality this was outweighed by the increased accessibility afforded by telephone delivery in this population. Without this option, it is likely that many children would not have accessed treatment; indeed, of those children receiving face-to-face treatment in the control condition of the pilot RCT, 60% accessed any treatment, and none completed a course of treatment [##UREF##12##23##].</p>", "<title>Benefits of lay counsellors</title>", "<p id=\"Par86\">The use of trained and supervised lay counsellors to deliver psychological therapies has been identified as a way of addressing the lack of mental health professionals in humanitarian settings [##UREF##9##14##, ##REF##34579750##17##]. However, the counsellors interviewed for this study highlighted further advantages to their involvement. Being local to the Beqaa region and with extensive experience working with Syrian refugees in case management roles, they were very familiar with the context and challenges that refugees experience. They were able to discuss mental health in culturally sensitive terms and build trust with families. The counsellors reported that they could take a less formal approach than a mental health professional might do; this was also facilitated by the fact that children were receiving therapy in their own home rather than in a clinical setting.</p>", "<p id=\"Par87\">The majority of children and caregivers talked about the comfortable and trusting relationship they had with the counsellors, and the ease with which many of them could talk to the counsellors about their problems, supporting the counsellors’ view that their less formal approach brought benefits. This included children talking about traumatic memories during imaginal gradual exposure to desensitize children to feared memories. It is of note that it was possible to build rapport between counsellors and children (and their caregivers) over the telephone, despite the challenges of communication highlighted above, and that this likely made it easier for children to talk about difficult memories and sensitive issues. Key to the involvement of lay counsellors was regular expert clinical supervision based on the apprenticeship model, involving a local psychotherapist with knowledge of the context and an experienced CETA supervisor based at Johns Hopkins University [##REF##22099582##26##]. This created space to reflect on challenges, jointly develop strategies to deal with them, and ensure that learning from each case was communicated to the wider team.</p>", "<p id=\"Par88\">During this study we employed Lebanese rather than Syrian counsellors. This was because of legal restrictions on Syrians working in Lebanon and because during focus group discussions as part of a linked study, Syrian refugees generally expressed a preference to see Lebanese researchers due to uncertainty about the identity or allegiance of other Syrians. This preference may not apply in other contexts or at other points in time, and the acceptability of using Syrian counsellors should be explored further.</p>", "<title>t-CETA works for many children, but not all</title>", "<p id=\"Par89\">There was agreement between counsellors and families that t-CETA works for many children, with a reduction in symptoms over the course of treatment, a finding backed up by quantitative symptom scores and reports by children, parents, and counsellors recorded as part of the pilot RCT [##UREF##11##22##, ##UREF##12##23##]. Some children and caregivers also described continuing to apply strategies that they had learned in real life situations, suggesting that at least some of the effects of the intervention persisted beyond the end of treatment. The success of t-CETA was unexpected to counsellors and some caregivers: several said that they were initially skeptical about whether talking over the phone could really help and were surprised by the outcomes. Research from other contexts has highlighted a discrepancy between clinicians and patients, with clinicians more concerned about establishing therapeutic alliance remotely [##REF##34061758##33##], so it is of note that both counsellors and families in the current study were generally positive about the experience.</p>", "<p id=\"Par90\">Reduced likelihood of effectiveness may relate to practical problems such as not being able to maintain an appropriate place for the calls or because children find it difficult to engage over the phone, and further research will be required to determine which factors (e.g., age, presenting problems, living conditions) might help in decision-making about when to use t-CETA. It should be noted that t-CETA involves caregivers throughout and this also contributes to outcomes. Caregivers were asked to be present at the beginning of each call to confirm consent, and at the end when the counsellor would recap the session content to them so that they could support their child in doing homework. Caregivers were also taught techniques such as cognitive restructuring and it is likely that some were able to use this to address their own problems. Furthermore, some also received the <italic>Parenting Skills</italic> component, which taught them techniques to manage their child’s behavior. Caregivers were required to engage with telephone sessions and support their child in understanding the content and practicing techniques, which may be particularly important for younger children [##REF##32594756##31##]. A mixed-methods analysis of the factors influencing treatment success in t-CETA provides further evidence of the importance of parental support during treatment [##UREF##11##22##].</p>", "<p id=\"Par91\">There was variation in the degree of support that caregivers provided to children during treatment. A significant barrier to engagement was experiencing stigma about mental health problems and caregivers who reported this were less likely to have participated in sessions. CETA includes a component on engagement and psychoeducation, which explores barriers such as stigma and normalizes current symptoms [##UREF##20##34##], but this may not always be sufficient. Involving family members, such as fathers, seemed to help in some cases but community-wide interventions to reduce stigma might be necessary alongside individual treatment approaches. One mother made clear that she was less concerned about stigma when she realized that t-CETA is a talking therapy; this was contrasted to medication, which carries with it a higher connotation of being “crazy”. Avoiding a medicalized approach to mental health interventions in this population may reduce the effects of stigma as a barrier to using services.</p>", "<p id=\"Par92\">When t-CETA did not work as well, this often related to unmet needs and structural factors that are not addressed by mental health services. Poverty, poor quality housing, physical illness, family separation, lack of access to education and employment, and numerous other stressors play a significant role in the lives of those living in informal settlements [##REF##33277944##35##]. This level of adversity will likely impact the success of any type of psychological therapy, regardless of the mode of delivery, and this was found to be particularly the case for children with a primary presentation of depressive symptoms linked to current living conditions [##UREF##11##22##]. A case management approach that aims to address a wider range of needs alongside mental health treatment may be of benefit to individual families, but major structural changes to address the challenging living conditions of refugees are necessary for optimal mental health.</p>", "<title>Strengths and limitations</title>", "<p id=\"Par93\">This study is the first to explore the feasibility and acceptability of delivering psychological therapy via telephone to children in a humanitarian setting. Conducting interviews separately with counsellors, children, and their caregivers enabled us to explore their different perspectives, building a more holistic picture and increasing confidence in the validity of the results. To look at what is possible in environments when both resources and mobility are severely constrained, we used only standard telephone calls and no other supporting materials, providing a baseline for what might be feasible in a range of resource-constrained contexts: the addition of workbooks or videocalls, where the situation allows, would likely facilitate therapy.</p>", "<p id=\"Par94\">This study is limited by a relatively small sample size, though we interviewed all the staff involved in delivering t-CETA (two lay counsellors and their local supervisor) and the majority of children and caregivers who received t-CETA; this included families who dropped out, to avoid biasing the results towards those with more favorable views. Twenty-five interviews were sufficient to draw out similarities and differences in experiences to draw preliminary conclusions from. We had to conduct interviews for this study either online or via telephone due to mobility restrictions at the time of the interviews. This made some interviews more difficult and may have reduced the quality of the data, though it should be noted that participants were familiar with talking about their experiences over the phone due to their experience of t-CETA.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"Par95\">There is promising evidence to suggest that trained lay counsellors delivering psychological treatments such as CETA via telephone is both feasible and acceptable in the challenging humanitarian context of informal tented settlements in Lebanon. It has the potential to increase access to mental health services in hard-to-reach populations who struggle to use existing services. While many services temporarily relied on remote delivery during the COVID-19 pandemic, there is the potential for tele-mental health to bridge the mental health treatment gap in vulnerable communities on an ongoing basis after the pandemic [##REF##33553090##36##]. Humanitarian settings engender specific challenges compared to high income settings, but with sufficient attention to potential challenges, and high-quality supervision of counsellors to support them in finding solutions, it is possible to deliver therapy via telephone to children as young as eight-years-old. Finally, using lay counsellors – carefully selected, trained, and under expert supervision – to deliver psychological treatment is not only feasible but potentially advantageous in terms of being able to recruit counsellors with the greatest familiarity with the population with whom they are to work. Further research in larger samples, and in a range of settings, will help to establish the potential for these approaches to address the significant gap that exists between the need for mental health treatment and available treatment in resource-constrained settings.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Refugee children are at high risk of mental health problems but face barriers to accessing mental health services, a problem exacerbated by a shortage of mental health professionals. Having trained lay counsellors deliver therapy via telephone could overcome these barriers. This is the first study to explore feasibility and acceptability of telephone-delivered therapy with refugee children in a humanitarian setting.</p>", "<title>Methods</title>", "<p id=\"Par2\">An evidence-based intervention, Common Elements Treatment Approach, was adapted for telephone-delivery (t-CETA) and delivered by lay counsellors to Syrian refugee children in informal tented settlements in the Beqaa region of Lebanon. Following delivery of t-CETA, semi-structured interviews were conducted with counsellors (N = 3) and with children who received t-CETA (N = 11, 45% female, age 8–17 years) and their caregivers (N = 11, 100% female, age 29–56 years) (N = 25 interviews). Thematic content analysis was conducted separately for interviews with counsellors and interviews with families and results were synthesized.</p>", "<title>Results</title>", "<p id=\"Par3\">Three themes emerged from interviews with counsellors and four themes from interviews with families, with substantial overlap between them. Synthesized themes were: counselling over the phone both solves and creates practical and logistical challenges; t-CETA is adapted to potential cultural blocks; the relationship between the counsellor and the child and caregiver is extremely important; the family’s attitude to mental health influences their understanding of and engagement with counselling; and t-CETA works and is needed. Counselling over the phone overcame logistical barriers, such as poor transportation, and cultural barriers, such as stigma associated with attending mental health services. It provided a more flexible and accessible service and resulted in reductions in symptoms for many children. Challenges included access to phones and poor network coverage, finding an appropriate space, and communication challenges over the phone.</p>", "<title>Conclusions</title>", "<p id=\"Par4\">Despite some challenges, telephone-delivered therapy for children shows promising evidence of feasibility and acceptability in a humanitarian context and has the potential to increase access to mental health services by hard-to-reach populations. Approaches to addressing challenges of telephone-delivered therapy are discussed.</p>", "<p id=\"Par5\"><italic>Trial Registration</italic> ClinicalTrials.gov ID: NCT03887312; registered 22nd March 2019.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s13031-023-00565-2.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>We warmly thank all participating families for their participation. Fieldwork was conducted with Médecins du Monde France (MdM) in Lebanon. We thank Patricia Moghames, Nicolas Chehade, Anas Mayya, Estefania Hanna, Stephanie Legoff, Nicolas Puvis, Alaa Hijazi, Zeina Hassan, and all other members of the research team (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.qmul.ac.uk/sbbs/about-us/our-departments/psychology/global-mental-health/meet-the-team/\">https://www.qmul.ac.uk/sbbs/about-us/our-departments/psychology/global-mental-health/meet-the-team/</ext-link>) for their dedication, hard work and insights.</p>", "<title>Author contributions</title>", "<p>HEK and KH conducted and transcribed the interviews and HEK translated transcripts from Arabic to English. HEK, KH, FM, and KP conducted thematic content analysis. MP, LM, RW, and EK secured funding for the projects, and MP, FM, NC, and TB were responsible for research administration and supervision. SS and NC were responsible for clinical supervision of counsellors. FM and KH drafted the manuscript and all authors discussed results, critically reviewed the manuscript, and agreed the final version.</p>", "<title>Funding</title>", "<p>This work was supported by Elrha’s Research for Health in Humanitarian Crises (R2HC) Programme, which aims to improve health outcomes by strengthening the evidence base for public health interventions in humanitarian crises. R2HC is funded by the UK Foreign, Commonwealth and Development Office (FCDO), Wellcome, and the UK National Institute for Health Research (NIHR). Visit elrha.org for more information about Elrha’s work to improve humanitarian outcomes through research, innovation, and partnership. Children were recruited from the BIOPATH study, which was funded by the Eunice Kennedy Shriver National Institute of Child Health &amp; Human Development [grant number R01HD083387]. The funders played no role in study design, in the collection, analysis or interpretation of data, or in the writing of this report. The study was sponsored by Queen Mary University of London (QMUL).</p>", "<title>Availability of data and materials</title>", "<p>Due to the sensitive nature of the data and the potential to identify individuals, original data (audio recordings and transcripts) are not publicly available.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par96\">Ethical approval was granted by the Institutional Review Board of the American University of Beirut (ref: SBS-2017-0429) and the study was approved by the Ministry of Public Health in Lebanon (ref: 2017/4/49165). Caregivers provided informed consent and children provided assent for participation in the RCT; counsellors and caregivers provided informed consent and children provided assent for participation in the interviews reported here.</p>", "<title>Consent for publication</title>", "<p id=\"Par97\">All participants provided consent for anonymized data to be published; pseudonyms have been used throughout to ensure anonymity.</p>", "<title>Competing interests</title>", "<p id=\"Par98\">The authors declare they have no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Results of thematic content analysis of interviews with counsellors and families (individual interviews with children and caregivers)</p></caption></fig>" ]
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[ "<disp-quote><p id=\"Par28\">Many of the families in the area that we work are in need therapy but cannot come to the clinic because of lack of transportation or because maybe the mother had many kids and they had to stay with them in the tent or maybe someone is sick and cannot move from the tent or maybe it’s far away from them.</p></disp-quote>", "<disp-quote><p id=\"Par32\">The mother refused at all to use her neighbour’s phone because sometimes think they will record it … She didn’t feel safe to use the neighbour’s phone, so we stopped. We cannot do anything so we stopped the counselling with the child, but as a good thing that happened, after one month and a half the father came back to their home and he started working beside the home so we continued again.</p></disp-quote>", "<disp-quote><p id=\"Par43\">I needed to provide therapy in itself but adapt it to the context because … I needed to take into consideration their beliefs, because for example, sometimes if I talked about depression that is relatively severe or they had … suicidal ideation, the parents think of demonic possession or jinn possession and they might … get help from a religious person, whereas me, I have to tread more carefully about this subject.</p></disp-quote>", "<disp-quote><p id=\"Par46\">Gives and takes with the person, and she gives them room to talk and say whatever they want, you know? I felt like when I spoke to her, I relaxed… like the most important thing is she knows how to reach out to a child and knows how to approach each person depending on their age.</p></disp-quote>", "<disp-quote><p id=\"Par49\">The conversation that would happen between her [counsellor] and Ayman [child], for example she would tell me it but not directly. Like, just so that I have an idea of what my son is bothered from… and everything is private between her and him. And I try not to tell my son so that he keeps opening his heart to her.</p></disp-quote>", "<disp-quote><p id=\"Par56\">I like the educated, I like to take other’s opinion bigger than me you know? … I studied till 6/7<sup>th</sup> grade… Others have done till baccalaureate or university, Arabic literature. So there are people who still know more than me… So I like this really. If something happened with me, I like to talk about it to someone who knows in this.</p></disp-quote>", "<disp-quote><p id=\"Par61\">At first, I used to say it’s possible he might not benefit on the phone… but on the contrary, he benefitted… Like I was not expecting for him to heal and like for this thing to go from him. Because I was telling you I used to take him to a mental health doctor/psychiatrist and he did not benefit from him… I kept taking him almost two months and he did not benefit, not even a little… Here on the phone, first week he was different to me.</p></disp-quote>", "<disp-quote><p id=\"Par66\">if she saw two people laughing, it does not mean that they are talking or laughing about you … for example someone passes by and doesn’t say ‘Hi’, that doesn’t mean he or she is upset with her. They might have other things on their mind or problems or something . . . that have nothing to do with us for example.</p></disp-quote>", "<disp-quote><p id=\"Par69\">Counsellor Farah spoke to me, she told me ‘You have to go along with him, a little from you, a little from me, we have to help each other’ … I feel that he got a bit relaxed . . . I no longer hit him, I no longer keep him in . . . I let him go outside and stuff, he is well with the kids.</p></disp-quote>", "<disp-quote><p id=\"Par74\">I can definitely work with this adolescent in therapy on so many things, but I have to take into consideration the huge limitations that the environment of this same adolescent is putting on our therapy … even if I, for example, adopted the CBT approach for behavior activation for therapy for depression, and I worked on the cognitive reprocessing, for example, I would still come off a bit as patronizing because for this adolescent, the main trigger for depression is something outside of his control.</p></disp-quote>", "<disp-quote><p id=\"Par77\">We are missing a lot. The biggest missing thing for us is financial. So, we see someone come to us and take down our names and tells us ‘we want to speak to you, and we want to comfort you’, we hope that this person gives us money.</p></disp-quote>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Fiona S. McEwen, Hania El Khatib, Kristin Hadfield are joint first authors.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"13031_2023_565_Fig1_HTML\" id=\"MO1\"/>" ]
[ "<media xlink:href=\"13031_2023_565_MOESM1_ESM.pdf\"><caption><p><bold>Additional file 1.</bold> Supplementary Materials.</p></caption></media>" ]
[{"label": ["1."], "mixed-citation": ["UNHCR. Global Trends: UNHCR; 2022. "], "ext-link": ["https://www.unhcr.org/globaltrends"]}, {"label": ["2."], "mixed-citation": ["UNHCR. Refugee Data Finder: UNHCR; 2022. "], "ext-link": ["https://www.unhcr.org/refugee-statistics/"]}, {"label": ["4."], "mixed-citation": ["UNHCR. Syria Regional Refugee Response: UNHCR; 2022. updated 31/03/2022. "], "ext-link": ["https://data2.unhcr.org/en/situations/syria"]}, {"label": ["5."], "mixed-citation": ["UNHCR. Syria Regional Refugee Response: Lebanon: UNHCR; 2022. updated 31/01/2022. "], "ext-link": ["https://data2.unhcr.org/en/situations/syria/location/71"]}, {"label": ["6."], "mixed-citation": ["UNHCR, UNICEF, WFP. 2021 Vulnerability Assessment for Syrian Refugees in Lebanon (VASyR). Lebanon; 2022 25 January 2022."]}, {"label": ["7."], "mixed-citation": ["RPW. \"I have not known the taste of safety for ten years.\" Syrians trying to survive in Lebanon and Syria. 2021 2021."]}, {"label": ["8."], "mixed-citation": ["McEwen FS, Biazoli CEJ, Popham CM, Moghames P, Saab D, Fayyad J, et al. Prevalence and predictors of mental health problems in refugee children living in informal settlements in Lebanon. Nature Mental Health. 2023;1:135\u2013144."]}, {"label": ["9."], "surname": ["Hansen", "Askgaard", "Abou-Diab"], "given-names": ["AKV", "R", "D"], "source": ["Lebanon: DIGNITY"], "year": ["2018"], "publisher-loc": ["DRC Lebanon"], "publisher-name": ["American University of Beirut"]}, {"label": ["10."], "surname": ["Hanna"], "given-names": ["EK"], "source": ["Predictors of pretreatment dropout in a sample of Syrian refugee children and adolescents with mental health difficulties in Lebanon: a mixed methods study"], "year": ["2022"], "publisher-loc": ["Beirut"], "publisher-name": ["American University of Beirut"]}, {"label": ["14."], "mixed-citation": ["WHO. Comprehensive mental health action plan 2013\u20132030. Geneva: World Health Organization; 2021"]}, {"label": ["16."], "surname": ["Cohen", "Yaeger"], "given-names": ["F", "L"], "article-title": ["Task-shifting for refugee mental health and psychosocial support: a scoping review of services in humanitarian settings through the lens of RE-AIM"], "source": ["Implem Res Pract"], "year": ["2021"], "volume": ["2"], "fpage": ["1"], "lpage": ["13"]}, {"label": ["22."], "mixed-citation": ["Bosqui T, McEwen FS, Chehade N, Moghames P, Skavenski S, Murray L, et al. What drives change in children receiving telephone-delivered Common Elements Treatment Approach (t-CETA)? A multiple n = 1 study with Syrian refugee children and adolescents in Lebanon. Child Abuse Negl. 2023:106388."]}, {"label": ["23."], "mixed-citation": ["Pluess M, McEwen FS, Biazoli C, Chehade N, Bosqui T, Skavenski S, et al. Delivering therapy over telephone in a humanitarian setting: a pilot randomized controlled trial of common elements treatment approach (CETA) with Syrian refugee children in Lebanon. In Preparation."]}, {"label": ["24."], "mixed-citation": ["Pluess M. Evaluation of Phone-Delivered Psychotherapy for Refugee Children: Elrha; 2019. "], "ext-link": ["https://www.elrha.org/project/evaluation-phone-delivered-psychotherapy-refugee-children/"]}, {"label": ["25."], "mixed-citation": ["CETAGlobal. What is CETA? : Johns Hopkins Bloomberg School of Public Health; 2021. "], "ext-link": ["https://www.cetaglobal.org/whatwedo1-index"]}, {"label": ["27."], "mixed-citation": ["McEwen FS, Bosqui T, Chehade N, Saad S, Abdul Rahman D, Skavenski S, et al. Delivering psychological treatment to children via phone: a set of guiding principles based on recent research with Syrian refugee children. London QMUL, editor: Queen Mary University of London; 2020. 10.17605/OSF.IO/JTF2V."]}, {"label": ["28."], "surname": ["Strauss", "Corbin"], "given-names": ["AL", "JM"], "source": ["Basics of qualitative research: techniques and procedures for developing grounded theory"], "year": ["1998"], "edition": ["2"], "publisher-loc": ["Thousand Oaks"], "publisher-name": ["Sage Publications, Inc."], "fpage": ["1998"]}, {"label": ["29."], "surname": ["Braun", "Clarke"], "given-names": ["V", "V"], "article-title": ["Using thematic analysis in psychology"], "source": ["Qual Res Psychol"], "year": ["2006"], "volume": ["3"], "issue": ["2"], "fpage": ["77"], "lpage": ["101"], "pub-id": ["10.1191/1478088706qp063oa"]}, {"label": ["30."], "surname": ["Bryman", "Burgess"], "given-names": ["A", "B"], "source": ["Analyzing qualitative data"], "year": ["1994"], "edition": ["1"], "publisher-loc": ["London"], "publisher-name": ["Routledge"], "fpage": ["1994"]}, {"label": ["32."], "mixed-citation": ["Hadfield K, Al-Hamad M, Bakhti R, Dajani R, El Kharouf A, Michalek J, et al. Predictors of literacy and attitudes toward reading among Syrian refugee children in Jordan. IJEC. 2022."]}, {"label": ["34."], "surname": ["Murray", "Dorsey", "Haroz", "Lee", "Alsiary", "Haydary"], "given-names": ["LK", "S", "E", "C", "MM", "A"], "article-title": ["A common elements treatment approach for adult mental health problems in low- and middle-income countries"], "source": ["Cogn Behav Pract"], "year": ["2013"], "volume": ["21"], "fpage": ["111"], "lpage": ["123"], "pub-id": ["10.1016/j.cbpra.2013.06.005"]}]
{ "acronym": [ "CETA", "CBT", "PTSD", "RCT", "t-CETA", "UNHCR" ], "definition": [ "Common Elements Treatment Approach", "Cognitive behavioral therapy", "Post-traumatic stress disorder", "Randomized controlled trial", "Telephone-Common Elements Treatment Approach", "United Nations High Commissioner for Refugees" ] }
36
CC BY
no
2024-01-14 23:43:47
Confl Health. 2024 Jan 13; 18:7
oa_package/42/28/PMC10787498.tar.gz
PMC10787499
38217005
[ "<title>Background</title>", "<p id=\"Par48\">Breast cancer (BC) is the most frequent malignancy and the leading cause of cancer-related deaths among women worldwide [##REF##33538338##1##]. Neoadjuvant chemotherapy (NAC) is currently the standard treatment for high-risk early-stage, locally advanced or inoperable BC. NAC is performed before surgery to reduce tumor burden and test the sensitivity of BC to treatment. Previous studies have indicated that response to NAC is significantly associated with the prognosis of BC patients [##REF##24529560##2##, ##UREF##0##3##]. However, the benefit varies from patient to patient.</p>", "<p id=\"Par49\">If tumors are sensitive to NAC, optimal treatment strategies can be used to improve the outcome. It has been demonstrated that patients with pathologic complete response (pCR) to NAC improve disease-free survival (DFS) and overall survival (OS) [##REF##24529560##2##, ##REF##9704717##4##–##REF##34902335##6##]. This has made achieving pCR one of the main objectives of NAC. Unfortunately, pCR occurs only in a small proportion of BC patients, and differs significantly according to tumor subtypes [##UREF##1##7##]. Therefore, it is critical to identify patients who are most likely to benefit from NAC. To date, several clinical biomarkers have been exploited in clinics to assess NAC response, including Ki-67 expression, tumor size and molecular subtype. Multiple predictive molecular biomarkers have also been investigated in clinical trials involving neoadjuvant therapies. It has been shown that <italic>BRCA1/2</italic> mutation status leads to a better response to NAC in BC whereas <italic>PIK3CA</italic> and <italic>TEKT4</italic> mutations are associated with resistance to neoadjuvant therapy, including chemotherapy and targeted therapy [##REF##29725888##8##–##REF##24823476##12##]. Previous studies provide predictive biomarkers for screening patients who benefit from NAC, and lay the foundation for exploring new therapeutic targets for BC. However, owing to high heterogeneity and insufficient precision of BC, the prediction for NAC response still remains a big challenge in BC management. Therefore, there is an urgent need to identify novel predictive molecular biomarkers that can further facilitate the selection of patients who are more likely to benefit from NAC.</p>", "<p id=\"Par50\">Studies in bladder cancer, gastric cancer, ovarian cancer, and esophageal squamous cell carcinoma have shown that NAC can change the omics characteristics of tumor cells, which may further affect responses to subsequent therapy and patient prognosis [##UREF##2##13##–##REF##35087175##16##]. In BC, similar studies have tended to focus on a single-level omics such as genomics or transcriptomics [##REF##31061067##17##–##REF##31919134##19##], rather than simultaneous multi-omics analyses, which are beneficial for a more comprehensive understanding of molecular changes in BC during NAC.</p>", "<p id=\"Par51\">In the present study, we first established the genomic and transcriptomic profiles of breast tumors before and after treatment using a multi-omics characterization strategy that combined whole exome sequencing (WES) and RNA sequencing (RNA-seq) analyses. Molecular features related to NAC sensitivity were further analyzed by integrating omics and clinical characteristics, followed by confirmation assays of potential biomarkers using in vitro cell line models or clinical validation cohorts.</p>" ]
[ "<title>Methods</title>", "<title>Patient population and samples</title>", "<p id=\"Par52\">This study included four datasets: three datasets enrolled BC patients who received NAC (the NACBC sequencing set, the internal NACBC validation set, and the external Gene Expression Omnibus (GEO) validation set) and the fourth dataset enrolled BC patients who received adjuvant chemotherapy (the external The Cancer Genome Atlas (TCGA) validation set). Their characteristics are as follows.</p>", "<p id=\"Par53\">In the NACBC sequencing set, eligible patients diagnosed with primary BC were treated with NAC, followed by surgery at The Second Hospital of Shandong University between March 2013 and August 2019. The inclusion criteria were: (1) patients were newly diagnosed with histologically confirmed non-metastatic BCs; (2) patients received at least two cycles of NAC before surgery; (3) Biopsies samples before NAC, and surgical samples after NAC (if there was residual disease) could be collected (Fig. ##FIG##0##1##A). Pre-NAC samples were collected by biopsy. For the post-NAC samples collection, immediately after the residual disease was resected, the specimens were delivered to the Department of Pathology for gross and microscopic examination. Post-NAC samples were collected without compromising the surgical pathological evaluation of the resection specimen. The tissues were submerged in RNAlater or frozen directly in liquid nitrogen until further use. In this set, samples from 50 patients were used for WES and/or RNAseq. Forty-seven pre- and 44 post-treatment tumor samples and matched germline DNA samples were analyzed by WES. Fifty pre- and 45 post-treatment tumor samples were analyzed by RNA-seq (Fig. ##FIG##0##1##B, Additional file ##SUPPL##0##1##: Fig. S1). This sequencing set was used to identify molecular changes following NAC and screen for molecular features associated with response to NAC in BC.</p>", "<p id=\"Par54\">After NAC, the abundance of residual tumor cells in the primary breast tumor bed site was evaluated according to the Miller–Payne (MP) histological grading system [##REF##14659147##20##]. It was performed on all patients according to the standard criteria by two independent, blinded pathologists. Tumors were classified into three groups: the responsive was defined when infiltrating cancer cells were significantly reduced by &gt; 90% with only small clusters or widely dispersed individual cancer cells, or there were no infiltrating cancer cells at the original tumor bed site (MP scores: 4 or 5); the middle responsive referred to those with a reduction of cancer cells between 30 and 90% (MP scores: 3); and the nonresponsive were those with a reduction of tumor cells between 0 and 30% (MP scores: 1 or 2) (Figs. ##FIG##0##1##C and D).</p>", "<p id=\"Par55\">Clinicopathological characteristics including age at initial diagnosis, tumor histologic type, tumor size, lymph node status, histologic grade, neoadjuvant therapies, and estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 status were collected. ER and PR status were assessed using immunohistochemistry (IHC), with positivity defined as ≥ 1% of tumor cells being positive immunostaining [##REF##20404251##21##]. HER2 status was assessed using IHC and in situ hybridization (ISH) analysis if necessary. Positive HER2 status was determined as an IHC score of 3 + (more than 10% tumor cells with intense, complete and homogeneous membrane staining of HER2) or a positive ISH result. Clinical tumor and lymph node stage before NAC were determined by an experienced physician through physical examination and ultrasonography by at least two independent radiologists.</p>", "<p id=\"Par56\">In the internal NACBC validation set, a tissue microarray (TMA) was constructed from formalin-fixed paraffin-embedded pre-treatment biopsies from patients who were diagnosed with primary BC and treated with NAC at the same center as the NACBC sequencing cohort between January 2013 and December 2018. Invasive cancer sites in donor paraffin blocks were identified by an experienced pathologist using matching hematoxylin and eosin reference slides. Then, the TMA was constructed using 2 mm cores by a tissue microarray facility (3DHISTECH, Budapest, Hungary). After the TMA was fabricated, it was sectioned into 4-μm-thick tissue slices and stained with hematoxylin–eosin. The quality of the TMA was evaluated by two experienced pathologists. In the subsequent IHC analysis, cores without invasive carcinoma were excluded. The patient inclusion criteria were as follows: (1) with newly diagnosed, histologically confirmed non-metastatic BC; (2) received at least two cycles of NAC before surgery; (3) received a standard treatment (including surgery and chemotherapy); (4) with complete follow-up information available; (5) whose tumor tissue on the TMA being confirmed as invasive carcinoma by hematoxylin–eosin staining; and (6) ADGRA2 and ADRB3 expression could be assessed. A final population of 156 patients was included in the NACBC validation set. Clinicopathological assessments of this validation set were the same as for the NACBC sequencing set. This validation set was used to analyze the relationship between ADGRA2 or ADRB3 protein expression and pathological response and prognosis of patients.</p>", "<p id=\"Par57\">In the GEO validation set, the GSE25066 dataset was used to study genomic predictors of response and survival following neoadjuvant taxane-anthracycline chemotherapy in BC [##REF##21558518##22##]. The GSE25066 is a combination of GSE25055 and GSE25065 datasets. Gene expression data were log2 transformed and scaled to a reference distribution of 1,322 BC specific genes. The GSE25066 dataset included a total of 508 patients with complete DFS event information; however, whether 20 of the patients had residual disease after NAC was unknown. We extracted the expression level of <italic>ADGRA2</italic> and <italic>ADRB3</italic> for analysis with the cut-off values being determined by using the maximum Youden Index [##REF##21868756##23##, ##REF##33042836##24##]. This set was used to validate the relationship between <italic>ADGRA2</italic> or <italic>ADRB3</italic> mRNA expression and pathological response and prognosis of patients.</p>", "<p id=\"Par58\">The TCGA validation set included 1,085 female BC patients. It was used to further validate the role of <italic>ADGRA2</italic> and <italic>ADRB3</italic> in chemotherapy response as did with the GSE25066 dataset. Therefore, only the 566 patients who received chemotherapy and had prognostic information available were analyzed for the mRNA expression in the present study. This validation set was used to analyze the relationship between <italic>ADGRA2</italic> or <italic>ADRB3</italic> mRNA expression and patient’s prognosis.</p>", "<title>Isolation of genomic DNA and RNA</title>", "<p id=\"Par59\">Total DNA was isolated from fresh frozen tissue samples using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and blood samples using the QIAamp DNA Blood Mini Kit (Qiagen). TRIzol reagents (Tiangen, Beijing, China) was used to extract RNA from fresh frozen tumor tissue. The purity of total DNA and RNA were estimated by measuring the absorbances at 260 nm (A<sub>260</sub>) and 280 nm (A<sub>280</sub>) using a NanoPhotometer® spectrophotometer (IMPLEN, Munich, Germany). The extracted DNA and RNA were considered pure and suitable for subsequent experiments when the A<sub>260</sub>/A<sub>280</sub> ratio was within the range of 1.8 to 2.0. A mass ≥ 3 µg was considered to meet the experimental requirements for sequencing sample library construction. RNA samples were also tested by formaldehyde denaturing gel electrophoresis, wherein the rRNA ratio (28S/18S) needed to be ≥ 1.5, otherwise it meant that the RNA had degraded.</p>", "<title>DNA sequencing</title>", "<p id=\"Par60\">Qualified genomic DNA samples were prepared from tissue and peripheral blood samples for WES. Briefly, 3 μg of DNA was sheared into short fragments of 150 to 200 bp using an ultrasonicator Covaris M220 (Thermo Fisher Scientific, Waltham, MA, USA). Quality control was performed using a 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) after fragmentation. The library was constructed using a KAPA Library Quantification kit (KAPA Biosystems, South Africa) and “SureSelectXT Human All Exon V6” (Agilent Technologies) according to the manufacturer’s protocol. The kit was used to enrich the 357,999 exons from the 21,522 genes, covering approximately 60 Mb of the human genome. Validated DNA libraries were sequenced with paired-end runs on an Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, USA) by the CapitalBio (Beijing, China).</p>", "<title>RNA sequencing</title>", "<p id=\"Par61\">Library construction for RNA-seq was performed as described in the TruSeq RNA Sample Preparation Kit. Briefly, isolated total RNA was reverse-transcribed into cDNA with poly-dT primers using the Hifair® kit (Yeasen Biotech, Shanghai, China). The RNA-seq library was prepared by cDNA synthesis, end repair, 3′ adenylation, adaptor ligation, amplification, and product purification. Quality control was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies) with a DNA chip. After quantification with a NanoPhotometer® spectrophotometer (IMPLEN), libraries were sequenced with paired-end runs on an Illumina NovaSeq 6000 (Illumina Inc.) by the CapitalBio.</p>", "<title>WES data analysis</title>", "<p id=\"Par62\">The fastp (v0.20.0) [##UREF##3##25##] was used to filter raw data. The specific conditions were as follows: the adapter in the sequence was identified and cut off in the read with a minimum length of the reserve being 100 bp. If a read with &gt; 5% “N” bases and/or &gt; 50% low-quality base, the entire pair of reads were removed. Valid sequencing data were aligned to the human reference genome (GRCh38) using the Burrows–Wheeler Aligner (v0.6.1) [##REF##19451168##26##], and the resulting BAM files were preprocessed using the Sentieon (v202010). Sequencing quality statistics were obtained using the fastp. The average target sequencing coverage depth of tumor and matched germline samples was approximately 100 × .</p>", "<p id=\"Par63\">To identify all somatic variants in the samples, we used two pipelines (Sentieon TNseq and TNscope) [##UREF##4##27##] to detect for single nucleotide variants (SNVs) and indels, and matched normal samples were used to exclude germline variations. Somatic mutations were annotated using the ANNOVAR (v20160201) [##REF##20601685##28##]. To obtain the accurate mutation call set, two caller consensus mutations were performed for additional filtering. The bcftools v1.10.2 [##UREF##5##29##] (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/samtools/bcftools\">https://github.com/samtools/bcftools</ext-link>) was used for further filtering to reduce false positive calls with the following criteria: (1) quality score ≥ 20; (2) FisherStrand ≤ 60.0; (3) StrandOddsRatio ≤ 3; (4) sequencing depth in the region ≥ 30; (5) sequence reads in support of the variant call ≥ 2; and (6) variant allele frequency (VAF) ≥ 0.05.</p>", "<p id=\"Par64\">Based on the somatic mutation data, we conducted somatic mutation signature analysis using the deconstructSigs1.9.0 R package with the default parameters [##REF##26899170##30##, ##REF##34875674##31##]. The COSMIC signatures were used as the reference to annotate the identified signatures. The MuSiC2 was used to explore significantly mutated genes (false discovery rate [FDR] &lt; 0.1) [##REF##22759861##32##]. Tumor mutation burden was calculated by the Maftools R package [##REF##30341162##33##]. When calculating tumor mutational burden and analyzing mutations related to chemotherapy sensitivity, only mutations with the following functional classifications were considered [##REF##31402092##34##–##REF##33295976##36##]: frame_shift_del, frame_shift_ins, in_frame_del, in_frame_ins, missense_mutation, nonsense_mutation, nonstop_mutation, splice_site, and translation_start_site. Somatic copy number alterations (SCNAs) were detected using the CNVkit [##REF##27100738##37##], and genomic regions with significant amplifications or deletions in the samples were summarized by the GISTIC2.0 [##UREF##6##38##]. Tumor purity was estimated by the ABSOLUTE [##REF##22544022##39##].</p>", "<p id=\"Par65\">Germline variants were identified using the Sentieon Haplotyper tool [##REF##31481971##40##]. The ClinVar database was used to annotate known pathogenic and likely pathogenic variants. The 28 cancer predisposition genes [##REF##33471974##41##] were evaluated. They include 12 established breast cancer–predisposition genes (<italic>ATM</italic>, <italic>BARD1</italic>, <italic>BRCA1</italic>, <italic>BRCA2</italic>, <italic>CDH1</italic>, <italic>CHEK2</italic>, <italic>NF1</italic>, <italic>PALB2</italic>, <italic>PTEN</italic>, <italic>RAD51C</italic>, <italic>RAD51D</italic>, and <italic>TP53</italic>) and 16 candidate predisposition genes (<italic>BLM</italic>, <italic>BRIP1</italic>, <italic>CDKN2A</italic>, <italic>ERCC3</italic>, <italic>FANCC</italic>, <italic>FANCM</italic>, <italic>MLH1</italic>, <italic>MRE11A</italic>, <italic>MSH2</italic>, <italic>MSH6</italic>, <italic>NBN</italic>, <italic>RAD50</italic>, <italic>RECQL</italic>, <italic>RINT1</italic>, <italic>SLX4</italic>, and <italic>XRCC2</italic>).</p>", "<title>RNA-seq analysis</title>", "<p id=\"Par66\">Raw data were filtered following standard pipelines, and reads that did not meet the analysis criteria were deleted by fastp. The HISAT2 [##REF##25751142##42##] was then used to map the filtered data to the human reference genome (GRCh38). Finally, the FeatureCounts [##REF##24227677##43##] and StringTie [##REF##25690850##44##] were used to perform transcript reconstruction and statistics on the basis of the reads-reply results.</p>", "<p id=\"Par67\">Differentially expressed genes between subgroups were identified using the DESeq2 R package [##REF##25516281##45##]. For comparisons between pre- and post-treatment samples, we performed a paired analysis on the basis of patient IDs. The WebGestalt 2019 [##UREF##7##46##] was used for the gene set enrichment analysis [##REF##16199517##47##]. Transcripts per million (TPM) was used to measure the expression levels of genes, and the composition of immune and stroma cells were calculated using the xCell [##REF##29141660##48##].</p>", "<title>Cell culture and chemicals</title>", "<p id=\"Par68\">The human BC cell line HCC1806 was purchased from the BeNa Culture Collection (Kunshan, China). BT-549, MDA-MB-231, MDA-MB-453, SK-BR-3, T47D, BT-474, and MCF-7 cells were purchased from the Zhong Qiao Xin Zhou Biotechnology Co. (Shanghai, China). HCC1806, MDA-MB-231, MDA-MB-453, T47D, and BT-474 cells were maintained in RPMI 1640 medium (Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China) and 1% penicillin and streptomycin (Solarbio, Beijing, China). MCF-7 cells were cultured in MEM medium (Corning Inc.) supplemented with 10% FBS, 1% penicillin and streptomycin, and 0.005 mg/mL bovine insulin (Solarbio). SK-BR-3 cells were cultured in McCoy’s 5a medium (Macgene, Beijing, China) supplemented with 10% FBS and 1% penicillin and streptomycin. BT-549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin, and 0.023 IU/mL insulin (Beyotime, Shanghai, China). All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO<sub>2</sub>. All cell lines were authenticated by the Shanghai Biowing Applied Biotechnology Co. Ltd. (China) using a short tandem repeat profiling analysis before conducting experiments. Assessments of mycoplasma contamination using the MycoBlue Mycoplasma Detector (Vazyme, Nanjing, China) were performed prior to performing experiments to confirm that the cells used for experiments were free of mycoplasma contamination.</p>", "<title>Cell infection</title>", "<p id=\"Par69\">The CDKAL1 wild type and mutant (CDKAL1<sub><italic>P409L</italic></sub>) cDNAs were cloned into the pCDH-CMV-MCS-EF1-BSD vector. The CENPT wild type, and CDKAL1<sub><italic>P409L</italic></sub> mutants (CENPT<sub><italic>R122G</italic></sub>, and CENPT<sub><italic>P442L</italic></sub>) cDNAs were cloned into the pLenti-C-Myc-DDK-IRES-Puro vector. These two lentiviral vectors were purchased from the BioSune Biotechnology Co. Ltd. (Shanghai, China). Viral particles were prepared by transfecting HEK293T cells with the constructed or control plasmids in combination with packaging vectors using Lipofectamine 3000 transfection reagents (Invitrogen, Waltham, MA, USA). The cell supernatant was collected at 48 and 72 h after transfection. After the supernatant was filtered through a 0.45-μm filter, it was ultracentrifuged at 11,000 × <italic>g</italic> for 3 h at 4 °C using an Optima XPN-80 ultracentrifuge (Beckman Coulter, Brea, CA, USA). After ultracentrifugation, virus pellets were resuspended in PBS. Finally, a concentrated virus solution (plus polybrene) was used to infect cells 48 h before selection with the appropriate antibiotic.</p>", "<title>Quantitative real-time PCR (qPCR)</title>", "<p id=\"Par70\">Total RNA was prepared from cells using a TRIzol reagent (Invitrogen) and reverse-transcribed to cDNA using the HiScript®III RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme). Primers are listed as follows: 5′-CTGCTGCATCTCAGTGTGAC-3′ (forward) and 5′-TCCTCAGCGCACAGTCTTGA-3′ (reverse) for <italic>CDKAL1</italic>; 5′-GCCTCTTCCCTCACCAGATCC-3′ (forward) and 5′-CACAATGTTTGGAGGAGCCAG-3′ (reverse) for <italic>CENPT</italic>; 5′- CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′- CTCCTTAATGTCACGCACGAT-3′ (reverse) for <italic>ACTB</italic>. qPCR was performed on a QuantStudio 5 Real-Time PCR Instrument (Thermo Fisher Scientific) using a 2 × Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China). <italic>ACTB</italic> was used as the internal control, and the relative expression of target genes was calculated using the 2<sup>−ΔΔCt</sup> method.</p>", "<title>Protein extraction and western blot analysis</title>", "<p id=\"Par71\">To obtain whole-cell protein extracts, cells were lysed with 1 × SDS-PAGE Sample Loading Buffer (Beyotime). The cell lysates were denatured for 5 min at 95 °C. Equal amounts of proteins from cell lysates were electrophoresed on SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated with the indicated primary antibodies overnight at 4 °C, and then with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 h. The membranes were washed three times (5 min per wash) with Tris-buffered saline containing Tween-20 (TBST) before and after antibody incubations. Finally, chemiluminescent HRP substrate (Millipore) was added to the membranes, and immunoreactive bands were detected by a chemiluminescent imaging system (Tanon, Shanghai, China). All experiments were repeated at least three times. The primary and secondary antibodies used in this study were as follows: CDKAL1 (Cat# ab169531, AbCam, Cambridge, UK), CENPT (Cat# ab86595, AbCam), β-actin (Cat# AC026, ABclonal), and HRP-AffiniPure Goat Anti-Rabbit IgG (H + L) (Cat# 111–035-003, Jackson ImmunoResearch, West Grove, PA, USA).</p>", "<title>Cell proliferation assays</title>", "<p id=\"Par72\">For proliferation assays, a CCK-8 cell counting kit (Dojindo, Kumamoto, Japan) was used to assay the cell viability. Infected cells were plated in 96-well plates with a final volume of 100 μL of growth medium and incubated overnight under 5% CO<sub>2</sub> at 37 °C. Ten drug concentrations were freshly prepared according to the half-log dilution method (10,000-fold range, docetaxel: 0–1 μM, epirubicin: 0–20 μM). The cells were treated with different concentrations of docetaxel (MedChemExpress, Houston, TX, USA) and epirubicin (MedChemExpress) with five replicates per condition. After 48 h, the CCK-8 assay was performed by incubating cells with a CCK-8 reagent for 2 h at 37 °C, and measuring the absorbance at 450 nm with an Infinite 200 PRO plate reader (TECAN, Männedorf, Switzerland). These data were used to calculate the cell viability at different drug concentrations. The growth and dose inhibition curves were plotted and analyzed using the GraphPad Prism 8.3.0 (GraphPad Software, Inc., San Diego, CA, USA). The IC<sub>50</sub> values were determined by nonlinear regression analysis of the plots of the percentage of growth inhibition vs. the log of inhibitor concentrations. All experiments were repeated at least three times, and data are expressed as mean ± SD.</p>", "<title>IHC</title>", "<p id=\"Par73\">In IHC analyses, the EnVision method was used to assess the expression of ADGRA2 and ADRB3. Briefly, the TMA was sectioned into 4-μm-thick tissue sections. After deparaffinization, rehydration, antigen retrieval using the PT Link for Pre-Treatment reagent (Agilent Technologies), and blockage of endogenous peroxidase activity, the sections were incubated with the rabbit anti-ADGRA2 (1:40; Cat# ab198817, Abcam) or rabbit anti-ADRB3 (1:50; Cat# ab140713, Abcam) antibodies for 1 h at room temperature, followed by incubation with a secondary antibody (Cat# SM802, DAKO, Glostrup, Denmark) for 20 min at room temperature. Negative controls only included the antibody dilution buffer (DAKO, Cat# DM830) without a primary antibody. The staining was assessed independently by two pathologists blinded to patient information. The IHC scoring was based on the proportion and intensity of positively stained invasive BC cells on slides. The proportion of positive tumor cells was recorded as a percentage. The intensity scores represent the average staining intensity of the positive tumor cells (negative = 0; weak staining = 1; moderate staining = 2; and strong staining = 3). The proportion and intensity scores were then multiplied to obtain a total IHC score, which ranges from 0 to 300. According to whether the patient had a DFS event (as a judgment standard), we analyzed the receiver operating characteristics (ROC) curve of ADGRA2 and ADRB3 expression. The maximum Youden Index was again used to determine the optimal cut-off value to divide patients into high and low expression groups. An IHC score ≥ 70 for ADGRA2 and ≥ 80 for ADGRA3 was considered high expression.</p>", "<title>Statistical analyses</title>", "<p id=\"Par74\">The Student’s t-test and Wilcoxon test were used to compare continuous variables, while the Pearson’s chi-square test and Fisher’s exact test were used to compare unordered categorical variables. The log-rank test was used to compare differences in breast cancer-specific survival (BCSS) and DFS between patients with a high and low expression of <italic>ADGRA2</italic> and <italic>ADRB3</italic>. Cox regression models were used to estimate the HRs at 95% CIs for BCSS and DFS events associated with the expression of <italic>ADGRA2</italic> and <italic>ADRB3</italic>. Age and Ki-67 level were adjusted as continuous variables; menopausal status, endocrine therapy, radiotherapy, and other clinical factors (cT, cN, histological grade, ER status, PR status, HER2 status) were adjusted as categorical variables. All statistical analyses were performed using the R packages version 4.2.0 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://cran.r-project.org/\">https://cran.r-project.org/</ext-link>) and the SPSS version 23.0 (IBM, Armonk, NY, USA). <italic>P</italic> &lt; 0.05 were considered statistically significant and <italic>P</italic> &lt; 0.1 marginally significant.</p>" ]
[ "<title>Results</title>", "<title>Characteristics of BC patients treated with NAC in the sequencing set</title>", "<p id=\"Par75\">To investigate the genomic and transcriptomic features of tumors before and after NAC, we enrolled 50 BC patients who received NAC in the NACBC sequencing set for this study (Figs. ##FIG##0##1##A and B). The median age at diagnosis was 49 years (range: 27–68 years). The stages of BCs at diagnosis were stage I (<italic>n</italic> = 4), stage II (<italic>n</italic> = 41), and stage III (<italic>n</italic> = 5). ER, PR, and HER2 positive patients accounted for 82%, 56%, and 40% of the cohort, respectively. Among them, 82% (41/50) patients received a taxane-based regimen as first-line treatment (Additional file ##SUPPL##1##2##: Tables S1 and S2).</p>", "<title>Changes in somatic mutation and copy number variation between paired pre- and post-treatment tumors</title>", "<p id=\"Par76\">There were no statistical differences in tumor purity among the 44 paired pre- and post-treatment tumors (Fig. ##FIG##1##2##A). We further analyzed the somatic mutation and copy number variation (CNV) landscape changes between tumors in response to NAC. We identified 15,499 somatic SNVs (median: 139.5) and 598 somatic small indels (median: 4) in the pre-treatment tumors, and 27,458 nucleotide substitutions (median: 134) and 770 small indels (median: 5) in the post-treatment tumors. SNV analysis showed that C &gt; T substitutions occurred more frequently than any other SNVs in all the tumors and that the fraction of transversion mutations (C &gt; A) was reduced after NAC (<italic>P</italic> = 0.020, Additional file ##SUPPL##0##1##: Fig. S2). We examined the mutational signature weights among the Catalogue of Somatic Mutations in Cancer (COSMIC) signatures based on the frequency of 96 different possible trinucleotide substitutions. However, we did not detect any statistically significant changes in the COSMIC mutational signatures in the cohort (Additional file ##SUPPL##0##1##: Fig. S3, Additional file ##SUPPL##1##2##: Table S3).</p>", "<p id=\"Par77\">In total, 4,433 and 6,767 nonsynonymous mutations were identified in the 44 paired pre- and post-treatment tumors, respectively. There were no statistically significant changes in mutation loads in the cohort (Fig. ##FIG##1##2##B), and the most frequently altered genes were <italic>TP53</italic>, <italic>TTN</italic>, and <italic>MUC16</italic> in both pre- and post-NAC samples although the change of these three genes was not statistically significant (Fig. ##FIG##1##2##C). However, compared with the pre-treatment tumors, <italic>CNR2</italic>, <italic>KIAA1549</italic>, and <italic>CCDC168</italic> gene mutations were solely observed in the post-treatment tumors under the pressure of chemotherapy (<italic>P</italic> &lt; 0.05, Additional file ##SUPPL##1##2##: Table S4). We next performed gene set enrichment analyses on the Molecular Signatures Database (MSigDB) of hallmark gene sets and identified that three pathways were significantly affected by mutations. The mutation rates of the DNA REPAIR and PROTEIN SECRETION pathways significantly decreased in the post-treatment tumors. Of the 44 paired pre- and post-treatment tumors, 29 pre-treatment and 16 post-treatment tumors contained gene mutations in the DNA REPAIR pathway (<italic>P</italic> = 0.006, Fig. ##FIG##1##2##D) while there were 14 pre-treatment and 4 post-treatment tumors contained gene mutations in the PROTEIN SECRETION pathway (<italic>P</italic> = 0.042, Fig. ##FIG##1##2##D). Conversely, a higher mutation rate of ANGIOGENESIS pathway was observed in the post-treatment group (4 out of 44) than in the pre-treatment group (11 out of 44) (<italic>P</italic> = 0.047, Fig. ##FIG##1##2##D).</p>", "<p id=\"Par78\">SCNA analyses identified 13 amplifications and 23 deletions in the pre-treatment tumors, and 13 amplifications and 18 deletions in the post-treatment tumors (Additional file ##SUPPL##0##1##: Fig. S4A). Of them, 4 amplifications (1q21.3, 11q13.3, 15q26.3, and 17p11.2) and 11 deletions (1p36.31, 1q44, 2q37.3, 3p14.1, 6p22.1, 6q27, 8p23.3, 11q12.1, 12p13.2, 13q34, and 15q13.3) only occurred in the pre-treatment tumors. In addition, 4 amplifications (2p11.2, 8q24.3, 11q13.4, and 11p15.4) and 6 deletions (1q43, 4q35.1, 5p15.33, 6p21.33, 6q22.33, and 16p13.11) were specifically detected in the post-treatment tumors (Additional file ##SUPPL##0##1##: Fig. S4B). The 4q35.1 region contains the <italic>CENPU</italic> gene, whose mRNA expression was also downregulated in post-treatment tumors (Additional file ##SUPPL##0##1##: Fig. S4C).</p>", "<title>Changes in gene expression and cell composition following NAC</title>", "<p id=\"Par79\">Differential gene expression analyses between the paired pre- and post-treatment tumors identified 1,130 differentially expressed genes (DEGs), including 705 upregulated and 425 downregulated genes (fold change &gt; 2, FDR &lt; 0.05, Fig. ##FIG##2##3##A). Compared with the pre-treatment tumors, gene enrichment analyses showed that gene sets associated with cell cycle progression (FDR &lt; 0.001) and DNA repair were significantly downregulated (FDR = 0.007), whereas gene sets associated with response to hypoxia/HIF1A targets (FDR &lt; 0.001) and KRAS signaling (FDR &lt; 0.001) were upregulated in the post-treatment tumors (FDR &lt; 0.01, Fig. ##FIG##2##3##B).</p>", "<p id=\"Par80\">We further analyzed the cell composition of each tumor using the xCell algorithm [##REF##29141660##48##] and compared the changes between the pre- and post-treatment tumors based on the Wilcoxon signed-rank test. The results indicated that after NAC, the fractions of B cells (<italic>P</italic> &lt; 0.001), activated dendritic cells (aDCs, <italic>P</italic> = 0.002), and gamma delta T cells (γδT cells, <italic>P</italic> &lt; 0.001) were decreased (Fig. ##FIG##2##3##C), whereas the fractions of M2 macrophages (<italic>P</italic> = 0.005), and endothelial cells (P &lt; 0.001) were increased in the post-treatment tumors (Fig. ##FIG##2##3##D). We next compared the expression of immune checkpoint molecules and genes in antigen processing/presentation, positive regulation of γδT cell activation, and angiogenesis pathways in the pre- and post-treatment tumors. The results showed that compared with the pre-treatment tumors, the expression of <italic>LAG3</italic> gene was significantly decreased (<italic>P</italic> = 0.024), while the expression of <italic>SIGLEC15</italic> was significantly increased (<italic>P</italic> &lt; 0.001) in the post-treatment tumors (Additional file ##SUPPL##0##1##: Figs. S5A and B). Marginal downregulation of <italic>CTLA</italic>4 (<italic>P</italic> = 0.05), <italic>PD-L1</italic> (<italic>P</italic> = 0.072), and <italic>PD-1</italic> (<italic>P</italic> = 0.067) genes were also observed in the post-treatment samples (Additional file ##SUPPL##0##1##: Figs. S5C-E). Most of the genes related to antigen processing and presentation (70%), and positive regulation of γδT cell activation (80%) were significantly downregulated (all <italic>P</italic> &lt; 0.05, Figs. ##FIG##2##3##E and F; Additional file ##SUPPL##1##2##: Table S5), while 84.62% of genes related to the angiogenesis pathway were upregulated in the post-treatment tumors (all <italic>P</italic> &lt; 0.05, Fig. ##FIG##2##3##G, Additional file ##SUPPL##1##2##: Table S5). We further analyzed the cell component fraction changes in subgroups with different degrees of NAC responses in the post-treatment tumors. A decrease in B cell fraction was observed in the middle responsive and nonresponsive groups, and a decrease in aDCs composition was only detected in the nonresponsive group (Additional file ##SUPPL##0##1##: Fig. S6).</p>", "<title>Somatic mutational analyses identify <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutation decreases NAC sensitivity in BC</title>", "<p id=\"Par81\">To screen for molecular features related to NAC susceptibility, we compared the genomic differences between the nonresponsive (<italic>n</italic> = 16) and responsive (<italic>n</italic> = 11) groups of the pre-treatment tumors by analyzing the WES data. We observed no statistically significant differences in mutational loads between the two groups (Fig. ##FIG##3##4##A). Among the six possible base pair substitutions, the proportion of C &gt; T substitutions was lower in the responsive group (39.35%) compared with the nonresponsive group (48.89%, <italic>P</italic> = 0.020, Fig. ##FIG##3##4##B), especially when the substitution site was flanked by C and G (responsive vs. nonresponsive: 3.93% vs. 6.87%, <italic>P</italic> = 0.022, Additional file ##SUPPL##0##1##: Fig. S7). Analyses of mutational signature weights for the COSMIC signatures demonstrated that a lower weight of signature 3, which is associated with failure of DNA double-strand break-repair by homologous recombination, in the nonresponsive group (range: 0%–29%) compared with the responsive group (range: 0%–72%); however, the difference was not statistically significant (<italic>P</italic> = 0.151, Fig. ##FIG##3##4##C). We further compared the differences in the expression of DNA repair pathway-related genes between the nonresponsive and responsive groups by analyzing the RNA-seq data. The expression of most DNA repair related genes was significantly upregulated in the nonresponsive group (all <italic>P</italic> &lt; 0.05, Fig. ##FIG##3##4##D, Additional file ##SUPPL##1##2##: Table S6).</p>", "<p id=\"Par82\">As germline mutations may affect pathological response, we analyzed the presence of pathogenic germline variants of the 28 cancer predisposition genes [##REF##33471974##41##]. We detected mutations in three of them in the NACBC sequence set (Fig. ##FIG##4##5##A): one <italic>BRCA1</italic> variant in the nonresponsive group, one <italic>BRCA2</italic> variant in the responsive group, and one <italic>BRCA1</italic> variant and one <italic>PALB2</italic> variant in the middle responsive group. No other pathogenic germline mutations were detected in the studied cohort. We found that the frequency of germline gene mutations did not differ between the nonresponsive (1 out of 16) and responsive (1 out of 11) groups (<italic>P</italic> = 1.0). Moreover, NAC sensitivity analyses including/excluding the patients carrying the aforementioned deleterious germline mutations did not alter the significance of the changes in the mutational signatures and the expression levels of DNA damage repair pathways (Fig. ##FIG##3##4##, Additional file ##SUPPL##0##1##: Figs. S7 and S8). Therefore, the pathological response observed in the sequencing set was not likely driven by the germline mutations in the breast cancer susceptibility genes.</p>", "<p id=\"Par83\">We next analyzed the WES data using the MuSiC2 [##REF##22759861##32##] and identified 43 significantly mutated genes (SMGs) in the 47 pre-treatment tumors (FDR &lt; 0.1, Fig. ##FIG##4##5##A, Additional file ##SUPPL##1##2##: Table S7). To identify mutated genes that are associated with chemosensitivity in BC, we compared the differentially mutated genes in the nonresponsive and responsive groups. Mutations in <italic>CDKAL1</italic>, <italic>ALPK2</italic>, <italic>EMILIN3</italic>, <italic>CENPT</italic>, <italic>OR51M1</italic>, <italic>THAP8</italic>, <italic>TTLL2</italic>, and <italic>ZFPM1</italic> genes were primarily detected in the nonresponsive group but not in the responsive group. These mutations occurred in at least 2 nonresponsive tumor samples (Additional file ##SUPPL##1##2##: Table S8). We further conducted SIFT 4G [##REF##26633127##49##] and PROVEAN [##REF##23056405##50##] analyses to predict whether these gene mutational variations affect protein functions. The <italic>CDKAL1</italic> missense variant P409L (p.Pro409Leu, c.1226 C &gt; T) and the <italic>CENPT</italic> missense variants R122G (p.Arg122Gly, c.364 A &gt; G) and P442L (p.Pro442Leu, c.1325 C &gt; T) were predicted to have a “deleterious” functional impact. These potentially deleterious mutations were also observed in at least 2 nonresponsive tumor samples (Fig. ##FIG##4##5##B, Additional file ##SUPPL##1##2##: Table S9).</p>", "<p id=\"Par84\">We subsequently conducted in vitro studies to validate the effects of the deleterious <italic>CDKAL1</italic> and <italic>CENPT</italic> mutations on the responsiveness of BC cells to chemotherapeutics. We examined the mutations of both <italic>CDKAL1</italic> and <italic>CENPT</italic> in different BC cell lines using the Cancer Cell Line Encyclopedia (CCLE) online database [##REF##31068700##51##]. All the cell lines tested did not harbor non-synonymous mutations except that MDA-MB-453 and BT-474 cells had nonsense and missense mutations of <italic>CENPT</italic>, respectively (Additional file ##SUPPL##0##1##: Fig. S9A). Western blots demonstrated that CDKAL1 and CENPT proteins were expressed in all cell lines tested (Fig. ##FIG##4##5##C, Additional file ##SUPPL##0##1##: Fig. S9B). HCC1806 and MDA-MB-231 for <italic>CDKAL1</italic>, and MDA-MB-231 and BT-549 cells for <italic>CENPT</italic> were selected for gene overexpression studies. Cells were infected with the lentiviruses that overexpress the wild type or mutants of the targeted genes <italic>CDKAL1</italic> (<italic>CDKAL1</italic><sub><italic>WT</italic></sub> and <italic>CDKAL1</italic><sub><italic>P409L</italic></sub>), or <italic>CENPT</italic> (<italic>CENPT</italic><sub><italic>WT</italic></sub>, <italic>CENPT</italic><sub><italic>R122G</italic></sub> and <italic>CENPT</italic><sub><italic>P442L</italic></sub>). The efficiency of gene expression in infected BC cells was confirmed by western blot and real-time PCR (Fig. ##FIG##4##5##D, Additional file ##SUPPL##0##1##: Fig. S9C). The genotypes of the infected cells were also verified by Sanger sequencing (Additional file 1: Fig. S9D). The sensitivity of infected BC cells to chemotherapy drugs were determined with CCK-8 assays. The results indicated that HCC1806 cells overexpressing the p.Pro409Leu <italic>CDKAL1</italic> variant (<italic>CDKAL1</italic><sub><italic>P409L</italic></sub>) decreased the sensitivity to docetaxel compared with cells overexpressing the wild type <italic>CDKAL1</italic> (<italic>CDKAL1</italic><sub><italic>WT</italic></sub>), with the IC<sub>50</sub> of docetaxel for <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> and <italic>CDKAL1</italic><sub><italic>WT</italic></sub> cells being 3.50 ± 0.45 nM and 1.69 ± 0.11 nM, respectively (<italic>P</italic> &lt; 0.05, Fig. ##FIG##4##5##E). A similar result was observed in MDA-MB-231 cells despite a higher expression of endogenous <italic>CDKAL1</italic><sub><italic>WT</italic></sub> compared with HCC1806 cells (<italic>P</italic> &lt; 0.01, Fig. ##FIG##4##5##E). However, <italic>CENPT</italic> mutations did not affect the IC<sub>50</sub> values of docetaxel in both MDA-MB-231 and BT-549 cells, and neither <italic>CDKAL1</italic> nor <italic>CENPT</italic> mutations affected the sensitivity of BC cells to epirubicin in the cell lines tested (Additional file ##SUPPL##0##1##: Fig. S9E).</p>", "<p id=\"Par85\">To delineate the in-depth mechanism underlying these findings, we further compared RNA-seq data from the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutant and <italic>CDKAL1</italic><sub><italic>WT</italic></sub> wild type groups. The results showed that the HALLMARK_APOPTOSIS set was significantly enriched in the <italic>CDKAL1</italic><sub><italic>WT</italic></sub> wild type group compared with the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutant group (Additional file ##SUPPL##0##1##: Fig. S10A). The expression of anti-apoptosis gene <italic>BCL2L2</italic> was significantly higher, while the expression of pro-apoptosis genes <italic>BAX</italic> and <italic>BID</italic> were lower in the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutant group (Additional file ##SUPPL##0##1##: Fig. S10B). These data suggested that the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutation induced docetaxel resistance possibly by inhibiting apoptosis in BC cells.</p>", "<title>SCNA analyses demonstrate ADRB3 or ADGRA2 amplification induces worse NAC response and BC prognosis</title>", "<p id=\"Par86\">We conducted SCNA analyses of the paired tumor and normal samples for copy number amplification or deletion peaks between the genomes of nonresponsive and responsive pre-treatment samples using the GISTIC2.0 (FDR &lt; 0.1). A unique amplification peak at 8p11.23 was identified in the nonresponsive group, which contained <italic>ADGRA2</italic> and <italic>ADRB3</italic> genes. Additionally, a unique deletion peak at 3p13 was observed in the responsive tumors, which contains the cancer related gene <italic>FOXP1</italic> (Fig. ##FIG##5##6##A).</p>", "<p id=\"Par87\">We next examined whether changes in gene copy number affected the mRNA expression of <italic>ADGRA2</italic>, <italic>ADRB3</italic>, and <italic>FOXP1</italic> by analyzing the RNA-seq data in the pre-treatment tumors. The results indicated that the mRNA levels of <italic>ADGRA2</italic>, <italic>ADRB3</italic>, and <italic>FOXP1</italic> were significantly downregulated in the responsive group (<italic>P</italic> &lt; 0.05, Figs. ##FIG##5##6##B-D). This was consistent with the SCNA analyses above.</p>", "<p id=\"Par88\">A previous study has revealed that cytoplasmic FOXP1 expression in BC is associated with worse outcomes [##REF##29848352##52##]. It is in agreement with our observation that <italic>FOXP1</italic> containing 3p13 region was deleted in the NAC-sensitive tumors. To validate the role of ADGRA2 and ADRB3 expression in chemotherapy response and prognosis, we used a NACBC validation set, which consisted of 156 pre-treatment tumor samples of BC patients who received NAC with follow-up information available. The baseline characteristics of this validation set are shown in Additional file ##SUPPL##1##2##: Table S1. ADGRA2 and ADRB3 protein expressions were examined by IHC on TMAs. The samples were divided into low and high expression groups based on the staining scores of ADGRA2 or ADRB3 (Fig. ##FIG##5##6##E). We demonstrated that a lower ADRB3 expression was significantly associated with a higher breast-only pCR rate (<italic>P</italic> = 0.031); no statistically significant correlations between ADGRA2 protein expression and baseline characteristics were found (Additional file ##SUPPL##1##2##: Table S10). Similar results were observed in an external GEO dataset [##REF##21558518##22##], in which a lower <italic>ADRB3</italic> expression was correlated with a higher pCR rate, albeit with only marginal significance (<italic>P</italic> = 0.075). However, a significant association between a lower <italic>ADGRA2</italic> expression and a higher pCR rate was observed in this dataset (<italic>P</italic> = 0.047, Additional file ##SUPPL##0##1##: Fig. S11A).</p>", "<p id=\"Par89\">We next determined whether ADGRA2 and ADRB3 expressions were associated with survival in our internal NACBC validation set, the external GEO and TCGA validation sets. In our NACBC validation set, Kaplan–Meier survival curve analysis showed that higher (versus lower) ADGRA2 protein levels were associated with a significantly reduced probability of DFS and BCSS (<italic>P</italic> = 0.017 and <italic>P</italic> = 0.018, respectively, Fig. ##FIG##5##6##F), and a high-level ADRB3 expression was associated with poor DFS (<italic>P</italic> = 0.026). In the GEO and TCGA validation sets, a lower <italic>ADGRA2</italic> expression was significantly associated with better prognosis (<italic>P</italic> &lt; 0.05, Additional file ##SUPPL##0##1##: Figs. S11B and C). In the multivariate Cox proportional hazards regression model, after adjusting for age at diagnosis, clinical characteristics, and treatment, a higher ADGRA2 expression in BC cells significantly increased the risk of BCSS (hazard ratio [HR]: 8.042, 95% confidence interval [CI]: 1.874–35.012, <italic>P</italic> = 0.005) and DFS (HR: 2.487, 95%CI: 1.193–5.183, <italic>P</italic> = 0.015) events in the NACBC validation set. However, ADRB3 expression levels were not associated with BCSS (HR: 1.49, 95%CI: 0.138–16.112, <italic>P</italic> = 0.742) and DFS (HR: 3.36, 95%CI: 0.434–26.032, <italic>P</italic> = 0.246). These findings suggested that the expression of ADGRA2 and/or ADRB3 may be potential biomarkers for predicting the NAC response and the outcomes of BC patients.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par90\">Through combination analyses of both the WES and RNA-seq data, we first evaluated the differences in gene mutations, CNVs, gene expression, signaling pathways, and cellular components between tumors before and after treatment in primary BC, then examined the key molecular features related to NAC sensitivity of BC, and successfully identified <italic>CDKAL1</italic><sub><italic>P409L</italic></sub>, ADGRA2 and ADRB3 as novel biomarkers for the selection of patients for NAC. These findings may help develop personalized treatments for BC.</p>", "<p id=\"Par91\">In our cohort, the most frequently altered genes were <italic>TP53</italic>, <italic>TTN</italic>, and <italic>MUC16</italic> in both paired pre- and post-treatment tumors of BC. Following NAC, we observed acquired genetic alterations in <italic>CNR2</italic>, <italic>KIAA1549</italic>, and <italic>CCDC168</italic>. We further analyzed the functional biological processes or pathways that the mutated genes may affect. The mutation rate of the DNA repair pathway was significantly decreased after NAC, together with an expression downregulation of this pathway-related genes. The exact mechanism of how NAC affects the DNA repair-related genes via mutations or expression remains to be further investigated.</p>", "<p id=\"Par92\">Our SCNA analysis demonstrated that <italic>CENPU</italic> was deleted in the post-treatment tumor samples in our cohort and that pathways related to cell cycle progression were downregulated in the RNA-seq data. <italic>CENPU</italic> has been shown to promote cell proliferation in various tumors [##REF##34872447##53##–##REF##35844791##55##], and previous studies have also found that tumors with a rapid growth rate are more sensitive to chemotherapy [##REF##26786263##56##, ##REF##22081974##57##]. Therefore, tumor cells with rapid proliferation are more likely to be eliminated by chemotherapy, while those with slow proliferation are more likely to remain. Our finding is in agreement with these studies in that cell cycle progression pathways were downregulated following NAC.</p>", "<p id=\"Par93\">Cancer immunotherapy has achieved remarkable successes in certain molecular subtypes of BC patients [##REF##35857659##58##]. The presence of immune cells and specific molecular expression patterns in the tumor microenvironment (TME) may affect the effectiveness of immunotherapy. The nature and composition of TME can vary over time with chemotherapy. The SWOG S0800 neoadjuvant trial showed no changes in tumor-infiltrating lymphocyte counts or <italic>PD-L1</italic> expression levels in residual disease (RD) cases [##REF##30967156##59##]. However, another study found that both stromal tumor-infiltrating lymphocytes and CD8<sup>+</sup> T cells were both decreased, while the expression of M2 macrophage-specific genes was significantly increased after treatment [##REF##31061067##17##]. Therefore, the reported results from different centers are inconsistent. In our study, we found that NAC affected TME. NAC altered not only the expression levels of immune-related genes in BC tumor tissues, but also the composition of immune and stromal cells, including B cells, M2 macrophages, aDCs, endothelial cells, and γδT cells. DCs [##REF##15122249##60##] and B cells [##REF##30967621##61##] are professional antigen-presenting cells (APCs) of the immune system that can efficiently generate immune responses against tumors, including effective activation and expansion of CD8<sup>+</sup> cytotoxic T lymphocytes that can specifically kill cancer cells [##REF##24998519##62##–##UREF##8##64##]. In our study, we demonstrated that patients in the nonresponsive group displayed reduced levels of aDCs and B cells in the TME after NAC, suggesting that NAC may further induce insensitivity to immunotherapy in nonresponsive patients. Therefore, more attention should be paid to this patient population, especially when using immunotherapy drugs. In this group, the timing of immunotherapy and chemotherapeutic drugs should be carefully considered.</p>", "<p id=\"Par94\">We also analyzed the molecular features associated with NAC response in the pre-treatment tumors. Different mutational processes often generate different combinations of single-nucleotide alterations, termed “signatures” [##REF##26551669##65##]. The pattern of mutation signatures is associated with tumor sensitivity to chemotherapy and prognosis [##UREF##2##13##, ##REF##29259186##15##]. In our cohort, there was a trend for higher levels of mutational signature 3 in the responsive group compared with the nonresponsive group. The signature 3 is associated with a failure of DNA double-strand break-repair by homologous recombination (<ext-link ext-link-type=\"uri\" xlink:href=\"https://cancer.sanger.ac.uk/signatures/signatures_v2/\">https://cancer.sanger.ac.uk/signatures/signatures_v2/</ext-link>). Our RNA-seq data confirmed that most of the genes related to the DNA repair pathway exhibited higher expression levels in the nonresponsive tumors than in the responsive tumors, suggesting that the nonresponsive tumors may have a stronger ability to repair DNA damage, which is conducive to the survival of tumor cells. This finding is consistent with a previous study that found a higher proportion of signature 3 was associated with a higher rate of pCR after NAC [##REF##31919134##19##]. Collectively, these results indicate that DNA repair deficiency confers increased chemotherapy sensitivity in BC.</p>", "<p id=\"Par95\">We have identified a <italic>CDKAL1</italic> mutation in the nonresponsive group. Using in vitro studies, we demonstrated that BC cells with the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutation were more resistant to docetaxel. <italic>CDKAL1</italic> is a mammalian methylthiotransferase that biosynthesizes 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNALys<sup>(UUU)</sup> for the accurate translation of AAA and AAG codons [##REF##21841312##66##]. Previous studies have shown that single nucleotide polymorphisms of <italic>CDKAL1</italic> are associated with susceptibility to and mortality from BC [##REF##24468095##67##–##REF##27314662##69##]. A germline genome-wide association study revealed that rs7453577 (located within <italic>CDKAL1</italic>) increased the pCR rate of NAC in HER2-negative BC patients who received bevacizumab [##REF##29768301##70##]. However, to our knowledge, no studies have reported a relationship between <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> and chemotherapy response. Our analysis of the RNA-seq data showed that the HALLMARK_APOPTOSIS gene set was significantly enriched in the <italic>CDKAL1</italic><sub><italic>WT</italic></sub> tumors compared with the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> tumors, resulting in a lower expression of the pro-apoptosis genes <italic>BAX</italic> and <italic>BID</italic>. Previous studies indicated that <italic>CDKAL1</italic> deficiency could induce the misreading of Lys codons and affect the synthesis of downstream proteins [##REF##21908934##71##, ##REF##33634165##72##]. We speculate that the <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutation may decrease the ms2t6A modification of tRNA<sup>Lys</sup> and downstream translation of pro-apoptotic proteins, thereby rendering mutant cells insensitive to docetaxel. Collectively, these results indicate that <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> could be a biomarker for predicting insensitivity to NAC.</p>", "<p id=\"Par96\">In the overall CNV analysis of the pre-treatment tumors, we found a gene amplification peak at 8p11.23 only in the nonresponsive subgroup. This chromosome region contains two genes <italic>ADGRA2</italic> and <italic>ADRB3</italic>, whose mRNA expression was higher in the nonresponsive group than in the responsive group, as shown in the RNA-seq analysis. <italic>ADGRA2</italic>, also known as <italic>GPR124</italic>, is an important member of the adhesion-type G protein-coupled receptor (aGPCR) family. <italic>ADGRA2</italic> was originally identified in the endothelial cells that form the neovasculature in invasive colorectal tumors [##REF##10947988##73##]. Aberrant expression of <italic>ADGRA2</italic> has also been found in other types of cancers. In glioblastoma, it affected cancer cell proliferation by regulating the duration of mitotic progression [##REF##31058365##74##]. In osteosarcoma, combination of β-elemene and paclitaxel inhibited bone neoplasm growth via downregulating <italic>ADGRA2</italic>, suggesting a potential role for <italic>ADGRA2</italic> in therapy response [##REF##30008912##75##]. <italic>ADRB3</italic> has been proven to be a poor prognostic factor that accelerates cell proliferation in a variety of human cancers [##REF##31477835##76##–##REF##32514619##78##]. Additionally, blocking <italic>ADRB3</italic> promoted apoptosis and reduced chemoresistance in leukemia cells [##UREF##9##79##]. However, the role of <italic>ADGRA2</italic> and <italic>ADRB3</italic> in NAC response in BC has not been previously reported. In our study, we observed that low level expressions of <italic>ADGRA2</italic> or <italic>ADRB3</italic> increased the pCR rate in the NACBC validation and GEO sets, suggesting a negative correlation between <italic>ADGRA2</italic> or <italic>ADRB3</italic> amplification and NAC response in BC. Further survival analyses of all three datasets—the NACBC validation, GEO, and TCGA datasets—identified that a higher <italic>ADGRA2</italic> expression significantly increased risks of BCSS and DFS events, and a higher ADRB3 expression was associated with poorer DFS in the NACBC validation set. These findings suggest that <italic>ADGRA2</italic> or <italic>ADRB3</italic> amplification could predict worse NAC responses and poor outcomes in BC patients.</p>", "<p id=\"Par97\">It is worth noting that our study is a single-center multi-omics analysis of BC before and after NAC. Conducting both genomic and transcriptomic studies in the same cohort has advantages to exploring the underlying mechanisms of genomic abnormalities. The consequence of any genomic abnormalities can be examined at a functional level. However, we also acknowledge that this study has limitations. Firstly, our study may suffer from potential biases introduced by the non-stratified population of molecular subtypes, such as ER, HER2 positive or triple negative, due to the relatively small sample size, which limited the power of our analyses. Secondly, although we conducted a series of in vitro studies and external dataset validations to confirm the key molecular features identified in the sequencing set, in vivo studies in animal models can be exploited next to provide further evidence. Therefore, future studies may focus on specific breast cancer subtypes with a big sample size for stronger evidence. A validation study using independent cohorts in other centers, perhaps on different ethnical populations, should also be considered.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"Par98\">In summary, our study has revealed the dynamic genomic and transcriptomic landscape before and after NAC in BC, and identified multi-omics molecular signatures and potential biomarkers associated with NAC responsiveness and prognosis that can be used to make informed therapeutic decisions or serve as potential therapeutic targets in this population.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Neoadjuvant chemotherapy (NAC) has become a standard treatment strategy for breast cancer (BC). However, owing to the high heterogeneity of these tumors, it is unclear which patient population most likely benefit from NAC. Multi-omics offer an improved approach to uncovering genomic and transcriptomic changes before and after NAC in BC and to identifying molecular features associated with NAC sensitivity.</p>", "<title>Methods</title>", "<p id=\"Par2\">We performed whole-exome and RNA sequencing on 233 samples (including matched pre- and post-treatment tumors) from 50 BC patients with rigorously defined responses to NAC and analyzed changes in the multi-omics landscape. Molecular features associated with NAC response were identified and validated in a larger internal, and two external validation cohorts, as well as in vitro experiments.</p>", "<title>Results</title>", "<p id=\"Par3\">The most frequently altered genes were <italic>TP53</italic>, <italic>TTN</italic>, and <italic>MUC16</italic> in both pre- and post-treatment tumors. In comparison with pre-treatment tumors, there was a significant decrease in C &gt; A transversion mutations in post-treatment tumors (<italic>P</italic> = 0.020). NAC significantly decreased the mutation rate (<italic>P</italic> = 0.006) of the DNA repair pathway and gene expression levels (FDR = 0.007) in this pathway. NAC also significantly changed the expression level of immune checkpoint genes and the abundance of tumor-infiltrating immune and stroma cells, including B cells, activated dendritic cells, γδT cells, M2 macrophages and endothelial cells. Furthermore, there was a higher rate of C &gt; T substitutions in NAC nonresponsive tumors than responsive ones, especially when the substitution site was flanked by C and G. Importantly, there was a unique amplified region at 8p11.23 (containing <italic>ADGRA2</italic> and <italic>ADRB3</italic>) and a deleted region at 3p13 (harboring <italic>FOXP1</italic>) in NAC nonresponsive and responsive tumors, respectively. Particularly, the <italic>CDKAL1</italic> missense variant P409L (p.Pro409Leu, c.1226C &gt; T) decreased BC cell sensitivity to docetaxel, and <italic>ADGRA2</italic> or <italic>ADRB3</italic> gene amplifications were associated with worse NAC response and poor prognosis in BC patients.</p>", "<title>Conclusions</title>", "<p id=\"Par4\">Our study has revealed genomic and transcriptomic landscape changes following NAC in BC, and identified novel biomarkers (<italic>CDKAL1</italic><sub><italic>P409L</italic></sub>, <italic>ADGRA2</italic> and <italic>ADRB3</italic>) underlying chemotherapy resistance and poor prognosis, which could guide the development of personalized treatments for BC.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s13073-024-01286-8.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank all participants involved in the study for their participation. We are also grateful to the Institute of Medical Sciences of The Second Hospital of Shandong University for their technical assistance and generous support. We would like to thank the TCGA, GEO, UCSC, and CCLE databases for providing open access to the datasets. Fei Wang is supported by Qilu Scholar-in-Training Award of Shandong University, and Young Elite Sponsorship Program of Shandong Provincial Medical Association.</p>", "<title>Authors’ contributions</title>", "<p>ZGY, SYH and FW conceived and designed the study. GSY, LYL, TY, WZZ, CMY, JLZ, LFZ, PZ, YXG, and XCM performed experiments and analyzed data. CJZ, XYW and CHJ performed TMA experiments. MF and GXT assessed the IHC scoring. LXY, ZBM, QYF, QZ, LL, FZ, CZ, YJX, MMG and YJW collected the samples. GSY, SYH and FW drafted the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This work was supported by the National Natural Science Foundation of China (82072914 and 82273701), the Major Scientific and Technological Innovation Project of Shandong Province (2017CXGC1212), the Special Foundation for Taishan Scholars, and the Fundamental Research Funds for the Central Universities (2022JC009).</p>", "<title>Availability of data and materials</title>", "<p>The WES and RNA-seq data of The NACBC sequencing datasets have been deposited in the Genome Sequence Archive [##REF##28387199##80##] in the National Genomics Data Center [##UREF##10##81##], the Beijing Institute of Genomics (China National Center for Bioinformation), the Chinese Academy of Sciences (<ext-link ext-link-type=\"uri\" xlink:href=\"https://ngdc.cncb.ac.cn/gsa-human/browse/HRA003759\">https://ngdc.cncb.ac.cn/gsa-human/browse/HRA003759</ext-link>) [##UREF##11##82##], and the processed datasets generated are deposited in the OMIX (<ext-link ext-link-type=\"uri\" xlink:href=\"https://ngdc.cncb.ac.cn/omix/release/OMIX002785\">https://ngdc.cncb.ac.cn/omix/release/OMIX002785</ext-link>) [##UREF##12##83##]. Based on the Regulation of the People's Republic of China on the Administration of Human Genetic Resources, the Human Genetic Resource Data are accessed via an application to the Data Access Committee for research purposes. Potential users need to complete and be approved for a data access request and then the data will be made available upon reasonable request according to the terms of the consent and the data use limitations for the subjects.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par99\">All procedures were conducted in accordance with the guidelines of the Declaration of Helsinki. This study was approved by the Institutional Review Board of The Second Hospital of Shandong University [Approval No.: KYLL-2020(KJ)P-0195], and informed consent was obtained from all participants of the NACBC sequencing and internal validation sets.</p>", "<title>Consent for publication</title>", "<p id=\"Par100\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par101\">The authors declare that they have no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Study overview. <bold>A</bold> A schematic diagram of sample collection in the context of neoadjuvant chemotherapy (NAC), followed by whole exome sequencing (WES), RNA sequencing (RNA-seq), and data analyses. <bold>B</bold> The final number of samples in the NACBC sequencing set for analysis. All samples were acquired from 50 patients. In the pre-treatment group, there were 47 tumor samples for WES and 50 for RNA-seq. In the post-treatment group, there were 44 tumor samples for WES and 45 for RNA-seq. <bold>C</bold> Representative pathological images of tumors by hematoxylin–eosin staining from the responsive, middle responsive, and nonresponsive patients. Bar, 250 μm. <bold>D</bold> The distribution of patients with different Miller–Payne scores in the responsive, middle responsive, and nonresponsive groups</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Changes in gene mutation, mutation burden, and the MSigDB pathway between the paired pre- and post-treatment tumor samples. Comparison of tumor purity (<bold>A</bold>) and mutation burden (<bold>B</bold>) between the 44 paired pre- and post-treatment tumors. <italic>P</italic> values are calculated based on the Wilcoxon signed-rank test. <bold>C</bold> The most frequently mutated genes before and after NAC. <bold>D</bold> Mutations associated with the MSigDB pathway in the pre- and post-treatment tumors. Bars on the top indicate the number of pathways affected in a given patient, and colored bars indicate if the variant was only found in the pre- or post-treatment tumors, or shared in both. <italic>P</italic> values in panels C and D are calculated based on the Pearson’s chi-square test; **<italic>P</italic> &lt; 0.01, *<italic>P</italic> &lt; 0.05</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Changes in gene expression, tumor-infiltrating immune and stromal cell composition following NAC. <bold>A</bold> Volcano plots showing differentially expressed genes (DEGs) between the matched pre- and post-treatment tumors. Significant DEGs are shown as red (upregulated) and blue (downregulated) dots (fold change &gt; 2, FDR &lt; 0.05). <bold>B</bold> Significantly down­ and up-regulated pathways following NAC (FDR &lt; 0.01). <bold>C</bold>, <bold>D</bold> The fractions of B cell, M2 macrophage, activated dendritic cell (aDC), endothelial cell, and gamma delta T (γδT) cell in the pre- and post-treatment tumors. <italic>P</italic> values are calculated based on the Wilcoxon signed-rank test. <bold>E</bold>–<bold>G</bold> The expression of DEGs was significantly related to positive regulations of γδT cell activation (<bold>E</bold>), antigen processing and presentation (<bold>F</bold>), and angiogenesis (<bold>G</bold>) between the pre- and post-treatment tumors. Values are presented as paired fold changes of post-/pre-treatment. <italic>P</italic> values were calculated by the Wilcoxon signed-rank test. ***<italic>P</italic> &lt; 0.001, **<italic>P</italic> &lt; 0.01, *<italic>P</italic> &lt; 0.05</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Mutation signatures in the pre-treatment tumors. Comparison of tumor mutation burden (<bold>A</bold>) and nucleotide substitutions (<bold>B</bold>) between the nonresponsive and responsive groups. <bold>C</bold> Distributions of the 10 main COSMIC signatures in the different NAC responsive groups across the 47 pre-treatment samples (<italic>left</italic>). Comparison of the relative weights of the signature 3 between the nonresponsive and responsive groups (<italic>right</italic>). <bold>D</bold> Heatmap comparison of the 22 genes statistically significantly related to the DNA repair pathway between the responsive and nonresponsive pre-treatment tumors. <italic>P</italic> values were calculated based on the Wilcoxon rank sum test. ***<italic>P</italic> &lt; 0.001, **<italic>P</italic> &lt; 0.01, *<italic>P</italic> &lt; 0.05</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p><italic>CDKAL1</italic><sub><italic>P409L</italic></sub> mutation decreased the sensitivity of cancer cells to docetaxel treatment. <bold>A</bold> Somatic and germline mutations in the 47 pre-treatment tumors and matched germline DNA. Samples were annotated for clinicopathological and molecular features (<italic>top panel</italic>). The types of somatic (<italic>middle panel</italic>) and germline (<italic>bottom panel</italic>) mutations of the indicated genes for each sample are displayed with colored squares. The histograms on the right-hand side show the accumulated number of alterations among the SMGs identified by the MuSiC2 (FDR &lt; 0.1) or the pathogenic germline mutations classified in the ClinVar database. <italic>AJCC</italic>, The American Joint Committee on Cancer. <bold>B</bold> The distribution of potentially deleterious mutations in <italic>CDKAL1</italic> and <italic>CENPT</italic> in the nonresponsive and responsive pre-treatment groups (<italic>left</italic>). Diagrams representing the protein domains of potentially deleterious mutations (<italic>right</italic>). The “lollipopPlots” were generated using the maftools R package and manually edited. <bold>C</bold> The CDKAL1 expression in different human breast cancer cell lines as indicated was examined by western blot. <bold>D</bold> The expression of <italic>CDKAL1</italic><sub><italic>WT</italic></sub> and <italic>CDKAL1P</italic><sub><italic>409L</italic></sub> in HCC1806 and MDA-MB-231 cells infected with empty vector, <italic>CDKAL1</italic><sub><italic>WT</italic></sub> and <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> lentiviruses by western blot and quantitative real-time PCR (qPCR) analyses. <bold>E</bold> IC<sub>50</sub> assays of docetaxel. The proliferation of HCC1806 and MDA-MB-231 cells as described in (<bold>D</bold>) were determined with a CCK-8 cell counting kit at an increasing dose of docetaxel as indicated. The significance of relative IC<sub>50</sub> values between <italic>CDKAL1</italic><sub><italic>WT</italic></sub> and <italic>CDKAL1</italic><sub><italic>P409L</italic></sub> cells with that of <italic>CDKAL1</italic><sub><italic>WT</italic></sub> cells as 1.0 were analyzed by paired t-test. Data represent mean ± SD (<italic>n</italic> = 3). *, <italic>P</italic> &lt; 0.05; **, <italic>P</italic> &lt; 0.01</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>High ADGRA2 or ADRB3 expression is associated with worse NAC response and prognosis of BC patients. <bold>A</bold> The SCNA signal profiles identified by the GISTIC2.0 in the nonresponsive and responsive pre-treatment tumors. The significantly altered chromosome regions (q &lt; 0.01) and the gene loci (<italic>ADGRA2</italic>, <italic>ADRB3</italic> and <italic>FOXP1</italic>) are annotated. The mRNA expression level of <italic>ADGRA2</italic> (<bold>B</bold>), <italic>ADRB3</italic> (<bold>C</bold>), and <italic>FOXP1</italic> (<bold>D</bold>) in the nonresponsive and responsive pre-treatment tumors from the RNA-seq data were shown as transcripts per million (TPM). <bold>E</bold> Representative immunohistochemistry staining of tumors with low and high expression of ADGRA2 and ADRB3 in the NACBC validation set (<italic>n</italic> = 156). Magnification: 400 × ; Bar, 100 μm. <bold>F</bold> Kaplan–Meier analyses of the DFS and BCSS in the NACBC validation set. Patients were stratified as high and low protein expression of ADGRA2 and ADRB3. <italic>P</italic> values were calculated based on the log-rank test</p></caption></fig>" ]
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[ "<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"13073_2024_1286_MOESM1_ESM.pdf\"><caption><p><bold>Additional file 1: Fig. S1.</bold> Sample information of the NACBC sequencing set. <bold>Fig. S2.</bold> Comparison of different types of base substitutions in the pre- and post-treatment tumors. <bold>Fig. S3.</bold> Distributions of the ten main COSMIC signatures in each pre- and post-treatment tumor. <bold>Fig. S4.</bold> Copy number alteration between the pre- and post-treatment tumors. <bold>Fig. S5.</bold> Changes in immune related gene expression between the pre- and post-treatment tumors. <bold>Fig. S6.</bold> Changes in the composition of immune and stroma cells between the pre- and post-treatment tumors in different NAC responsive subgroups. <bold>Fig. S7.</bold> Comparison of 96 base substitution classifications in the pre-treatment tumors of responsive and nonresponsive groups. <bold>Fig. S8.</bold> Mutational signatures in the pre-treatment tumors with samples containing germline mutations removed. <bold>Fig. S9.</bold> Sensitivity of CDKAL1 and CENPT mutation to chemotherapy drugs in BC cells. <bold>Fig. S10.</bold> RNA-seq data analysis between the <italic>CDKAL1</italic><sub>WT</sub> and <italic>CDKAL1</italic><sub>P409L</sub> tumors. <bold>Fig. S11.</bold> Associations between <italic>ADGRA2</italic> or <italic>ADRB3</italic> expression and pCR or prognosis of BC patients. </p></caption></media>", "<media xlink:href=\"13073_2024_1286_MOESM2_ESM.xlsx\"><caption><p><bold>Additional file 2: Table S1.</bold> The summary of clinical information and sequencing data of the NACBC sequencing set. <bold>Table S2.</bold> Characteristics of patients included in the NACBC sequencing and validation sets. <bold>Table S3.</bold> The relative weights of the COSMIC mutational signatures. <bold>Table S4.</bold> Changed somatic variants in CNR2, KIAA1549, or CCDC168 after NAC. <bold>Table S5.</bold> Differences in the expression levels of genes related to KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION, GOBP_POSITIVE_REGULATION_OF_GAMMA_DELTA_T_CELL_ACTIVATION, and HALLMARK_ANGIOGENESIS pathway between pre- and post-treatment. <bold>Table S6.</bold> Differences in the expression levels of genes related to HALLMARK_DNA_REPAIR pathway between the nonresponsive and responsive groups. <bold>Table S7.</bold> Significantly mutated genes in the pre-treatment tumors. <bold>Table S8.</bold> The frequency of non-synonymous SMG between the nonresponsive and responsive tumors.<bold>Table S9.</bold> Prediction of biological functional impact of somatic variants on protein functions. <bold>Table S10.</bold> Association of ADGRA2 or ADRB3 protein expression with clinicopathological parameters in the NACBC validation set.</p></caption></media>" ]
[{"label": ["3."], "mixed-citation": ["Pusztai L, Foldi J, Dhawan A, DiGiovanna MP, Mamounas EP. Changing frameworks in treatment sequencing of triple-negative and HER2-positive, early-stage breast cancers. Lancet Oncol. 2019;20(7):e390\u2013390e396. 10.1016/S1470-2045(19)30158-5."]}, {"label": ["7."], "mixed-citation": ["Derouane F, van Marcke C, Berli\u00e8re M, Gerday A, Fellah L, Leconte I, et al. Predictive Biomarkers of Response to Neoadjuvant Chemotherapy in Breast Cancer: Current and Future Perspectives for Precision Medicine. Cancers (Basel). 2022;14(16). 10.3390/cancers14163876."]}, {"label": ["13."], "mixed-citation": ["Li Z, Gao X, Peng X, May Chen MJ, Li Z, Wei B, et al. Multi-omics characterization of molecular features of gastric cancer correlated with response to neoadjuvant chemotherapy. Sci Adv. 2020;6(9):eaay4211. 10.1126/sciadv.aay4211."]}, {"label": ["25."], "mixed-citation": ["Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884\u2013884i890. 10.1093/bioinformatics/bty560."]}, {"label": ["27."], "mixed-citation": ["Pei S, Liu T, Ren X, Li W, Chen C, Xie Z. Benchmarking variant callers in next-generation and third-generation sequencing analysis. Brief Bioinform. 2021;22(3). 10.1093/bib/bbaa148."]}, {"label": ["29."], "mixed-citation": ["Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10(2). 10.1093/gigascience/giab008."]}, {"label": ["38."], "mixed-citation": ["Mermel CH, Schumacher SE, Hill B, Meyerson ML, Beroukhim R, Getz G. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol. 2011;12(4):R41. 10.1186/gb-2011-12-4-r41."]}, {"label": ["46."], "mixed-citation": ["Liao Y, Wang J, Jaehnig EJ, Shi Z, Zhang B. WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs. Nucleic Acids Res. 2019;47(W1):W199\u2013199W205. 10.1093/nar/gkz401."]}, {"label": ["64."], "mixed-citation": ["Thompson JC, Davis C, Deshpande C, Hwang WT, Jeffries S, Huang A, et al. Gene signature of antigen processing and presentation machinery predicts response to checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma. J Immunother Cancer. 2020;8(2). 10.1136/jitc-2020-000974."]}, {"label": ["79."], "mixed-citation": ["Calvani M, Dabraio A, Bruno G, De Gregorio V, Coronnello M, Bogani C, et al. \u03b23-Adrenoreceptor Blockade Reduces Hypoxic Myeloid Leukemic Cells Survival and Chemoresistance. Int J Mol Sci. 2020;21(12). 10.3390/ijms21124210."]}, {"label": ["81."], "mixed-citation": ["Database\u00a0Resources of the National Genomics Data Center, China National Center for Bioinformation in 2021. Nucleic Acids Res. 2021;49(D1):D18\u201318D28. 10.1093/nar/gkaa1022."]}, {"label": ["82."], "mixed-citation": ["Yin G, Liu L, Yu T, Yu L, Feng M, Zhou C, et al. Genomic and Transcriptomic Landscape of Breast Cancer with Neoadjuvant Chemotherapy. HRA003759, NGDC Genome Sequence Archive for Human. 2023. "], "ext-link": ["https://ngdc.cncb.ac.cn/gsa-human/browse/HRA003759"]}, {"label": ["83."], "mixed-citation": ["Yin G, Liu L, Yu T, Yu L, Feng M, Zhou C, et al. Genomic and Transcriptomic Landscape of Breast Cancer with Neoadjuvant Chemotherapy. OMIX002785, NGDC OMIX. 2023. "], "ext-link": ["https://ngdc.cncb.ac.cn/omix/release/OMIX002785"]}]
{ "acronym": [ "aDC", "aGPCR", "AJCC", "APC", "BC", "BCSS", "CCLE", "CI", "CNV", "COSMIC", "DEG", "DFS", "ER", "FBS", "FDR", "GEO", "γδT cell", "HER2", "HR", "HRP", "IHC", "ISH", "MP", "MSigDB", "ms2t6A", "NAC", "OS", "pCR", "PR", "qPCR", "RD", "RNA-seq", "ROC", "SCNA", "SMG", "SNV", "TBST", "TCGA", "TMA", "TME", "TPM", "VAF", "WES" ], "definition": [ "Activated dendritic cell", "Adhesion-type G protein-coupled receptor", "The American joint committee on cancer", "Antigen-presenting cell", "Breast cancer", "Breast cancer-specific survival", "The cancer cell line encyclopedia", "Confidence interval", "Copy number variation", "The catalogue of somatic mutations in cancer", "Differentially expressed gene", "Disease-free survival", "Estrogen receptor", "Fetal bovine serum", "False discovery rate", "The Gene Expression Omnibus", "Gamma delta T cell", "Human epidermal growth factor receptor 2", "Hazard ratio", " Horseradish peroxidase", "Immunohistochemistry", "In situ hybridization", "Miller–Payne", "The molecular signatures database", "2-methylthio-N6-threonylcarbamoyladenosine", "Neoadjuvant chemotherapy", "Overall survival", "Pathologic complete response", "Progesterone receptor", "Quantitative real-time PCR", "Residual disease", "RNA sequencing", " Receiver operating characteristics", "Somatic copy-number alteration", "Significantly mutated gene", "Single nucleotide variant", "Tris-buffered saline containing Tween-20", "The Cancer Genome Atlas", " Tissue microarray", "Tumor microenvironment", "Transcripts per million", "Variant allele frequency", "Whole exome sequencing" ] }
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CC BY
no
2024-01-14 23:43:47
Genome Med. 2024 Jan 12; 16:11
oa_package/ac/9d/PMC10787499.tar.gz
PMC10787500
0
[ "<title>Introduction</title>", "<p id=\"Par7\">In 2021, an estimated 38.4 million people were living with Human Immunodeficiency Virus (HIV) globally, and of these, 2.73 million were children and adolescents below 19 years [##UREF##0##1##]. The majority of children and adolescents living with HIV are in sub-Saharan Africa. Similarly, in 2020, sub-Saharan African countries accounted for about 89% of new HIV paediatric infections. Malawi is one of the countries worst burdened by HIV pandemic and HIV/AIDS remains a major public health problem in Malawi despite a significant reduction in the number of new infections in recent years [##UREF##0##1##]. In 2020, approximately, one out of 18 million people were living with HIV in Malawi, and approximately 6% of those affected were children under the age of 15 years [##UREF##0##1##].</p>", "<p id=\"Par8\">HIV/AIDS is the second leading cause of death among adolescents aged 10–19 years globally and in sub-Saharan Africa [##UREF##1##2##] partly due to inadequate HIV testing and counselling and substandard follow up for HIV infected children and adolescents on antiretroviral therapy (ART), causing higher mortality and morbidity rates [##UREF##2##3##]. For example, out of 650,000 people who died of AIDS-related illnesses globally in 2021, 110,000 (17%) of them were children and adolescents under 20 years of age [##UREF##3##4##]. This situation needs curbing to prevent worsening, especially with the prolonged COVID-19 pandemic, global economic crisis, overloaded health care systems and constrained access to life-saving services [##UREF##4##5##].</p>", "<p id=\"Par9\">While the provision of triple ART has increased the life expectancy of children and adolescents living with HIV, these children are at risk of poor developmental outcomes due to both the direct and indirect impacts of HIV infection [##UREF##5##6##]. One of the impacts of HIV infection is psychosocial problems, which intensify in adolescence or early adulthood. In addition to trying to navigate the world while living with HIV, these children and adolescents grow to deal with social pressures associated with adolescence for emotional regulation and social development [##UREF##6##7##, ##UREF##7##8##]. Therefore, children and adolescents living with HIV need support to have a positive sense of identity, manage thoughts and emotions, build social relationships, acquire education, and to actively integrate in the society [##UREF##8##9##].</p>", "<p id=\"Par10\">Authors of systematic reviews have reported high rates of common mental disorders among children, adolescents and adults living with HIV in sub-Saharan Africa [##UREF##9##10##–##REF##29304757##12##]. The most common mental health disorders among children and adolescents in sub-Saharan Africa are attention deficit hyperactivity disorder (ADHD), mood, depression, anxiety, conduct problems, violent behaviour, and emotional and behavioural problems [##UREF##5##6##, ##UREF##11##13##, ##UREF##12##14##]. Previous studies have shown that mental and behavioural health challenges are prevalent in HIV-infected adolescents and pose an enormous burden than HIV-negative peers especially in resource-limited settings [##UREF##10##11##, ##UREF##13##15##]. Besides, these children may experience neurocognitive complications like deficits in cognitive, speech, gross motor and fine motor functioning which can greatly impact their social relationships, academic achievements, general health, and risk of substance abuse [##REF##18647794##16##–##REF##16510653##18##]. Authors of recent studies have found that these cognitive deficits persists, despite early initiation of ART and viral suppression [##REF##28169874##19##, ##REF##32116995##20##].</p>", "<p id=\"Par11\">Among HIV positive children and adolescents, mental health problems have serious implication for later physical health through its impact on health-related behaviour such as smocking, substance abuse, unsafe sex and non-adherence to medication which are detrimental to their health [##UREF##15##21##]. To prevent these issues there is need to have a differentiated ART service delivery model with tailored, layered, and combined prevention packages that are age-appropriate offered in venues that are acceptable and patronized by the adolescents [##REF##29528850##22##].</p>", "<p id=\"Par12\">To meet the unique needs of the children and adolescents living with HIV and thereby increase treatment adherence and achieve viral suppression, the Malawi Ministry of Health, uses a Teen Club model which was adapted from the Baylor College of Medicine International Paediatric AIDS Initiative-Centre of Excellence Curriculum [##UREF##16##23##]. The “ART teen club” program, a peer support group for adolescents living with HIV, is a globally recognized model that empowers adolescents to build positive relationships, improve self-esteem and ultimately improve both clinical and mental health outcomes [##UREF##17##24##]. The “ART teen club” is a targeted psychosocial support intervention which uses different strategies to address the barriers faced by adolescents living with HIV to achieve optimal treatment outcomes [##UREF##18##25##]. The “ART teen club” mission is to provide a safe and nurturing environment for HIV-infected adolescents to build supportive relationships, increase their self-esteem and develop and reinforce good habits [##UREF##17##24##]. According to MacKenzie, Lettow, Gondwe et al., 2017 at “ART teen clubs”, teens do ART refills, are assessed for adherence, and are provided with individualized peer counselling and support as necessary. These children and adolescents are also stratified by age and gender into small group sessions for sexual and reproductive health education sessions in order to encourage comfort and privacy. In addition, these teens participate in recreational activities like facilitated sports, arts and games for peer interaction [##UREF##18##25##]. “ART teen club” provides a forum to address other issues affecting HIV-infected adolescents, such as nutrition and other psychological needs. Adolescents are eligible for participation in the “ART teen club” if they are between the ages of 10 and 19, on ART, and have had their HIV status disclosed to them. These adolescents are referred to the Teen club by either the ART clinician or a nurse after the HIV disclosure process has been completed. Teen club sessions are held monthly on a weekend (usually Saturday) to avoid school absenteeism which is one of the challenges to HIV treatment adherence amongst adolescents in low socio-economic settings like Malawi [##UREF##16##23##, ##UREF##19##26##]. Teens are supposed to graduate to adult ART clinics after their 19th birthday. The adolescents recruited in this study were in “ART teen clubs” run by regular ART clinicians and nurses who have been trained in the paediatric HIV care curriculum. These health workers work on a roaster and are compensated accordingly for the extra hours in line with the Ministry of Health or non-governmental organization (NGO) policies.</p>", "<p id=\"Par13\">Studies on the teen club model have shown that the model is more effective towards attaining virological suppression among adolescents living with HIV in Malawi [##UREF##18##25##, ##UREF##20##27##]. However, there is a dearth of knowledge on the impact of this model on mental wellness of its consumers. This population is particularly vulnerable to mental health and conduct problems because of age, HIV diagnosis and poor social economic factors, which if left unidentified or treated could persist to adulthood [##REF##25587353##28##]. Understanding the emotional and behaviours difficulties among this population has a potential to provide valuable information on the subject to policy makers and first line health workers. This information can be used to develop polices and interventions that can better support the need of adolescents living with HIV. We, therefore, conducted the study to establish the prevalence of emotional and behavioural problems among children and adolescents living with HIV. Specifically, the study had two research questions namely (1) What is the prevalence of emotional and behavioural difficulties among children and adolescents attending ART teen club and (2) What demographic and clinical factors are associated with emotional and behavioural problems among this population? Identifying the prevalence of emotional and behavioural problems among the study sample provides a baseline understanding of the scope and magnitude of the issue within the target population. If a significant portion of the population is affected, it signals the need for interventions on a larger scale. Understanding the demographic and clinical factors associated with emotional and behavioural difficulties helps in identifying high-risk groups within the sample. For instance, if a certain demographic group or those with specific demographic characteristics are more prone to these problems, interventions can be targeted towards these groups. In addition, resources for interventions are often limited. Understanding the associated factors helps in efficient resource allocation, directing efforts towards the factors that have the most significant impact on emotional and behavioural difficulties in this population.</p>" ]
[ "<title>Methods</title>", "<title>Research design, study population and recruitment criteria</title>", "<p id=\"Par14\">This was cross-sectional study conducted with children and adolescents. We targeted children and adolescents living with HIV in Mzuzu city. Potential participants were recruited in the study if they were: (1) living in Mzuzu and enrolled in the “ART teen club”; (2) aware of their HIV status; (3) had informed consent to participate in the study. Since all of them were below 18 years old, their parents signed informed consent forms for them to participate in the study. We planned to exclude children and adolescents who had an illness with a potential to impact on informed consenting process and completion of the study questionnaire such as psychiatric and severe HIV related illnesses. However, none was excluded based on these criteria.</p>", "<title>Research setting</title>", "<p id=\"Par15\">This study was undertaken in three “ART teen clubs” each at Mzuzu Central Hospital, Mzuzu Health Centre and St John’s Mission Hospital in Mzuzu City, Northern Malawi. All these facilities provided HIV services to teens one weekend in the month. Mzuzu Central Hospital ART teen clinic has a cumulative total of 324 clients, Mzuzu Health Centre 195 clients and St John’s Hospital 184 Clients. These sites were purposively selected for this study because they were the only facilities with “ART teen clubs” in Mzuzu City.</p>", "<title>Sampling and sample size</title>", "<p id=\"Par16\">Participants to this study were recruited through systematic sampling between April and May 2022. We used an online sample size calculator (Raosoft, 2020) to determine the required sample size for this study [##UREF##21##29##]. We found that the three health facilities had in total 703 children and adolescents enrolled in “ART Teen Clubs.” Based on the estimated population, and setting the margin error and confidence level at 0.05 and 95% respectively, a sample size of 249 children and adolescents was needed. To include a non-response rate of 15% a total sample size of 286 was considered adequate.</p>", "<title>Data collection instruments</title>", "<p id=\"Par17\">Data were collected using a questionnaire that had two sections: (1) Socio demographic variables and (2) child emotional and behavioural difficulties. The sociodemographic sections contained questions about age, gender, educational level of parents/primary caregivers, and availability of parents. The child emotional and behavioural difficulties was assessed by the youth version of the Strengths and Difficulties Questionnaire (SDQ-Y) [##UREF##22##30##]. The SDQ-Y is a self-report tool, designed to be completed by children and adolescents aged 11–17 years and used to detect childhood emotional and behavioural problems. The SQD-Y is a well-validated instrument. The scale has adequate internal consistency (Cronbach’s alpha ranging from 0.78 to 0.82) and predictive validity [##UREF##22##30##]. This questionnaire has been used in more than 50 studies across 12 African countries including Malawi [##UREF##23##31##] and is translated in 60 different languages including Chichewa, the Malawian local national language [##UREF##24##32##]. The tool has 25 items divided in five subscales comprising of five domains of conduct problems, hyperactivity/inattention, emotional symptoms, peer problems and prosocial behavior. While four of the subscales refers to problem behaviours, the fifth (prosocial behaviour) refers to positive behaviour. We also collected data from participants clinical records on HIV disclosure status, and WHO clinical stage of HIV.</p>", "<p id=\"Par18\">Data were collected by research assistants with a Bachelor of Science in Nursing qualification. The research assistants were trained in the data collection process. We used the original Chichewa-translated version of the SDQ-Y to collect data from all participants. For participants who were illiterate, the research assistants read the questions to the participants and marked the answers selected by the participants on the questionnaire.</p>", "<title>Recruitment and data collection procedures</title>", "<p id=\"Par19\">Potential participants were assigned numbers from one to last person based on the time they arrived at the “ART teen club”. Those with even numbers were screened based on the inclusion criteria (see Sect. <xref rid=\"Sec3\" ref-type=\"sec\">2.1</xref> above). Research assistants with a degree or diploma in Nursing were recruited to assist with data collection. These research assistants were trained on the research procedure prior to data collection. In March, 2022, our research assistants, who were not part of the teen club staff, informed potential participants about the aims, procedure, outcomes, benefits, and associated risks as well as their rights with regard to consent, confidentiality, anonymity and withdrawal from the study. The research assistant further distributed the informed consent/assent forms and study information sheets to potential participants. Participants brought their signed informed consent forms to the clinic on their next visit. The research assistants confirmed the understanding of the study purpose, reminded participant that of voluntary participation, assured participant of their confidentiality and verified if non coerced consenting was made prior to data collection. Participants were asked to complete their questionnaires in a private room at the clinic soon after clinic activities. Data collection took place between April and May 2022. Completion of the questionnaire took approximately 20 min.</p>", "<title>Ethical consideration</title>", "<p id=\"Par20\">This research was granted ethical approval by the Malawi National Health Sciences Research Committee (Ref: #17/05/1804). Written permission to conduct the study was obtained from three participating facilities. The participants were informed that they had the right to withdraw from the study at any time without any prejudice. All participants provided consent/assent prior to data collection.</p>", "<p id=\"Par21\">During the research process, respondents were not subjected to any physical harm as the study did not involve any invasive procedures. Prior arrangements for counselling and support were made for anticipated emotional reactions by the participants considering the emotional nature of the topic under study. The respondents answered the questions in a comfortable environment with privacy assured and no public interference.</p>", "<title>Statistical analysis</title>", "<p id=\"Par22\">Descriptive statistics were used to understand the general characteristics of participants. Bivariate analysis was conducted using binary logistic regression to identify individual factors that are associated with emotional and behavioural difficulties. All variables that were significant at <italic>P</italic> ≤ 0.05 in bivariate analysis were entered into multivariate logistic regression to identify factors that were independently associated with emotional and behavioural difficulties among the study population. All analyses were considered statistically significant at <italic>P</italic> ≤ 0.05. Data analyses were conducted using statistical Package for Social Sciences (SPSS) version 22.</p>" ]
[ "<title>Results</title>", "<title>Response rates and demographic characteristics</title>", "<p id=\"Par23\">A total number of 286 questionnaires were distributed and 277 were returned resulting in 96.8% response rate. This study recruited participants whose ages ranged from 10 to 17 years with a mean of 14 years. The majority of the participants were female (71.5%, n = 198) and recruited from three health facilities in Malawi of which majority came from Mzuzu Central Hospital (46.9%, n = 130) and the least came from St John’s Hospital (18.8%, n = 52). Results further indicate that the majority of children sampled were doing their studies at primary school followed by those who were at secondary school. Regarding parents’ education, 70.3% of children in the study had parents who had secondary education or above with only 10% whose parents had no formal education. Among the sampled children, the majority were protestants (49.5%, n = 137) and 51.6% of them had both parents. The household size of the sampled children ranged from 2 to 11 individuals with a mean of 5 individuals per household (See Table ##TAB##0##1##).</p>", "<p id=\"Par24\">\n\n</p>", "<title>Prevalence of emotional and behavioural difficulties of participants</title>", "<p id=\"Par25\">Basing on the four-band categorization of the SDQ, higher scores for the total difficulties scale score was observed in 72.9% of the children. According to the subscales of the SDQ, results show that children had higher scores in emotional (68.2%), conduct (68.6%), peer problems (62.8%) and prosocial behaviour (57.8%) subscales while a lower score was identified in the hyperactivity (16.6%) subscale (see Table ##TAB##1##2##).</p>", "<p id=\"Par26\">\n\n</p>", "<p id=\"Par27\">Distribution of total difficult scores based on four levels shows some trends by variable gender. Results show that within each level, males are having lower frequencies as compared to females. Among the four levels of total difficult scoring, level high has the highest frequency in general and by gender (See Fig. ##FIG##0##1##).</p>", "<p id=\"Par28\">\n\n</p>", "<title>Factors associated with emotional and behavioural difficulties of participants</title>", "<p id=\"Par29\">Binary logistic regression was run to identify factors associated with the emotional and behavioural difficulties of participants of the children. Results from multivariate binary logistic regression indicate children and adolescents who were catholic (AOR 4.114; 95% CI:1.502–11.262, <italic>p</italic> = 0.006) or Pentecost (AOR 3.730; 95% CI: 1.155–12.043, <italic>p</italic> = 0.028) are more likely to have abnormal emotional and behavioural difficulties as compared to those children who were protestant. Results further demonstrate that children who do not have a single or both parents (AOR 3.404; 95% CI:1.563–7.416, <italic>p</italic> = 0.002) were more likely to have abnormal emotional and behavioural difficulties as compared to those with single or both parents. Lastly, results indicate that children who were in WHO HIV clinical stage 2 (AOR 2.536; 95% CI:1.005–6.395, <italic>p</italic> = 0.049) or 3 and 4 (AOR 8.459; 95% CI:1.5.820-10.544, <italic>p</italic> &lt; 0.001) had more odds of having the mental disorder as compared with those children in WHO clinical stage 1.</p>", "<p id=\"Par30\">\n\n</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par31\">To the best of our knowledge this is the first study to assess the emotional and behavioural difficulties of children and adolescents living with HIV who completed a disclosure process and were attending ART teen club in Malawi. The findings of the current study show that about three-quarters (72.9%) of the children and adolescents living with HIV had emotional or behavioural problems as revealed by self-rated using the SDQ-Y (Table ##TAB##1##2##). With regard to the SDQ-Y scoring categories, more than two-thirds of the participants had higher scores (slightly raised, higher, and very high) in the emotional (68.2%), conduct (68.6%), prosocial (57.8%) and peer relationship problem scales (62.8%) except for hyperactivity scoring which had (16.6%).</p>", "<p id=\"Par32\">The higher score (slightly raised/ high/very high) of 68.2% for emotional problems found in the current study is surprising for this group of “ART teen club” as these participants would have been counselled and are aware of their diagnosis. One of the main benefit of disclosure of HIV status to HIV-infected children and adolescents is to enhance mental health status [##UREF##25##33##]. In Malawi, parents or guardians are encouraged to disclose their HIV status to their perinatally acquired HIV children gradually [##UREF##26##34##]. Perhaps reviewing to evaluate and revise the post disclosure counselling system can benefit the psychosocial wellbeing of this population. The high rate could also be attributed to participant’s everyday worries regarding their future, health as well as feelings of resentment an blame towards their caregivers [##REF##30606141##35##]. Authors of previous studies have reported found few mental health challenges in children with full HIV status disclosure than those who were unaware of their HIV status [##UREF##27##36##, ##UREF##28##37##]. This calls for open dialogue about “teenage challenges” with a special focus mental health especially for those who are transitioning from a Teen Club to the adult clinic as they are at risk of attrition [##UREF##26##34##]. This is important because mental health ultimately influences retention in the HIV program and medication adherence [##UREF##29##38##].</p>", "<p id=\"Par33\">The prevalence of conduct problems as measured by the SDQ-Y(68.6%) was extremely high in this study compared with 38% reported previously in selected facilities in Malawi [##REF##30606141##35##]. This could be attributed to the sample size and the age of the participants recruited in these studies. The previously reported study recruited children as young as 6 years old and a parental version of SDQ was used to collect data. Differing results were also reported in Zambia (10.2%) [##UREF##30##39##] and the United Kingdom (3%) [##UREF##31##40##]. Similarly, the parent version of SDQ-Y was used in both studies. Children are well known to report more symptoms on SDQ-Y than parents [##UREF##32##41##]. Future studies should consider using the parental version of SDQ-Y as well to supplement data for an in-depth understanding of the subject and to make real time inferences. Even though there were a lot of children and adolescents of unemployed guardians (n = 119) compared to employed guardians (n = 86) who had emotional and behavioural problems in the current study (Table ##TAB##2##3##), the association did not reach the level of significance. This is in line with studies in developed and developing countries which reported that unemployed parents were more likely to report emotional and behavioural problems in their children compared to those who were employed [##REF##19084312##42##, ##UREF##33##43##]. In addition, HIV in the family may escalate economic hardship as unemployment is increased and scarce family resources may be diverted to meet the needs of parents or guardians. In an earlier cross-sectional study done in Malawi and South Africa to explore cash grants on children’s cognitive development, results showed that helping families infected with HIV with cash receipts was associated with enhanced child cognitive outcomes [##REF##28056921##44##]. Finding ways to assist socio-economically disadvantaged families with children and adolescents living with HIV with financial aid through social grants like cash transfers may reduce emotional and behavioural difficulties in these children and adolescents.</p>", "<p id=\"Par34\">The current study has shown that children and adolescents who were in WHO HIV clinical stage 2 (AOR-2.536 95% CI, CL:1.005-6,395 <italic>p</italic> = 0.049) and stage 3 or 4 (AOR 8.459; 95% CI:1.5.820-10.544, <italic>p</italic> &lt; 0.001) were more likely to score higher scores on SQD as compared with children in WHO HIV clinical stage 1. This finding is consistent with a previous study which reported that immunosuppression was associated with poor emotional and behavioural outcomes [##UREF##34##45##]. In a recent study done in Botswana, HIV congenitally infected adolescents were almost four times more likely to present with externalizing disorders like ADHD which may have occurred partly due to the effect of HIV on the brain [##UREF##35##46##] or immunosuppression resulting from prolonged period of poor ART adherence [##UREF##36##47##]. Researchers have found a strong association between externalizing disorders and viral load above 400 copies [##UREF##35##46##]. In another study children with low CD4 count &lt; 500 cells/mm<sup>3</sup> were more likely to develop emotional and behavioural difficulties as compared to children with CD4 count &gt; 500 cells/mm<sup>3</sup>(<italic>p</italic> = 0.015) [##UREF##37##48##]. In a study by Kim, Mazenga, Yu et al., (2015), no significant correlation was found between mental health difficulties, and CD4 Count [##UREF##38##49##]. Rigorous longitudinal studies are therefore recommended to resolve the discrepancy as well are highlight the direction of the observed effect.</p>", "<p id=\"Par35\">In this study caregivers’ level of education was not associated with the children and adolescents’ mental health challenges. Previous studies have reported caregivers with low levels of education had a higher likelihood of having children with a mental health problem [##REF##30606141##35##]. In a study by Cortia, Fazel, Hlungwani et al. (2013) in rural Southern Africa mothers with lower educational levels (i.e. not completed high school) were more likely to report poor psychological outcomes for their children like hyperactivity, anxiety and learning difficulties [##UREF##39##50##]. Low education may also mean having low socio-economic status. Support for caregivers from socio-economic backgrounds is critical in a holistic treatment plan.</p>", "<p id=\"Par36\">About half of the respondents (51.6%) of the children and adolescents in “ART Teen Club” in Mzuzu City had both parents. The results have shown that those who did not live with both parents had high chance of developing emotional and behavioural difficulties (AOR 3.404; 95% CI: 1.563–7.416, <italic>P</italic> = 0.002). Similar findings were found in other parts of Africa [##UREF##40##51##]. Research suggests that presence of parent is a protective factor for emotional problems. However, other researchers have recorded emotional problems in both double and non-orphans [##REF##19121218##52##]. Perhaps a lack of socioeconomic support following the demise of parents or perceived collective family HIV- associated stigma magnifies the problem. Health workers in these clinics, should pay more attention to the orphans to timely identify and refer or manage emotional and difficult behaviours.</p>", "<p id=\"Par37\">Our study has found that children and adolescents who were catholic (AOR 4.114; 95% CI:1.502–11.262, <italic>p</italic> = 0.006), Pentecost (AOR 3.730; 95% CI: 1.155–12.043, <italic>p</italic> = 0.028) or Muslim (4.742: 95% CI: 10.534–42.100, <italic>p</italic> = 0.02) are more likely to have abnormal emotional and behavioural difficulties as compared to those children who were protestant. This could be because of issues to do with spirituality or religiousness. Roman Catholics, Pentecost and Muslims are regarded to be of higher spirituality or religiousness than protestants. Several studies confirm our findings that low spirituality was associated with low emotional and behavioural challenges [##UREF##41##53##, ##REF##23674243##54##]. For example, a study by Michealson, Robinson and Pickett (2014) in Canada found that Canadian youth with regular church involvement had poorer levels of emotional well-being compared to those who did not participate in religious services [##REF##23674243##54##]. In contrast, a study by Lyona, D’Angeloa, Chengd et al., (2020) found that religiousness moderates adolescent medical decision-making. The authors recommended that religious adolescents should be encouraged to practice their religion to develop resilient and enhance mental well-being, which consequently, promote medication adherence [##REF##31535560##55##].</p>" ]
[ "<title>Conclusion</title>", "<p id=\"Par43\">The findings of this research underscore the multifaceted nature of mental well-being among children and adolescents living with HIV. Elevated scores in total difficulties, emotional, conduct, and peer problems signify areas of concern, while disparities in hyperactivity and prosocial behavior highlight the nuanced nature of their behavioral challenges. Recognizing the inadequacy of a one-size-fits-all approach, the research emphasizes the necessity of a comprehensive strategy, incorporating factors like religious background, family structure, and clinical HIV stage. Furthermore, the role of Teen support clubs in this context is pivotal. Beyond addressing identified risk factors, these clubs must actively foster resilience. Creating an inclusive environment, tapping into individual strengths, and nurturing a sense of community are vital components. By adopting such a holistic approach, Teen support clubs can significantly contribute to the overall mental well-being of adolescents living with HIV, enabling them to navigate challenges effectively and thrive amidst their circumstances.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">While triple anti-retroviral therapy (ART) has improved HIV-infected children surviving into adolescence and adulthood, these children remain vulnerable to HIV-related psychological disturbance due to both the direct HIV infection effects on the brain and indirect effects related to coping with a range of medical, psychological and social stresses associated with HIV, which makes it vital to identify their mental health needs. This study assessed the emotional and behavioural challenges of HIV perinatally infected children and adolescents with a completed disclosure process attending “ART teen club” in Malawi.</p>", "<title>Methods</title>", "<p id=\"Par2\">A cross-sectional descriptive study design was conducted to obtain quantitative descriptive descriptions of emotional and behavioural challenges among HIV-infected children and adolescents between 10 and 22 years of age. They were interviewed on their family socio-demographic characteristics, clinical characteristics as well as emotional, conduct, hyperactivity, peer and prosocial problems using the Chichewa version of the Strengths and Difficulties Questionnaire. Data were analyzed using descriptive analysis and logistic regression.</p>", "<title>Results</title>", "<p id=\"Par3\">Based on the four-band categorization of the SDQ, higher scores for total difficulties score were observed in 72.9% of the children. According to the subscales of the SDQ, results show that children had higher scores in peer problems (62.8%), emotional (68.2%), conduct (68.6%) and prosocial (57.8%) subscales while lower scores were identified in the hyperactivity (16.6%) subscale. Results show that within each level, males are having lower frequencies as compared to females. Results from multivariate binary logistic regression indicate that those with a single parent or not as well as the WHO HIV clinical stage had an impact on the mental health status of the children. Children who do not have a single parent (AOR 3.404; 95% CI:1.563–7.416, <italic>p</italic> = 0.002) had 3.404 odds of having abnormal mental health status unlike those children with a single parent and children who were in WHO HIV clinical stage 2 (AOR 2.536; 95% CI:1.005–6.395, <italic>p</italic> = 0.049) or 3 and 4 (AOR 8.459; 95% CI:1.5.820-10.544, <italic>p</italic> &lt; 0.001) had more odds of having the mental disorder as compared with those children in WHO HIV clinical stage 1.</p>", "<title>Conclusion</title>", "<p id=\"Par6\">The findings of this research underscore the multifaceted nature of mental well-being among children and adolescents living with HIV. Elevated scores in total difficulties, emotional, conduct, and peer problems signify areas of concern, while disparities in hyperactivity and prosocial behavior highlight the nuanced nature of their behavioral challenges. Recognizing the inadequacy of a one-size-fits-all approach, the research emphasizes the necessity of a comprehensive strategy, incorporating factors like religious background, family structure, and clinical HIV stage. Furthermore, the role of “ART teen clubs” in this context is pivotal. Beyond addressing identified risk factors, these clubs must actively foster resilience. Creating an inclusive environment, tapping into individual strengths, and nurturing a sense of community are vital components. By adopting such a holistic approach, Teen support clubs can significantly contribute to the overall mental well-being of adolescents living with HIV, enabling them to navigate challenges effectively and thrive amidst their circumstances.</p>", "<title>Keywords</title>" ]
[ "<title>Strength of the study</title>", "<p id=\"Par38\">The study used a well-established emotional and behavioural tool with strong psychometric properties validated for several African countries including Malawi. However, this tool heavily relies on self-report of children and adolescent, which has a potential bias of providing socially acceptable responses. In addition, this tool has been previously critiqued and is not recommended in children below 11 years [##UREF##42##56##]. Collecting data from other informants such parent and/or teacher would not only strengthen the rigour of the study, but also credibility of the emotional and behavioural difficulties evaluation [##UREF##42##56##]. The findings of the study should be interpreted with a consideration of limitations associated with cross-sectional studies such as limitation in determining casuality and direction of correlations.</p>", "<title>Implications of the study findings</title>", "<p id=\"Par39\">The findings from this research study have several implications for practice, research, and policy, particularly in the context of child and adolescent emotional and behavioral difficulties.</p>", "<title>Implication for practice</title>", "<p id=\"Par40\">In light of the study findings, there are several practical implications that merit consideration. Firstly, the Ministry of Health needs to intensify mental health screening in ART teen clubs as undiagnosed mental health problems may decrease antiretroviral therapy adherence leading to a lowering of immunity and acceleration of disease progression in these children and adolescents [##UREF##29##38##]. This approach facilitates the early identification of at-risk individuals, allowing for timely interventions and support. Secondly, healthcare providers operating in HIV clinical settings need to integrate mental health assessments and interventions. Implementing collaborative care models, involving both medical and mental health professionals, ensures a holistic approach to healthcare. This integration is crucial for promoting resilience by addressing both mental and physical health needs concurrently. Furthermore, given the association between abnormal emotional and behavioural difficulties and Catholic or Pentecostal affiliation, practitioners must integrate cultural and religious sensitivity into therapeutic approaches. For instance, incorporating spiritual practices within therapy for children and adolescents from these religious backgrounds may enhance engagement and improve outcomes. Lastly, interventions targeting children without one or both parents should concentrate on building robust support systems. Practitioners could collaborate with community organizations to establish mentorship programs, contributing to fostering resilience in children facing familial challenges. Strengthening protective factors, including supportive adult relationships, is vital for promoting resilience.</p>", "<title>Implications for Research</title>", "<p id=\"Par41\">The study findings underscore the need for further research to deepen our understanding of risk factors contributing to emotional and behavioral difficulties. Qualitative studies exploring the lived experiences of children from diverse religious backgrounds can provide nuanced insights, informing targeted interventions and resilience-building strategies. In addition, longitudinal studies are imperative to track the developmental trajectories of emotional and behavioural difficulties in high-risk groups. Identifying critical periods for intervention contributes to resilience research by shedding light on factors that buffer against mental health challenges across the lifespan. Moreover, researchers should adopt an intersectional lens to explore the interplay of various risk factors contributing to emotional and behavioral difficulties. Investigating how religious affiliation intersects with familial structure, for example, provides a nuanced understanding. This approach contributes to resilience frameworks by identifying protective factors in diverse contexts.</p>", "<title>Implications for policy</title>", "<p id=\"Par42\">Policy makers must consider allocating resources to implement evidence-based mental health programs targeting high-risk groups. Funding initiatives like Teen club and community-based mental health programs enhances accessibility and fosters resilience among children and adolescents, particularly those living with HIV. Furthermore, policies related to HIV care should explicitly integrate mental health components. Implementing guidelines that mandate routine mental health screenings within HIV clinical settings, including Teen clubs, ensures a comprehensive approach to healthcare. This aligns with resilience-oriented policies by addressing mental health needs alongside physical health. The findings of this study also underscore the need to formulate policies that specifically support vulnerable families, recognizing the unique challenges they face. Implementing financial assistance programs or community-based support services strengthens familial resilience. Policies aimed at supporting vulnerable families contribute to broader societal resilience.</p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors would like to thank the clinical staff and patients in the three study sites: Mzuzu Central Hospital, St John Hospital and Mzuzu Health Centre especially the “ART teen club” coordinators for helping to systematically sample the participants and providing their rooms for data collection. Research assistants for their dedication in interviewing and listening to the youth participating in the study. Sincere thanks to the “ART teen club” attendees who gave their time and provided their responses to the emotional questions surrounding mental health.</p>", "<title>Author contributions</title>", "<p>FWK, PUK, BCM and MZ participated in the design of the study. PUK, FWK, MZ, BMC, ON and ML collected data. ML, FWK, PUK, MZ, ON and BCM analyzed data. PUK, FWK, MZ, ML, ON and BCM participated in writing the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This research did not receive any funding.</p>", "<title>Data availability</title>", "<p>The dataset used and/or analyzed during the current study are available from the corresponding author on a reasonable request.</p>", "<title>Declarations</title>", "<title>Ethical approval and consent to participate</title>", "<p id=\"Par45\">This study was approved by the Malawi National Health Sciences Research and Ethics Committee (Protocol number:17/05/1804)). This was done to fulfill the council for international organization of medical sciences (CIOMS) guidelines when carrying out research on human beings. The guidelines state that any research carried out on human beings should be approved by the ethics research committee to make sure that it meets all ethical issues to protect the population. Prior to approval, all participating sites wrote support letters accepting the study to be done in their respective sites as ethics clearance requirement. Upon approval, letters of introduction were submitted to authorities of the participating facilities. The “ART teen clubs” in-charges were then asked to provide names of the children and adolescents that had come to the clinic on that day. The names were given numbers from the first to the last. Only even number were randomly selected. The participants were then informed of the study and they were also informed that they could withdraw from participating in the study at any time without fear of any penalty. Parents and/or legal guardians signed the informed consent forms on behalf of all study participants before data collection, expressing their children willingness to participate in the study as all participants were below 18 years. Only those whose parents or legal guardian signed the consent forms were interviewed. The questionaries were put in envelopes and handed out to the respondents. Questionnaires were filled with the help of research assistants without any identifying information. All study methods were performed in accordance with Malawi National Health Sciences Research and Ethics Committee ethical guidelines.</p>", "<title>Consent for publication</title>", "<p id=\"Par46\">This manuscript does not contain any identifying image, therefore consent for publication was not required.</p>", "<title>Competing interests</title>", "<p id=\"Par47\">The authors declare that they have no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Gender differences in SDQ-Y scores based on the 4-band-categorisation</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Demographic characteristics of the participants</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Demographic Characteristics</th><th align=\"left\">Frequency</th><th align=\"left\">Percent</th></tr></thead><tbody><tr><td align=\"left\">\n<bold>Sex</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Male</td><td align=\"left\">79</td><td align=\"left\">28.5</td></tr><tr><td align=\"left\"> Female</td><td align=\"left\">198</td><td align=\"left\">71.5</td></tr><tr><td align=\"left\">\n<bold>Health Facility</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Mzuzu Central Hospital</td><td align=\"left\">130</td><td align=\"left\">46.9</td></tr><tr><td align=\"left\"> Mzuzu Health Centre</td><td align=\"left\">95</td><td align=\"left\">34.3</td></tr><tr><td align=\"left\"> St John’s Hospital</td><td align=\"left\">52</td><td align=\"left\">18.8</td></tr><tr><td align=\"left\">\n<bold>Participant’s level of education</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> None</td><td align=\"left\">5</td><td align=\"left\">1.8</td></tr><tr><td align=\"left\"> Primary</td><td align=\"left\">153</td><td align=\"left\">55.2</td></tr><tr><td align=\"left\"> Secondary</td><td align=\"left\">110</td><td align=\"left\">39.7</td></tr><tr><td align=\"left\"> Tertiary</td><td align=\"left\">9</td><td align=\"left\">3.2</td></tr><tr><td align=\"left\">\n<bold>Guardian/parent’s level of education</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> None</td><td align=\"left\">10</td><td align=\"left\">3.6</td></tr><tr><td align=\"left\"> Primary</td><td align=\"left\">72</td><td align=\"left\">26</td></tr><tr><td align=\"left\"> Secondary</td><td align=\"left\">145</td><td align=\"left\">52.3</td></tr><tr><td align=\"left\"> Tertiary</td><td align=\"left\">50</td><td align=\"left\">18</td></tr><tr><td align=\"left\">\n<bold>Guardian/parents occupation</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Unemployed</td><td align=\"left\">31</td><td align=\"left\">11.2</td></tr><tr><td align=\"left\"> Informal employed</td><td align=\"left\">30</td><td align=\"left\">10.8</td></tr><tr><td align=\"left\"> Formal employed</td><td align=\"left\">79</td><td align=\"left\">28.5</td></tr><tr><td align=\"left\"> Business</td><td align=\"left\">115</td><td align=\"left\">41.5</td></tr><tr><td align=\"left\"> Others</td><td align=\"left\">22</td><td align=\"left\">7.9</td></tr><tr><td align=\"left\">\n<bold>Participant’s religion</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Protestant</td><td align=\"left\">137</td><td align=\"left\">49.5</td></tr><tr><td align=\"left\"> Roman Catholic</td><td align=\"left\">53</td><td align=\"left\">19.1</td></tr><tr><td align=\"left\"> Pentecostal</td><td align=\"left\">48</td><td align=\"left\">17.3</td></tr><tr><td align=\"left\"> Traditional</td><td align=\"left\">14</td><td align=\"left\">5.1</td></tr><tr><td align=\"left\"> Muslim</td><td align=\"left\">19</td><td align=\"left\">6.9</td></tr><tr><td align=\"left\"> Apostolic</td><td align=\"left\">6</td><td align=\"left\">2.1</td></tr><tr><td align=\"left\">\n<bold>Availability of both parents</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">143</td><td align=\"left\">51.6</td></tr><tr><td align=\"left\"> No</td><td align=\"left\">134</td><td align=\"left\">48.4</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Psychosocial characteristics of the study participants</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Psychosocial characteristics</th><th align=\"left\">Frequency</th><th align=\"left\">Percent</th></tr></thead><tbody><tr><td align=\"left\">\n<bold>Total Difficult Scoring</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Close to average</td><td align=\"left\">75</td><td align=\"left\">27.1</td></tr><tr><td align=\"left\"> Slightly raised/high/very</td><td align=\"left\">202</td><td align=\"left\">72.9</td></tr><tr><td align=\"left\">\n<bold>Emotional Problem Scoring</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Close to average</td><td align=\"left\">88</td><td align=\"left\">31.8</td></tr><tr><td align=\"left\"> Slightly raised/high/very</td><td align=\"left\">189</td><td align=\"left\">68.2</td></tr><tr><td align=\"left\">\n<bold>Conduct Problem Scoring</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Close to average</td><td align=\"left\">87</td><td align=\"left\">31.4</td></tr><tr><td align=\"left\"> Slightly raised/high/very</td><td align=\"left\">190</td><td align=\"left\">68.6</td></tr><tr><td align=\"left\">\n<bold>Hyperactivity Scoring</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Close to average</td><td align=\"left\">231</td><td align=\"left\">83.4</td></tr><tr><td align=\"left\"> Slightly raised/high/very</td><td align=\"left\">46</td><td align=\"left\">16.6</td></tr><tr><td align=\"left\">\n<bold>Peer Problem Scoring</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Close to average</td><td align=\"left\">103</td><td align=\"left\">37.2</td></tr><tr><td align=\"left\"> Slightly raised/high/very</td><td align=\"left\">174</td><td align=\"left\">62.8</td></tr><tr><td align=\"left\">\n<bold>Prosocial Scoring</bold>\n</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Close to average</td><td align=\"left\">117</td><td align=\"left\">42.2</td></tr><tr><td align=\"left\"> Slightly lowered/low/very</td><td align=\"left\">160</td><td align=\"left\">57.8</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Factors associated with emotional and behavioural difficulties in simple and multiple binary logistic regression</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Factors</th><th align=\"left\" colspan=\"2\">Mental Health Status</th><th align=\"left\" colspan=\"2\">Crude Odds Ratio</th><th align=\"left\" colspan=\"2\">Adjusted Odds Ratio</th></tr><tr><th align=\"left\">Normal</th><th align=\"left\">Abnormal</th><th align=\"left\">OR[95%]</th><th align=\"left\"><italic>P</italic>-Value</th><th align=\"left\">AOR[95%]</th><th align=\"left\"><italic>P</italic>-Value</th></tr></thead><tbody><tr><td align=\"left\">\n<bold>Sex</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Male</td><td align=\"left\">25(31.6)</td><td align=\"left\">54(68.4)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\"> Female</td><td align=\"left\">50(25.3)</td><td align=\"left\">148(74.7)</td><td align=\"left\">1.370(0.773–2.429)</td><td align=\"left\">0.281</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">\n<bold>Religion</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Protestant</td><td align=\"left\">53(38.7)</td><td align=\"left\">84(61.3)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\">1</td><td align=\"left\"/></tr><tr><td align=\"left\"> Roman Catholic</td><td align=\"left\">10(18.9)</td><td align=\"left\">43(81.1)</td><td align=\"left\">2.713(1.257–5.855)</td><td align=\"left\">0.011</td><td align=\"left\">4.113(1.502–11.262)</td><td align=\"left\">0.006</td></tr><tr><td align=\"left\"> Pentecost</td><td align=\"left\">8(16.7)</td><td align=\"left\">46(84.3)</td><td align=\"left\">3.155(1.371–7.259)</td><td align=\"left\">0.007</td><td align=\"left\">3.730(1.155–12.043)</td><td align=\"left\">0.028</td></tr><tr><td align=\"left\"> Tradition</td><td align=\"left\">2(14.3)</td><td align=\"left\">12(85.7)</td><td align=\"left\">3.786(0.815–17.587)</td><td align=\"left\">0.089</td><td align=\"left\">2.130(0.399–11.379)</td><td align=\"left\">0.377</td></tr><tr><td align=\"left\"> Muslim</td><td align=\"left\">2(10.5)</td><td align=\"left\">17(89.5)</td><td align=\"left\">5.363(1.191–24.155)</td><td align=\"left\">0.02</td><td align=\"left\">4.742(0.534–42.100)</td><td align=\"left\">0.162</td></tr><tr><td align=\"left\">\n<bold>Participant’s Education Level</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Up to primary</td><td align=\"left\">48(30.4)</td><td align=\"left\">110(69.6)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\">1</td><td align=\"left\"/></tr><tr><td align=\"left\"> Secondary and above</td><td align=\"left\">27(22.7)</td><td align=\"left\">92(77.3)</td><td align=\"left\">1.606(0.920–2.803)</td><td align=\"left\">0.096</td><td align=\"left\">1.632(0.747–3.566)</td><td align=\"left\">0.219</td></tr><tr><td align=\"left\">\n<bold>Guardian’s Education Level</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> None</td><td align=\"left\">2(20.0)</td><td align=\"left\">8(80.0)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Primary</td><td align=\"left\">23(31.9)</td><td align=\"left\">49(68.1)</td><td align=\"left\">0.533(0.105–2.710)</td><td align=\"left\">0.448</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\"> Secondary</td><td align=\"left\">44(30.3)</td><td align=\"left\">101(69.7)</td><td align=\"left\">0.574(0.117–2.812)</td><td align=\"left\">0.493</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\"> Tertiary</td><td align=\"left\">6(12.0)</td><td align=\"left\">44(88.0)</td><td align=\"left\">2.050(0.337–12.481)</td><td align=\"left\">0.436</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">\n<bold>Guardian’s Occupation</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Unemployed</td><td align=\"left\">44(27.0)</td><td align=\"left\">119(73.0)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Employed</td><td align=\"left\">31(26.5)</td><td align=\"left\">86(73.5)</td><td align=\"left\">1.035(0.599–1.786)</td><td align=\"left\">0.903</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">\n<bold>Single/both parents</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">42(33.3)</td><td align=\"left\">84(66.7)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\">1</td><td align=\"left\"/></tr><tr><td align=\"left\"> No</td><td align=\"left\">33(21.9)</td><td align=\"left\">118(78.1)</td><td align=\"left\">1.880(1.047–3.374)</td><td align=\"left\">0.034</td><td align=\"left\">3.404(1.563–7.416)</td><td align=\"left\">0.002</td></tr><tr><td align=\"left\">\n<bold>WHO Clinical Stages</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Stage 1</td><td align=\"left\">49(40.2)</td><td align=\"left\">73(59.8)</td><td align=\"left\">1</td><td align=\"left\"/><td align=\"left\">1</td><td align=\"left\"/></tr><tr><td align=\"left\"> Stage 2</td><td align=\"left\">16(23.5)</td><td align=\"left\">52(76.5)</td><td align=\"left\">2.222(1.088–4.538)</td><td align=\"left\">0.028</td><td align=\"left\">2.536(1.005–6.395)</td><td align=\"left\">0.049</td></tr><tr><td align=\"left\"> Stage 3/4</td><td align=\"left\">10(11.5)</td><td align=\"left\">77(88.5)</td><td align=\"left\">6.556(2.752–15.615</td><td align=\"left\">&lt; 0.001</td><td align=\"left\">8.459(5.820-10.544</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\"> Age</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">0.995(0.869–1.050)</td><td align=\"left\">0.341</td><td align=\"left\">-</td><td align=\"left\">-</td></tr></tbody></table></table-wrap>" ]
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[{"label": ["1."], "mixed-citation": ["Joint United Nations Programme on HIV/AIDS (UNAIDS). 2021. UNAIDS Data 2021. UNAIDS. [Internet]. Available from: "], "ext-link": ["https://www.unaids.org/sites/default/files/media_asset/JC3032_AIDS_Data_book_2021_En.pdf"]}, {"label": ["2."], "mixed-citation": ["Bain R, Beise J, Benali N, Cappa C, Carvajal-Aguirre L, Diallo M et al. Progress for Every Child in the SDG Era. 2018; Available from: www.data.unicef.org."]}, {"label": ["3."], "mixed-citation": ["WHO. Guidance for HIV testing and counselling and care for adolescents living with HIV: Recommendations for a public health approach and considerations for policy-makers and managers. 2013; Available from: "], "ext-link": ["https://apps.who.int/iris/handle/10665/94334"]}, {"label": ["4."], "mixed-citation": ["United Nations Programme on HIV/aids. UNAIDS. UNAIDS data 2021. 2021;4\u201338."]}, {"label": ["5."], "mixed-citation": ["SeyedAlinaghi SA, Mirzapour P, Pashaei Z, Afzalian A, Tantuoyir MM, Salmani R et al. The impacts of COVID-19 pandemic on service delivery and treatment outcomes in people living with HIV: a systematic review. AIDS Res Ther [Internet]. 2023;20(1):1\u201317. 10.1186/s12981-022-00496-7"]}, {"label": ["6."], "surname": ["Nassen", "Donald", "Walker", "Paruk", "Vujovic", "Duncan"], "given-names": ["R", "K", "K", "S", "M", "W"], "article-title": ["Management of mental health disorders and central nervous system sequelae in HIV-positive children and adolescents"], "source": ["South Afr J HIV Med"], "year": ["2014"], "volume": ["15"], "issue": ["3"], "fpage": ["81"], "lpage": ["96"], "pub-id": ["10.4102/sajhivmed.v15i3.7"]}, {"label": ["7."], "mixed-citation": ["Zgambo M, Arabiat D, Ireson D. \u201cWe just do it \u2026 we are dead already\u201d: Exploring the sexual behaviors of youth living with HIV. 2022;(December 2021):34\u201344"]}, {"label": ["8."], "mixed-citation": ["Merikangas KR, Nakamura EF, Kessler RC. Epidemiology of mental disorders in children and adolescents. Dialogues Clin Neurosci [Internet]. 2009;11(1):7\u201320. Available from: 10.1001/jamapediatrics.2013.192"]}, {"label": ["9."], "mixed-citation": ["World Health Organization (WHO). Comprehensive Mental Health Action Plan 2013\u20132030 [Internet]. Available from: "], "ext-link": ["https://www.who.int/publications/i/item/9789240031029"]}, {"label": ["10."], "mixed-citation": ["Brandt R. The mental health of people living with HIV/AIDS in Africa: a systematic review. African J AIDS Res [Internet]. 2009;8(2):123\u201333. 10.2989/AJAR.2009.8.2.1.853"]}, {"label": ["11."], "surname": ["Vreeman", "McCoy", "Lee"], "given-names": ["RC", "BM", "S"], "article-title": ["Mental health challenges among adolescents living with HIV"], "source": ["J Int AIDS Soc"], "year": ["2017"], "volume": ["20"], "issue": ["Suppl 3"], "fpage": ["100"], "lpage": ["9"]}, {"label": ["13."], "mixed-citation": ["Betancourt T, Scorza P, Kanyanganzi F, Smith Fawzi MC, Sezibera V, Cyamatare F et al. HIV and child mental health: a case-control study in Rwanda. Pediatrics. 2014;134(2)."]}, {"label": ["14."], "surname": ["Mellins", "Xu", "Nestadt", "Knox", "Kauchali", "Arpadi"], "given-names": ["CA", "Q", "DF", "J", "S", "S"], "article-title": ["Screening for Mental Health among Young South African children: the Use of the strengths and difficulties Questionnaire (SDQ)"], "source": ["Glob Soc Welf"], "year": ["2019"], "volume": ["5"], "issue": ["1"], "fpage": ["29"], "lpage": ["38"], "pub-id": ["10.1007/s40609-018-0110-3"]}, {"label": ["15."], "mixed-citation": ["Bankole KO, Bakare MO, Edet BE, Igwe MN, Ewa AU, Bankole IA et al. Psychological complications associated with HIV/AIDS infection among children in South-South Nigeria, sub-Saharan Africa. Cogent Med [Internet]. 2017;4(1):1372869. 10.1080/2331205X.2017.1372869"]}, {"label": ["17."], "mixed-citation": ["Paramesparan Y, Garvey LJ, Ashby J, Foster CJ, Fidler S, Winston A. High rates of asymptomatic neurocognitive impairment in vertically acquired HIV-1-infected adolescents surviving to adulthood. Vol.\u00a055, Journal of acquired immune deficiency syndromes (1999). United States; 2010. p.\u00a0134\u20136."]}, {"label": ["21."], "surname": ["Lyambai", "Mwape"], "given-names": ["K", "L"], "article-title": ["Mental Health problems experienced by HIV positive adolescents; a case of Choma District, Zambia"], "source": ["Open J Psychiatry"], "year": ["2018"], "volume": ["08"], "issue": ["02"], "fpage": ["97"], "lpage": ["114"], "pub-id": ["10.4236/ojpsych.2018.82009"]}, {"label": ["23."], "mixed-citation": ["Baylor College of Medicine International Pediatric AIDS Initiative (BIPAI). Baylor College of Medicine International Pediatric AIDS Initiative-Malawi Teen Club Curriculum Part 1: Content A Resource for Groups working with Adolescents Living with HIV. 2012;(February):1\u201376. Available from: "], "ext-link": ["https://toolkits.knowledgesuccess.org/toolkits/alhiv/bipai-malawi-teen-club-life-skills-curriculum"]}, {"label": ["24."], "mixed-citation": ["Management Sciences for Health. District Health System Strengthening and quality improvement for service delivery retain: Teen clubs help retain adolescents in HIV care and treatment. Mpawa H, Mchacha INC, Ngwalo C, Betha R, Birse S, Konings E, editor. [Internet]. Malawi: Management Sciences for Health. 2018. Available from: "], "ext-link": ["https://msh.org/wp-content/uploads/2018/02/cdc_-_teen_club_brief_revised.pdf"]}, {"label": ["25."], "mixed-citation": ["Mackenzie RK, van Lettow M, Gondwe C, Nyirongo J, Singano V, Banda V et al. Greater retention in care among adolescents on antiretroviral treatment accessing Teen Club an adolescent-centred differentiated care model compared with standard of care: a nested case\u2013control study at a tertiary referral hospital in Malawi. J Int AIDS Soc. 2017;20(3)."]}, {"label": ["26."], "surname": ["van Wyk", "Davids"], "given-names": ["BE", "LAC"], "article-title": ["Challenges to HIV treatment adherence amongst adolescents in a low socio-economic setting in Cape Town"], "source": ["South Afr J HIV Med"], "year": ["2019"], "volume": ["20"], "issue": ["1"], "fpage": ["1"], "lpage": ["7"]}, {"label": ["27."], "mixed-citation": ["Alibi M, Mwapasa V, Gwalangwa F. Retrospective cohort study comparing antiretroviral treatment outcomes among adolescents in Teen Clubs and Standard Care Clinics: Blantyre, Malawi. Res Sq [Internet]. 2023;1\u201315. 10.21203/rs.3.rs-2337996/v1%0ALicense"]}, {"label": ["29."], "mixed-citation": ["Raosoft I. (2020). "], "italic": ["Sample size calculator by Raosoft"], "ext-link": ["http://www.raosoft.com/samplesize.html"]}, {"label": ["30."], "surname": ["Goodman", "Meltzer", "Bailey"], "given-names": ["R", "H", "V"], "article-title": ["The strengths and difficulties Questionnaire: a pilot study on the validity of the self-report version"], "source": ["Eur Child Adolesc Pyschiatry"], "year": ["1998"], "volume": ["7"], "issue": ["3"], "fpage": ["125"], "lpage": ["30"], "pub-id": ["10.1007/s007870050057"]}, {"label": ["31."], "mixed-citation": ["Hoosen N, Davids EL, de Vries PJ, Shung-King M. The Strengths and Difficulties Questionnaire (SDQ) in Africa: A scoping review of its application and validation. Child Adolesc Psychiatry Ment Health [Internet]. 2018;12(1):1\u201339. 10.1186/s13034-017-0212-1"]}, {"label": ["32."], "mixed-citation": ["Strengths and Difficulties Questionnaire. : Chichewa Version. Available from: "], "ext-link": ["https://sdqinfo.org/py/sdqinfo/b3.py?language=Chichewa"]}, {"label": ["33."], "mixed-citation": ["Amankwah-Poku M, Klutsey DA, Asante KO. Disclosure and health-related outcomes among children living with HIV and their caregivers. AIDS Res Ther [Internet]. 2021;18(1):1\u20138. 10.1186/s12981-021-00337-z"]}, {"label": ["34."], "mixed-citation": ["Malawi Ministry of Health. Clinical Management of HIV in Children and Adults. 2018 [Internet]. Available from: "], "ext-link": ["https://differentiatedservicedelivery.org/wp-content/uploads/malawi-clinical-hiv-guidelines-2018-1.pdf"]}, {"label": ["36."], "mixed-citation": ["Vreeman RC, Scanlon ML, Marete I, Mwangi A, Inui TS, McAteer CI et al. Characteristics of HIV-infected adolescents enrolled in a disclosure intervention trial in western Kenya. AIDS Care - Psychol Socio-Medical Asp AIDS/HIV [Internet]. 2015;27(00):6\u201317. 10.1080/09540121.2015.1026307"]}, {"label": ["37."], "mixed-citation": ["Abegaz BF, Walle TA, Tilahun AD. HIV positive status disclosure and associated factor among HIV infected children in pediatric ART clinics in Gondar town public health facilities, North West Ethiopia, 2018. J Infect Public Health [Internet]. 2019;12(6):873\u20137. 10.1016/j.jiph.2019.05.018"]}, {"label": ["38."], "surname": ["Kim", "Ecoff", "Brown", "Gallo", "Stichler"], "given-names": ["SC", "RNL", "NCE", "CNSA", "RJF"], "article-title": ["Benefits of a Regional evidence-based Practice Fellowship Program: a test of the ARCC Model"], "source": ["Worldviews Evidence-Based Nurs"], "year": ["2017"], "volume": ["14"], "issue": ["2"], "fpage": ["90"], "lpage": ["8"], "pub-id": ["10.1111/wvn.12199"]}, {"label": ["39."], "surname": ["Menon", "Glazebrook", "Ngoma"], "given-names": ["A", "C", "MS"], "article-title": ["Mental Health of HIV positive adolescents in Zambia"], "source": ["Med J Zambia [Internet]"], "year": ["2009"], "volume": ["36"], "issue": ["4"], "fpage": ["151"], "lpage": ["6"]}, {"label": ["40."], "surname": ["Melvin", "Krechevsky", "Divac", "Tacconelli", "Miah", "Waugh"], "given-names": ["D", "D", "A", "E", "J", "S"], "article-title": ["Parental reports of emotional and behavioural difficulties on the SDQ for school-age children with vertically acquired HIV Infection living in London"], "source": ["Psychol Heal Med"], "year": ["2007"], "volume": ["12"], "issue": ["1"], "fpage": ["40"], "lpage": ["7"], "pub-id": ["10.1080/13548500600731708"]}, {"label": ["41."], "mixed-citation": ["Van Roy B, Groholt B, Heyerdahl S, Clench-Aas J. Understanding discrepancies in parent-child reporting of emotional and behavioural problems: effects of relational and socio-demographic factors. BMC Psychiatry. 2010;10."]}, {"label": ["43."], "surname": ["Mendoza", "Hernandez-Reif", "Castillo", "Burgos", "Zhang", "Shor-Posner"], "given-names": ["R", "M", "R", "N", "G", "G"], "article-title": ["Behavioural symptoms of children with HIV Infection living in the Dominican Republic"], "source": ["West Indian Med J"], "year": ["2007"], "volume": ["56"], "issue": ["1"], "fpage": ["53"], "lpage": ["9"], "pub-id": ["10.1590/S0043-31442007000100010"]}, {"label": ["45."], "mixed-citation": ["Ruise\u00f1or-Escudero H, Familiar I, Nakasujja N, Bangirana P, Opoka R, Giordani B et al. Immunological correlates of behavioral problems in school-aged children living with HIV in Kayunga, Uganda. Glob Ment Heal. 2015;2."]}, {"label": ["46."], "mixed-citation": ["Olashore AA, Paruk S, Akanni OO, Chiliza B. Psychiatric disorders in adolescents living with HIV in Botswana. AIDS Res Ther [Internet]. 2023;20(1):1\u201310. 10.1186/s12981-022-00490-z"]}, {"label": ["47."], "mixed-citation": ["Rosenstein LD. ADHD as a Potential Risk Factor in Poor Antiretroviral Adherence Rates in HIV: A Brief Narrative Review and Suggestions for Future Research. Neuropsychol Rev [Internet]. 2021;31(4):683\u20138. 10.1007/s11065-021-09483-7"]}, {"label": ["48."], "mixed-citation": ["Joshi D, Tiwari MK, Kannan V, Dalal SS, Mathai SS. Emotional and behavioral disturbances in school going HIV positive children attending HIV clinic. Med J Armed Forces India [Internet]. 2017;73(1):18\u201322. 10.1016/j.mjafi.2016.12.002"]}, {"label": ["49."], "mixed-citation": ["Kim MH, Mazenga AC, Yu X, Devandra A, Nguyen C, Ahmed S et al. Factors associated with depression among adolescents living with HIV in Malawi. BMC Psychiatry [Internet]. 2015;15(1):1\u201312. 10.1186/s12888-015-0649-9"]}, {"label": ["50."], "mixed-citation": ["Cortina MA, Fazel M, Hlungwani TM, Kahn K, Tollman S, Cortina-Borja M et al. Childhood psychological problems in School settings in Rural Southern Africa. PLoS ONE. 2013;8(6)."]}, {"label": ["51."], "mixed-citation": ["Cluver L, Orkin M, Boyes ME, Sherr L, Makasi D, Nikelo J. Pathways from parental AIDS to child psychologicducational and sexual risk: Developing an empirically-based interactive theoretical model. Soc Sci Med [Internet]. 2013;87:185\u201393. 10.1016/j.socscimed.2013.03.028"]}, {"label": ["53."], "surname": ["Lyon", "Kimmel", "Cheng", "Wang", "Medicine"], "given-names": ["ME", "AL", "YI", "J", "YA"], "article-title": ["Of Life among adolescents with HIV: A Latent Profile Analysis"], "source": ["J Reli Heal"], "year": ["2017"], "volume": ["55"], "issue": ["5"], "fpage": ["1688"], "lpage": ["99"], "pub-id": ["10.1007/s10943-016-0238-3"]}, {"label": ["56."], "mixed-citation": ["Hall CL, Guo B, Valentine AZ, Groom MJ, Daley D, Sayal K et al. The validity of the strengths and difficulties Questionnaire (SDQ) for children with ADHD symptoms. PLoS ONE. 2019;14(6)."]}]
{ "acronym": [], "definition": [] }
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2024-01-14 23:43:47
BMC Pediatr. 2024 Jan 13; 24:41
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38216909
[ "<title>Introduction</title>", "<p id=\"Par2\">Peanuts are extensively cultivated in over 100 countries and are cherished by consumers worldwide [##REF##31131506##1##]. In 2020, global peanut cultivation covered 32 million hectares, yielding approximately 54 million metric tons, with China contributing more than 18 million metric tons [##REF##36172561##2##].Recognized as a vital cash and oil crop, peanuts contain a substantial oil content in their kernels (40-60%). Moreover, they serve as a staple in the food industry, utilized for butter, soup thickening, nuts, chocolate beans, and sprouts, owing to their rich protein content (20-40%), carbohydrates (10-20%), and bioactive compounds such as flavonoids, vitamins, resveratrol, and anthocyanins [##REF##34829585##3##–##UREF##0##5##].</p>", "<p id=\"Par3\">Nitrogen (N) stands out as a crucial nutrient and a limiting factor in agricultural systems worldwide [##UREF##1##6##]. N fertilizer application is a common practice to sustain crop productivity, supporting the rapid global population growth [##UREF##2##7##], however, excessive use can reduce N utilization efficiency, leading to environmental issues like soil degradation and water contamination [##UREF##3##8##].</p>", "<p id=\"Par4\">As a legume crop, peanuts acquire nitrogen from both soil through their roots and atmospheric nitrogen fixation via nodules. Nitrogen fixation in peanuts can contribute over 60% of the total nitrogen requirement in poor soil fertility conditions and around 40% in high-yield cultivation [##UREF##4##9##, ##UREF##5##10##]. Adequate nitrogen fertilizer application becomes essential for optimizing yield [##UREF##6##11##].</p>", "<p id=\"Par5\">Typically, the nitrogen fertilizer management strategy for peanuts involves applying all nitrogen fertilizer as basal fertilizer at planting, providing plant nutrient requirements before nodule formation. However, this approach can lead to excessive vegetative growth in early phases due to nitrogen excess and premature senility in reproductive phases due to nitrogen deficiency [##UREF##7##12##]. In addition, early-stage excess nitrogen inhibits root nodule formation and rhizobium activities, limiting potential symbiotic nitrogen fixation [##UREF##8##13##]. Excessive nitrogen fertilizer application reduces nitrogen-fixation traits by approximately 50% in each peanut growing season, with more significant effects on J11 and Gangapuri (Spanish types) than NC17 and Robut33-1 (Virginia types) [##UREF##9##14##]. Virginia cultivars outperform Spanish types in nodulation, nitrogen fixation, specific activity, growth, pod yield, and harvest index. Nitrogen assimilation and nitrogen use efficiency (NUE) in peanuts increase within a certain nitrogen application range but decrease with excess nitrogen fertilizer application [##UREF##10##15##]. This highlights the necessity for efficient nitrogen transport and assimilation to drive growth and development when enhancing nitrogen acquisition [##UREF##11##16##]. There are notable differences in nitrogen uptake, transport, and assimilation efficiency among different peanut cultivars [##UREF##12##17##]. Previous researches mainly focused on root uptake and nodule fixation, however, nitrogen fertilizer assimilation and application by pods have been scarcely explored.</p>", "<p id=\"Par6\">Peanuts are geocarpic crops, exhibiting the unique trait of pushing the ovary into the soil for pod development after fertilization [##UREF##13##18##]. During pod development, both gynophores and underground pods participate in nutrient uptake. At the pod filling stage, over 90% of Ca and 40% of Zn are directly absorbed by pods from the soil [##UREF##14##19##, ##UREF##15##20##], highlighting the crucial role of sufficient calcium in the pod zone for pod development, yield formation, and nutritional quality [##REF##28769950##21##]. The late podding stage represents a critical period for nitrogen (N) accumulation, where the capacity of root N uptake and nodule N fixation significantly decreases, limiting pod production [##UREF##16##22##]. Previous studies have demonstrated that a substantial portion of N fertilizer applied to the pod zone can be absorbed by the pod, with a notable proportion remaining in the fruit parts [##UREF##17##23##]. Our earlier investigation into peanut pod N uptake, involving soil separation between roots and pods, revealed that the application of 60 kg/hm² of N in the pod zone resulted in the highest pod yield for FH1 (large-seeded type) [##UREF##18##24##].</p>", "<p id=\"Par7\">Effective management and appropriate use of nitrogen fertilizers in the pod zone present a promising strategy to boost peanut productivity and optimize nitrogen utilization efficiency.Peanut cultivars are commonly categorized into large-seeded and small-seeded based on seed size/weight. Large-seeded cultivars generally have a more extended pod filling period, leading to increased nitrogen uptake and accumulation compared to small-seeded ones [##UREF##19##25##]. However, information regarding the responses of pod yield in cultivars with diverse seed sizes to pod zone N fertilizer application is currently lacking, necessitating further investigation. Our study aims to explore the responses of pod yield and nitrogen accumulation in two peanut cultivars with distinct pod sizes to pod zone N fertilizer application. Additionally, we seek to unravel the physiological mechanisms underlying nitrogen absorption. The outcomes of this research will furnish valuable insights for advancing nitrogen precision management in peanut cultivation.</p>" ]
[ "<title>Materials and methods</title>", "<title>Experimental design and treatments</title>", "<p id=\"Par8\">The pot experiments were carried out at the Field Crop Research Station of Shandong Academy of Agriculture Sciences, Jinan, China. The soil type was a silty loam, with organic matter 13.7 g kg<sup>− 1</sup>, alkali-hydrolyzable nitrogen 80.2 mg kg<sup>− 1</sup>, available phosphorus 42.2 mg kg<sup>− 1</sup>, available kalium 238.5 mg kg<sup>− 1</sup>, exchangeable calcium 5.2 g kg<sup>− 1</sup>, in 0 ~ 20 cm tillage layer, and with pH 6.5.</p>", "<p id=\"Par9\">Cylindrical pots with the height of 35 cm and diameter of 40 cm were used. Each pot was filled with air-dried soil from the 0 ~ 20 cm tillage layer, resulting in a soil weight of 25 kg per pot.Nitrogen fertilizer as urea at the rate of 1.51 g N pot<sup>− 1</sup>, phosphorus fertilizer in the form of triple superphosphate at the rate of 1.13 g P<sub>2</sub>O<sub>5</sub> pot<sup>− 1</sup> and potassium fertilizer in the form of potassium sulphateat 1.88 g K<sub>2</sub>O pot<sup>− 1</sup> were applied. Then water was supplied to saturation for each pot before planting.</p>", "<p id=\"Par10\">Two peanut cultivars, SH11 (LS) and HY23 (SS), were used in this study (Fig. ##FIG##0##1##). Three plump seeds were sown in each pot, and two uniform seedlings were retained per pot at seven days after germination. Before flowering, designed plastic boxes were placed into the pots, filled with 4 kg of clean river sand, serving to separate the pod zone from the rooting zone (Fig. ##FIG##0##1##). This setup allowed for the growth of peanut pods and roots in different nutritional environments. The experiment utilized a split-plot design, with peanut cultivars as the main plots and nitrogen treatments in the pod zone as the sub-plots. Before the gynophore penetrated into the soil, nitrogen fertilizer (urea) was applied in the pod zone at rates of 0 g (control), 0.38 g, 0.75 g, and 1.13 g N per pot, corresponding to 0, 30, 60, and 90 kg·hm-2, respectively. Nitrogen-free nutrient solution was applied to the pod zone along with fertilization, following the methods described by Inanaga et al. [##UREF##17##23##]. Each treatment consisted of ten replicate pots. The trials were sown on May 15th, 2020, and the plants were harvested on September 20th, 2020.</p>", "<p id=\"Par11\">For gene expression analysis and enzyme activity study, pod samples were collected at 10, 20, and 30 days after gynophores penetrated into the soil (S1, S2, and S3), frozen with liquid nitrogen, and stored at -80 °C. Plant samples were taken at 20 days after gynophores penetrated soil (20DAP), 40 DAP, and 60 DAP, representing three key stages of pod development: pod expanding, rapid filling, and harvest stages, respectively. The plants were divided into root, stem, leaf, shell, seed, and gynophore. All samples were washed, fixed, dried at 105 °C for 0.5 h, and then dried at 80 °C to constant weight in a drying oven. Dry weight and total nitrogen in each part were determined.</p>", "<p id=\"Par12\">\n\n</p>", "<title>Determination of nitrogen content</title>", "<p id=\"Par13\">The dried samples were finely ground using a plant grinder, and the nitrogen content was subsequently assessed using the Kjeltec 2300 automatic nitrogen determination instrument.</p>", "<title>Determination of protein and total amino acids content</title>", "<p id=\"Par14\">The protein and total amino acid contents were analyzed using near-infrared spectroscopy (DA7250, Perten, Stockholm, Sweden). The instrument, equipped with data acquisition and analysis software, allows for the creation of standard curve models to analyze four quality indexes, including crude fat, protein, fatty acids, and amino acids. Plump and intact peanut kernels were placed in a spinning ring cup with a 7.5 diameter, ensuring a flat surface. To mitigate the impact of kernel size, each sample underwent two scans, involving emptying and refilling the ring cup before the second scan.</p>", "<title>Determination of NR enzyme activity</title>", "<p id=\"Par15\">NR activity was assessed following the method of Khator and Shekhawat [##UREF##20##26##], with minor adjustments. Fresh samples (0.5 g) were crushed using a pre-cooled mortar in 4 mL of 50 mM potassium phosphate buffer (pH 8.0), which contained 25 mM cysteine, 1 mM EDTA, and 3% (w/v) BSA at 0 ~ 4℃. The homogenate was then centrifuged at 12,000×g for 20 min at 4 °C, and the resulting supernatant was utilized for NR activity analysis. The assay mixture, composed of 0.4 mL enzyme solution, 1.2 mL potassium phosphate buffer (0.1 M), 0.2 mL potassium nitrate (50 mM), and 0.2 mL NADH (3 mM), was incubated for 30 min at 25 °C. Following incubation, 1 mL sulphanilamide (58 mM) and 1 mL α-naphthylamine (14 mM) were added to the reaction mixture and incubated for an additional 15 min. The solution was then centrifuged at 4000×g for 5 min, and the supernatant was collected for absorbance measurement at 540 nm. A blank assay mixture without NADH was included for reference.</p>", "<title>Determination of GS and GOGAT enzyme activities</title>", "<p id=\"Par16\">Fresh samples (0.5 g) were homogenized in a pre-chilled mortar with 5 mL Tris–HCl buffer (pH 8.0) containing 50 mM Tris-HCl, 10 mM MgSO<sub>4</sub>, 10 mM EDTA, and 10 mM cysteine. Extracts were centrifuged at 12,000×g for 15 min at 4 ℃, and the resulting supernatants were utilized for the assay of GS and GOGAT.</p>", "<p id=\"Par17\">GS activity was determined following the method of O’Neal and Joy [##REF##4150338##27##]. The reaction mixture consisted of 1 ml reaction system (50 mM Tris-HCl, 20 mM MgSO<sub>4</sub>, 5 mM NH<sub>2</sub>OH, 8 mM ATP, 1 mM EDTA, 80 mM α-glutamate) and 0.5 ml enzyme solution. The mixture was incubated at 35 ºC for 30 min and terminated by adding 0.5 mL FeCl<sub>3</sub>. After centrifugation at 5000×g for 15 min, the absorbance was recorded at 540 nm using a UV-spectrophotometer. GS activity was calculated based on γ-glutamyl hydroxamate, with one unit of enzyme activity defined as the production of 1 µmol γ-glutamyl hydroxamic acid per mg of fresh tissue per hour per mL of the reaction system.</p>", "<p id=\"Par18\">For GOGAT activity, 0.3 mL enzyme solution was added to 1.2 mL reaction solution consisting of 0.1 M potassium phosphate (0.1 mL), 2 mM α-oxoglutarate (0.5 mL), 0.2 mM NADH (0.2 mL), and 10 mM L-glutamine (0.4 mL). The mixture was incubated at 30 ºC for 30 min and terminated by a boiling water bath for 30 s. The absorbance was immediately read at 340 nm. The reaction solution without L-glutamine served as a control, with one unit of GOGAT activity defined as the decrease of 1 µmol NADH per min.</p>", "<title>Expression analysis of genes for nitrogen uptake and metabolism in pod</title>", "<p id=\"Par19\">Total RNA extraction was conducted using the Trizol Reagent kit (TaKaRa, Inc., Dalian, China) following the manufacturer’s instructions. Three biological replications were employed, and RNA samples underwent DNase I treatment to eliminate genomic DNA contamination. Agilent 2100 and NanoDrop were utilized to assess RNA quality and purity. Total RNA from each sample was employed for mRNA enrichment with Oligo (Dt) and subsequent cleavage into short fragments (~ 200 nt) in a fragmentation buffer. Reverse transcription, using a random hexamer primer to obtain the first-strand cDNA, and synthesis of the second strand of cDNA using buffer, dNTPs, RNaseH, and DNA polymerase were performed. After end repair and the addition of sequencing adaptors, the cDNA fragments underwent PCR amplification with gene-specific primers. The gene-specific primers, designed using Perl Primer software, are listed in Table ##TAB##0##1##. qRT-PCR was executed using the SYBR Green Pro Taq HS premixed qPCR kit in the 7500 Real-Time PCR System. The thermal cycle parameters were 94 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min in a 20 mL volume. Three biological replications were conducted for each reaction, with the peanut actin gene serving as an internal reference. The relative gene expression level was calculated using the 2<sup>−ΔΔCT</sup> method.</p>", "<p id=\"Par20\">\n\n</p>", "<title>Statistical analysis</title>", "<p id=\"Par21\">All parameters were measured in a minimum of three replications and are presented as means ± standard deviation. The average for each trait was computed using Microsoft Excel 2010 and visualized with Sigmaplot 10.0. Duncan’s multiple range test, facilitated by SPSS Statistics 23, was employed to identify significant differences between treatments (<italic>P</italic> &lt; 0.05). Correlation analysis was conducted using OmicShare tools for data analysis.</p>" ]
[ "<title>Results</title>", "<title>Application of nitrogen in pod zone increased dry matter accumulation and yield</title>", "<p id=\"Par22\">To investigate the impact of nitrogen on peanut yield, a specialized container was devised to segregate the peanut’s underground portions into two zones: the pod zone and the root zone (Fig. ##FIG##0##1##). Varied concentrations of nitrogen fertilizer were applied to the pod zone of two peanut cultivars. Our findings revealed that nitrogen application enhanced dry matter accumulation throughout the pod development stage for both peanut cultivars, with a more pronounced positive effect on SH11 compared to HY23 (Table ##TAB##1##2##). In comparison to the control, dry matter accumulation per plant increased by 5.93–9.44% for SH11 and 4.22–7.65% for HY23 at 60 DAP. Notably, the application of 90 kg·hm<sup>− 2</sup> and 60 kg·hm<sup>− 2</sup> resulted in the maximum dry matter accumulation for SH11 and HY23, respectively. Both cultivars exhibited a significant increase in pod yield under pod zone nitrogen application compared to the control (Table ##TAB##1##2##). The pod yield of SH11 and HY23 increased by 8.11–11.30% and 4.68–8.76%, respectively, in comparison to the control. The 60 kg·hm<sup>− 2</sup> treatment yielded the maximum pod yield for both SH11 and HY23 (Table ##TAB##1##2##).</p>", "<p id=\"Par23\">\n\n</p>", "<title>Application of nitrogen in pod zone affected the nitrogen contentof whole plants</title>", "<p id=\"Par24\">We observed a significant rise in nitrogen content for entire plants with the application of nitrogen in the pod zone (Table ##TAB##2##3##). Nitrogen accumulation consistently increased during the pod filling process in SH11, while remaining relatively stable in HY23, compared to the control. The surge in nitrogen accumulation for HY23 (7.29%~16.58%) was slightly higher than that for SH11 (8.79%~12.14%) at 20 DAP. Notably, a more substantial increase in nitrogen accumulation was noted in SH11 compared to HY23 at 60 DAP, with an increase of 13.30%~27.83% in SH11, while HY23 experienced a rise of 8.05%~15.64%. The maximum increases in nitrogen accumulation for SH11 and HY23 were observed with treatments of 90 kg·hm<sup>− 2</sup> and 60 kg·hm<sup>− 2</sup>, respectively.</p>", "<p id=\"Par25\">\n\n</p>", "<title>Application of nitrogen in pod zone increased the protein content of seeds</title>", "<p id=\"Par26\">Protein and total amino acid content are crucial quality indicators for peanuts. Our observations revealed a consistent increase in protein and total amino acid content in the kernels of both cultivars with the application of nitrogen in the pod zone, ranging from 0 to 90 kg·hm<sup>− 2</sup> (Fig. ##FIG##1##2##). In comparison to the control, SH11 exhibited an increase of 10.43%~17.56% in protein content and 12.41%~21.51% in total amino acids content. The patterns of increase in these two nutritional components in HY23 were essentially parallel to those in SH11.</p>", "<p id=\"Par27\">\n\n</p>", "<title>Application of nitrogen in pod zone affected the activities of NR, GS, GOGAT</title>", "<p id=\"Par28\">With pod development, NR activity exhibited a declining trend in both the control and nitrogen-applied treatments (Fig. ##FIG##2##3##-A, D). In comparison to the control, pod zone nitrogen application led to a significant increase in NR activity at all three stages, with higher levels observed in SH11 than in HY23. At S1, NR activities increased by 12.34%~46.99% in SH11 and 23.20%~38.83% in HY23; at S2, the increases were 16.34%~22.24% in SH11 and 8.28%~14.52% in HY23; at S3, the increases were 18.54%~36.40% in SH11 and 8.64%~24.23% in HY23, with the maximum increases observed at the 60 kg·hm<sup>-2</sup> treatment in both cultivars (Fig. ##FIG##2##3##-A, D).</p>", "<p id=\"Par29\">For GS activity, it was significant induced by application of nitrogen in pod zone for both cultivars (Fig. ##FIG##2##3##-B, E). At S1, the highest increase in GS activity was observed in SH11 (35.06%) and HY23 (52.38%) under the 60 kg·hm<sup>− 2</sup> and 30 kg·hm<sup>− 2</sup> treatments. At S2, GS activities in both cultivars gradually increased with the rising application of nitrogen in the pod zone. At S3, GS activities in the 60 kg·hm<sup>− 2</sup> treatment were significantly higher than in others for both cultivars, increasing by 49.94% in SH11 and 38.64% in HY23 compared to the control.</p>", "<p id=\"Par30\">The changing patterns of GOGAT activity were similar to that of the GS activity (except for the values at S1 in HY23) (Fig. ##FIG##2##3##-C, F). Compared to the control, the maximum increases in GOGAT activity in SH11 (76.02%~133.27%) were observed at S3, while in HY23 (24.62%~106.59%), they occurred at S2.</p>", "<title>Relative gene expression levels of <italic>NR</italic>,<italic>NIR</italic>,<italic>ABC</italic>,<italic>NRT2</italic>,<italic>GS</italic> and <italic>NADH-GOGAT</italic></title>", "<p id=\"Par31\">The qRT-PCR method was employed to assess the expression of genes related to nitrogen metabolism. In general, the application of nitrogen in the pod zone induced the expression of genes involved in nitrogen metabolism, particularly at S2 and S3 (Fig. ##FIG##3##4##). The relative expression levels of <italic>NR</italic>, <italic>NIR</italic>, <italic>ABC</italic>, <italic>NRT2</italic>, <italic>GS</italic>, and <italic>NADH-GOGAT</italic> in SH11 were up-regulated at S1 under pod zone nitrogen application compared to the control. However, they exhibited slight down-regulation at the 90 kg·hm<sup>− 2</sup> treatment. Similar results were observed in HY23 at the same stage. The increasing trends in the relative expressions of the six genes were consistently observed with the rising nitrogen application at S2, showing up-regulation of more than 10 folds in both cultivars compared to the control at the 90 kg·hm<sup>− 2</sup> treatment. By S3, the expression of the six genes continued to be up-regulated in SH11. However, in HY23, there was an extreme decrease compared to those at S2, especially without the 60 kg·hm<sup>− 2</sup> treatment. At S3, the maximum up-regulations of the relative expressions of the six genes were observed with the 60 kg·hm<sup>− 2</sup> pod zone nitrogen application in both cultivars, all up-regulated 27 folds in SH11 and 7.99 ~ 11.35 folds in HY23. The 90 kg·hm<sup>− 2</sup> treatment up-regulated gene expression by 14.26 ~ 23.30 folds in SH11; however, their expression level decreased in HY23 compared to the control.</p>", "<p id=\"Par32\">\n\n</p>", "<title>Correlation analysis</title>", "<p id=\"Par33\">To further assess the impact of nitrogen application in the pod zone on yield, we conducted correlation analysis between pod yield and nitrogen metabolism traits, encompassing the enzyme activities of <italic>NR</italic>, <italic>GS</italic>, <italic>GOGAT</italic>, and gene expression (Fig. ##FIG##4##5##-A). A significant positive correlation (<italic>P</italic> &lt; 0.05) was identified between nitrogen metabolism traits and pod yield in SH11 at S2 and S3, and in HY23 at S3 only. The correlation coefficients between nitrogen metabolism traits and pod yield were highest at S3 for both cultivars. However, there was no significant correlation between nitrogen metabolism traits and plant nitrogen accumulation under pod zone nitrogen application conditions in the two cultivars at S1 (Fig. ##FIG##4##5##-B). Remarkably, nitrogen metabolism traits exhibited a robust positive correlation (<italic>P</italic> &lt; 0.01) with plant nitrogen accumulation in SH11 at S2 and S3, while this correlation was observed in HY23 at S2 only. Pod zone nitrogen applications appeared to stimulate nitrogen uptake and transformation in pods, potentially contributing to the elevated plant nitrogen accumulation and pod yield.</p>", "<p id=\"Par34\">\n\n</p>", "<p id=\"Par35\">\n\n</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par36\">As the fourth-largest edible oil and the third most important vegetable protein globally [##UREF##21##28##], peanuts are nutrient-intensive crops. The supply of nitrogen (N) fertilizer and its balance with plant development requirements are key factors in peanut production [##UREF##22##29##]. The accumulation of dry matter and pod yield in peanuts is closely linked to total N accumulation, displaying a quadratic relationship with N application rates under N deficiency conditions [##UREF##23##30##]. However, excessive N application can significantly reduce yields and dry matter [##UREF##24##31##]. Achieving a 50% N split ratio at flowering-pegging or pod-filling stages has been shown to significantly enhance peanut production and pod yield, ensuring sufficient N for reproductive growth [##UREF##16##22##, ##UREF##25##32##]. Despite symbiotic N fixation, there is still insufficient N to meet the demands of plant growth and development after flowering. Previous research has indicated that pod zone N application increases peanut yield, particularly addressing root zone N deficiency [##UREF##18##24##]. Our study further confirms that applying N in the pod zone significantly increases pod yield, with the 60 kg·hm<sup>− 2</sup> treatment achieving maximum yield. Excessive N application might lead to an imbalance in the C/N ratio, promoting vegetative development [##UREF##26##33##]. Larger-seeded cultivar SH11 exhibited higher pod yield and a more substantial increase than the smaller-seeded cultivar HY23 under the same pod zone N application conditions. This suggests that larger-seeded peanut cultivars are more sensitive to N, maintaining higher yields and N accumulation than smaller-seeded varieties [##UREF##9##14##, ##UREF##19##25##]. Our findings align with those in rice, where N application had a more pronounced effect on high-yield hybrid rice cultivars than on straight cultivars [##UREF##27##34##].</p>", "<p id=\"Par37\">Peanut shells can absorb mineral elements from the soil and translocate them to vegetative tissues [##UREF##14##19##, ##UREF##15##20##]. When N and calcium (Ca) are deficient in the fruiting zone, their contents in the shell and seed decrease [##REF##28769950##21##, ##UREF##28##35##]. More than 10% of total N in peanut seeds is absorbed by the pod [##UREF##18##24##]. However, the behavior of N absorbed through the pod in different peanut cultivars is not well understood. In our study, SH11 showed higher N accumulation than HY23 throughout pod filling and had more N uptake compared to HY23 under the same pod zone N application conditions, except during the first 20 days. N accumulation reached a maximum in SH11 and HY23 at 90 kg·hm<sup>− 2</sup> and 60 kg·hm<sup>− 2</sup> pod zone N application, respectively. This may be attributed to the smaller-seeded peanut cultivar having a shorter pod-filling period than the larger-seeded cultivar, resulting in lower yield [##UREF##29##36##]. Similar results were found in cotton, where large-seeded cultivars were more sensitive in response to N than smaller-seeded cultivars [##UREF##30##37##].</p>", "<p id=\"Par38\">Nitrate reductase (NR) is a rate-limiting enzyme in the pathway of N absorption and utilization in plants, accelerating N metabolism activity and protein synthesis [##REF##37625793##38##]. Glutamine synthetase (GS) plays a major role in fixing ammonium to form the amino acid glutamine, while glutamate synthase (GOGAT) catalyzes the conversion of glutamine to glutamate, providing glutamate for ammonium assimilation. The activities of GS/GOGAT directly affect the efficiency of N assimilation and play a key role in N metabolism [##UREF##31##39##, ##UREF##32##40##]. The activities of NR, NiR, GS, and GOGAT significantly decrease in N deficiency but rapidly recover with supplemental ammonium nitrate.Moderate N application notably promotes yield and enzymatic activities related to N metabolism throughout the growing period in most crops [##UREF##33##41##, ##UREF##34##42##]. Excessive N application has adverse effects on NR, GS, and GOGAT activities, leading to reductions in N use efficiency [##UREF##35##43##, ##REF##30897177##44##]. The activities of NR, GS, GOGAT in the flag leaf were proposed as candidate indicators for rice N accumulation and yield [##UREF##35##43##]. The Virginia type (large-seeded) peanut cultivar exhibited higher NR activity and N fixation levels than the Spanish type (small-seeded) [##UREF##36##45##]. In our current study, the increase in pod NR activity was greater in SH11 than in HY23 under pod zone N application compared to control, reaching a maximum at the 60 kg·hm<sup>− 2</sup> treatment in both cultivars. Pod GS activity in SH11 showed less increase than in HY23 at S1 and S2 compared to control but increased more at S3. The maximum increase in pod GS activities in both cultivars was found under the highest pod zone N application at S2. This might be attributed to the pod development of larger-seeded peanuts being later than that of smaller-seeded peanuts [##UREF##29##36##], requiring more N for pod development [##UREF##19##25##]. These results also suggest that N uptake in the pod is most sensitive at S2. GOGAT activity showed a trend similar to GS activity but exhibited an observable decrease in HY23 at S1 compared to control. Our findings are consistent with a field N trial in peanuts [##UREF##37##46##], where N application enhanced the activities of GS and GOGAT in the root, leaf, and stem, increasing the protein content in the kernel. Larger-seeded peanuts require more N application to maintain high N metabolism activity.</p>", "<p id=\"Par39\">Plant nitrogen (N) nutrition status is regulated by a series of genes associated with N uptake and utilization, with gene expression levels primarily activated by soil N content [##UREF##38##47##]. In spinach roots and leaves, the expression levels of <italic>NR</italic> and <italic>NiR</italic> genes decrease during N deprivation and increase after optimal N replenishment, but further decrease with excessive N supplementation [##UREF##39##48##]. N-efficient rice cultivars exhibit higher transcription levels of <italic>OsNRT </italic>in roots and leaves than N-inefficient ones, accumulating more N under N-deficient conditions [##UREF##40##49##]. N application significantly up-regulates the expression of <italic>NRT</italic>, <italic>NR</italic>, <italic>NiR</italic>, <italic>GS</italic>, and <italic>GOGAT</italic> in rootstock-grafted watermelon compared to self-grafted, enhancing N uptake and utilization [##UREF##41##50##]. In the present research, the expressions of <italic>NR</italic>, <italic>NiR</italic>, <italic>GS</italic>, <italic>NADH-GOGAT</italic>, <italic>ABC</italic>, and <italic>NRT2</italic> genes in the pod were minimally affected by pod zone N application at S1. These results align with transcriptome expressions during different pod development stages in peanuts [##UREF##13##18##]. This lack of impact at S1 may be attributed to the immature pod’s weak absorption capacity, which is not sensitive to N response. Maximum up-regulated expressions of the mentioned genes were observed at S3 in SH11 and at S2 in HY23 compared to the control, under 90 kg·hm<sup>− 2</sup> and 60 kg·hm<sup>− 2</sup> pod zone N application at S2 and S3 in the two cultivars, respectively. SH11 exhibited higher expressions with these genes than HY23, suggesting a longer duration of N-efficient pod absorption in large-seeded peanut cultivars compared to small-seeded cultivars. These findings are consistent with our pod transcriptome analysis under pod zone N application, indicating increased absorption activity at S2 [##UREF##18##24##].</p>", "<p id=\"Par40\">Correlation analysis revealed significant positive correlations between yield and the mentioned enzymatic activities and relative gene expressions in SH11 at S2 and S3, and in HY23 at S3 only. Remarkable positive correlations were observed between N accumulation and the mentioned enzymatic activities and relative gene expressions in SH11 at S2 and S3, and in HY23 at S2 only. This suggests that pod zone N supplementation promotes pod N uptake and yield formation to some extent by activating pod nitrogen metabolism. These results align with previous studies that demonstrated significant positive correlations between yield and transcript levels and activities in <italic>NR</italic>, <italic>NiR</italic>, <italic>GS</italic>, and <italic>GOGAT</italic> in wheat [##UREF##33##41##] and citrus [##REF##30897177##44##]. Over-expression of <italic>NR</italic> and <italic>GS</italic> genes in transgenic wheat [##REF##24040315##51##] and rice [##REF##34814097##52##] significantly enhanced <italic>NR</italic> and <italic>GS</italic> activities in roots and leaves, promoting root capacity to obtain N, increasing grain protein content, yield, and 1000-grain weight compared to the wild type. SH11 exhibited a closer correlation than HY23 between yield and N accumulation in response to pod zone N application at different pod developmental stages, possibly due to the longer pod development period and higher N requirement in large-seeded peanut cultivars compared to small-seeded cultivars [##UREF##29##36##, ##UREF##37##46##].</p>" ]
[ "<title>Conclusions</title>", "<p id=\"Par41\">In summary, the application of nitrogen in the pod zone increased enzyme activities and gene expressions related to nitrogen metabolism in the pod, enhancing yield and nitrogen accumulation in plants. The peanut varieties, SH11 (Var. <italic>hypogaea</italic> type) and HY23 (Var. <italic>vulgaris</italic> type), exhibited distinct responses to nitrogen application in the pod zone. HY23 demonstrated the highest up-regulation in physical traits in response to pod zone N application at S2 compared to the control. On the other hand, SH11 maintained a close sensitivity to pod zone N application at both S2 and S3.The optimal pod zone N application was the 60 kg·hm<sup>− 2</sup> treatment, as indicated by the positive effects on yield and nitrogen accumulation, with a more pronounced impact on the large-seeded cultivar than the small-seeded one. This knowledge will be valuable for the high-efficiency nitrogen management of different peanut cultivars.</p>" ]
[ "<p id=\"Par1\">Cultivated peanut (<italic>Arachis hypogaea</italic> L.) represents one of the most important oil and cash crops world-widely. Unlike many other legumes, peanuts absorb nitrogen through their underground pods. Despite this unique feature, the relationship between yield and nitrogen uptake within the pod zone remains poorly understood. In our pot experiment, we divided the underground peanut part into two zones—pod and root—and investigated the physiological and agronomic traits of two peanut cultivars, SH11 (large seeds, LS) and HY23 (small seeds, SS), at 10 (S1), 20 (S2), and 30 (S3) days after gynophores penetrated the soil, with nitrogen application in the pod zone. Results indicated that nitrogen application increased pod yield, kernel protein content, and nitrogen accumulation in plants. For both LS and SS peanut cultivars, optimal nitrogen content was 60 kg·hm<sup>− 2</sup>, leading to maximum yield. LS cultivar exhibited higher yield and nitrogen accumulation increases than SS cultivar. Nitrogen application up-regulated the expression of nitrogen metabolism-related genes in the pod, including nitrate reductase (<italic>NR</italic>), nitrite reductase (<italic>NIR</italic>), glutamine synthetase (<italic>GS</italic>), glutamate synthase (<italic>NADH-GOGAT</italic>), ATP binding cassette (<italic>ABC</italic>), and nitrate transporter (<italic>NRT2</italic>). Additionally, nitrogen application increased enzyme activity in the pod, including <italic>NR</italic>, <italic>GS</italic>, and <italic>GOGAT</italic>, consistent with gene expression levels. These nitrogen metabolism traits exhibited higher up-regulations in the large-seeded cultivar than in the small-seeded one and showed a significant correlation with yield in the large-seeded cultivar at S2 and S3. Our findings offer a scientific basis for the judicious application and efficient utilization of nitrogen fertilization in peanuts, laying the groundwork for further elucidating the molecular mechanisms of peanut nitrogen utilization.</p>", "<title>Keywords</title>" ]
[]
[ "<title>Acknowledgements</title>", "<p>We appreciate the help provided by research staff on the field experimental station and laboratory.</p>", "<title>Author contributions</title>", "<p>Li GH and Zhao CZ conceived and designed the experiment. Guo X,Li GH and Qu CJ performed the experiments and data collection. Hou L, Tian RZ and Sun W conducted data analysis. Li GH wrote the manuscript. Li GH, Wang GH, Wang XJ, Qu CJ and Zhao CZ revised and edited the manuscript. All authors have read and agreed to the published version of the manuscript.</p>", "<title>Funding</title>", "<p>National Key Research and Development Program (2022YFD1200400), Key Research and Development Project of Shandong Province (2022LZGC022, 2022LZGC007, 2023TZXD078), Agricultural scientific and technological innovation project of Shandong Academy of Agricultural Sciences, Taishan Scholar Project of Shandong Province.</p>", "<title>Data availability</title>", "<p>Data are contained within the article.</p>", "<title>Declarations</title>", "<title>Ethical approval and consent to participate</title>", "<p id=\"Par44\">This research on peanut was approved by Institute of Crop Germplasm Resources (Institute of Biotechnology), Shandong Academy of Agricultural Sciences. The authors confirmed that the experimental research and field studies on plants, including the collection of plant material comply with relevant institutional, national, and international guidelines and legislation.</p>", "<title>Consent for publication</title>", "<p id=\"Par45\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par42\">The authors declare no competing interests.</p>", "<title>Informed consent statement</title>", "<p id=\"Par43\">Not applicable.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>The seeds of two peanut cultivars and schematic diagram of the separation of the rooting zone and the pod zone</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Contents of protein and total amino acids of peanut kernels under different nitrogen applicationin pod zone in SH11 and HY23. Values are shown as means ± SD of three independent biological sample. Different lowercase letters in the bar graph indicate significantly different between different nitrogen application ratesin the same cultivar at <italic>P</italic> &lt; 0.05 according to Duncan’s multiple range test. N0, N30, N60, N90 meaning that nitrogen application ratesin pod zone were 0, 30, 60 and 90 kg·hm<sup>− 2</sup>, respectively</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>The pod enzymatic activities of NR, GS, GOGAT under different nitrogen applicationin pod zone in SH11(<bold>A</bold>, <bold>B</bold>, <bold>C</bold>) and HY23 (<bold>D</bold>, <bold>E</bold>, <bold>F</bold>). Values are shown as means ± SD of three independent biological sample. Different lowercase letters in the bar graph indicate significantly different between different nitrogen application ratesin the same cultivar at <italic>P</italic> &lt; 0.05 according to Duncan’s multiple range test. N0, N30, N60, N90 meaning that nitrogen application ratesin pod zone were 0, 30, 60 and 90 kg·hm<sup>− 2</sup> respectively. S1, S2, S3 represent 10, 20, 30 days after peanut gynophores penetration soil</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Relative genes expressions of <italic>NR</italic>, <italic>NIR</italic>, <italic>GS</italic>, <italic>NADH-GOGAT</italic>, <italic>ABC</italic> and <italic>NRT2</italic> under different nitrogen applicationin pod zone in SH11(<bold>A</bold>, <bold>B</bold>, <bold>C</bold>, <bold>D</bold>, <bold>E</bold>, <bold>F</bold>) and HY23 (<bold>G</bold>, <bold>H</bold>, <bold>I</bold>, <bold>J</bold>, <bold>K</bold>, <bold>L</bold>). Values are shown as means ± SD of three independent biological sample. Different lowercase letters in the bar graph indicate significantly different between different nitrogen application ratesin the same cultivar at <italic>P</italic> &lt; 0.05 according to Duncan’s multiple range test. N0, N30, N60, N90 meaning that nitrogen application ratesin pod zone were 0, 30, 60 and 90 kg·hm<sup>− 2</sup> respectively. S1, S2, S3 represent 10, 20, 30 days after peanut gynophores penetration soil</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>The Heatmap of correlation analysis between key genes and enzyme activities related to nitrogen metabolism in peanut pod and yield per plant (<bold>A</bold>), and nitrogen accumulation per plant (<bold>B</bold>). SH11 and HY23 were two peanut cultivars. S1, S2, S3 represent 10, 20, 30 days after peanut gynophores penetration soil</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Real-time PCR primers information</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Gene name</th><th align=\"left\">Primer Sequence(5’-3’)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\">NR</td><td align=\"left\">F: GAAACGCATCATAGTTACACC</td></tr><tr><td align=\"left\">R: GATCAAATACTCTGGCTTGTACC</td></tr><tr><td align=\"left\" rowspan=\"2\">NiR</td><td align=\"left\">F: CGGAAATCTTGAAAGGTCTG</td></tr><tr><td align=\"left\">R: TATCAACAATCTCACGAGGG</td></tr><tr><td align=\"left\" rowspan=\"2\">NRT2</td><td align=\"left\">F: TAACTACACCCTCCTTCAATTCTC</td></tr><tr><td align=\"left\">R: GTTGGTTTCTTGCTTGTGTC</td></tr><tr><td align=\"left\" rowspan=\"2\">GS</td><td align=\"left\">F: TTCTGTTGGTATCTCTGCTG</td></tr><tr><td align=\"left\">R: TGTGCTGTAGTTAGTGTGAG</td></tr><tr><td align=\"left\" rowspan=\"2\">NADH-GOGA</td><td align=\"left\">F: ATAACACAACCTTCCTTTCCAC</td></tr><tr><td align=\"left\">R: TCAGATAACTTCTCACCACCC</td></tr><tr><td align=\"left\" rowspan=\"2\">ABC</td><td align=\"left\">F: TCTACTTCTGCTTCACTTCGG</td></tr><tr><td align=\"left\">R: CATCTTTGCTATTTGCCTTCGT</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Dry matter accumulation and pod yield per plant under different nitrogen applicationin pod zone</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Cultivars</th><th align=\"left\" rowspan=\"2\">Pod zone nitrogen application (kg·hm<sup>− 2</sup>)</th><th align=\"left\" colspan=\"3\">Dry matter accumulation per plant(g)</th><th align=\"left\" rowspan=\"2\">Yield per plant (g)</th></tr><tr><th align=\"left\">20 DAP</th><th align=\"left\">40 DAP</th><th align=\"left\">60 DAP</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"4\">SH11</td><td align=\"left\">0</td><td align=\"left\">34.62 ± 2.37a</td><td align=\"left\">43.54 ± 0.99b</td><td align=\"left\">48.82 ± 0.68c</td><td align=\"left\">22.60 ± 0.64b</td></tr><tr><td align=\"left\">30</td><td align=\"left\">37.00 ± 1.84a</td><td align=\"left\">45.68 ± 1.77ab</td><td align=\"left\">51.72 ± 0.64b</td><td align=\"left\">24.43 ± 0.78a</td></tr><tr><td align=\"left\">60</td><td align=\"left\">37.62 ± 1.52a</td><td align=\"left\">46.65 ± 0.56a</td><td align=\"left\">53.38 ± 0.27a</td><td align=\"left\">25.15 ± 0.25a</td></tr><tr><td align=\"left\">90</td><td align=\"left\">36.25 ± 0.48a</td><td align=\"left\">45.91 ± 0.96a</td><td align=\"left\">53.43 ± 1.13a</td><td align=\"left\">24.53 ± 0.59a</td></tr><tr><td align=\"left\" rowspan=\"4\">HY23</td><td align=\"left\">0</td><td align=\"left\">30.78 ± 0.26b</td><td align=\"left\">40.50 ± 0.67a</td><td align=\"left\">40.24 ± 0.99b</td><td align=\"left\">19.74 ± 0.60b</td></tr><tr><td align=\"left\">30</td><td align=\"left\">31.76 ± 0.70b</td><td align=\"left\">40.94 ± 0.62a</td><td align=\"left\">41.94 ± 0.71a</td><td align=\"left\">20.67 ± 0.46ab</td></tr><tr><td align=\"left\">60</td><td align=\"left\">33.76 ± 1.29a</td><td align=\"left\">41.85 ± 0.71a</td><td align=\"left\">43.32 ± 0.68a</td><td align=\"left\">21.47 ± 0.61a</td></tr><tr><td align=\"left\">90</td><td align=\"left\">33.15 ± 1.07a</td><td align=\"left\">41.96 ± 0.96a</td><td align=\"left\">42.90 ± 0.79a</td><td align=\"left\">20.81 ± 0.37ab</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Nitrogen accumulation per plant under different nitrogen application in pod zone</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Cultivars</th><th align=\"left\" rowspan=\"2\">Pod zone nitrogen application (kg·hm<sup>− 2</sup>)</th><th align=\"left\" colspan=\"3\">Nitrogen accumulation per plant (mg)</th><th align=\"left\" colspan=\"3\">Increase over CK (%)</th></tr><tr><th align=\"left\">20 DAP</th><th align=\"left\">40 DAP</th><th align=\"left\">60 DAP</th><th align=\"left\">20 DAP</th><th align=\"left\">40 DAP</th><th align=\"left\">60 DAP</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"4\">SH11</td><td align=\"left\">0</td><td align=\"left\">690.6 ± 32.6b</td><td align=\"left\">950.4 ± 27.7c</td><td align=\"left\">1017.4 ± 40.8c</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">30</td><td align=\"left\">751.3 ± 41.4ab</td><td align=\"left\">1043.6 ± 41.8b</td><td align=\"left\">1152.7 ± 15.8b</td><td align=\"left\">8.79 ± 0.84a</td><td align=\"left\">9.81 ± 2.56b</td><td align=\"left\">13.30 ± 4.23b</td></tr><tr><td align=\"left\">60</td><td align=\"left\">774.1 ± 45.7a</td><td align=\"left\">1148.3 ± 21.2a</td><td align=\"left\">1282.8 ± 10.0a</td><td align=\"left\">12.09 ± 2.22a</td><td align=\"left\">20.83 ± 3.75a</td><td align=\"left\">26.08 ± 4.09a</td></tr><tr><td align=\"left\">90</td><td align=\"left\">774.4 ± 10.3a</td><td align=\"left\">1141.6 ± 34.1a</td><td align=\"left\">1300.6 ± 43.4a</td><td align=\"left\">12.14 ± 4.46a</td><td align=\"left\">20.13 ± 3.79a</td><td align=\"left\">27.83 ± 2.27a</td></tr><tr><td align=\"left\" rowspan=\"4\">HY23</td><td align=\"left\">0</td><td align=\"left\">577.3 ± 20.3b</td><td align=\"left\">761.6 ± 25.9c</td><td align=\"left\">832.6 ± 16.1c</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">30</td><td align=\"left\">619.4 ± 29.2ab</td><td align=\"left\">820.6 ± 22.0b</td><td align=\"left\">899.6 ± 15.3b</td><td align=\"left\">7.29 ± 2.62b</td><td align=\"left\">7.75 ± 3.08b</td><td align=\"left\">8.05 ± 0.64b</td></tr><tr><td align=\"left\">60</td><td align=\"left\">669.3 ± 38.1a</td><td align=\"left\">871.2 ± 8.2a</td><td align=\"left\">962.8 ± 43.2a</td><td align=\"left\">15.95 ± 2.70a</td><td align=\"left\">14.40 ± 3.16ab</td><td align=\"left\">15.64 ± 4.16a</td></tr><tr><td align=\"left\">90</td><td align=\"left\">673.0 ± 30.8a</td><td align=\"left\">894.3 ± 10.22a</td><td align=\"left\">952.6 ± 38.6a</td><td align=\"left\">16.58 ± 1.73a</td><td align=\"left\">17.43 ± 3.97a</td><td align=\"left\">14.41 ± 2.45a</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p><italic>Note</italic>: Data are means ± SD of three replicates each containing five plants independently. Different lowercase letters in the same column indicate significantly different between different nitrogen application ratesin the same cultivarat <italic>p</italic> &lt; 0.05 according to Duncan’s multiple range test. DAP means the days after peanut gynophores penetration soil</p></table-wrap-foot>", "<table-wrap-foot><p><italic>Note</italic>: Data are means ± SD of three replicates.Different lowercase letters in the same column indicate significantly different between different nitrogen application ratesin the same cultivar at <italic>p</italic> &lt; 0.05 according to Duncan’s multiple range test. DAP means the days after peanut gynophores penetration soil</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[{"label": ["5."], "surname": ["Attree", "Du", "Xu"], "given-names": ["R", "B", "B"], "article-title": ["Distribution of phenolic compounds in seed coat and cotyledon, and their contribution to antioxidant capacities of red and black seed coat peanuts ("], "italic": ["Arachis hypogaea"], "source": ["Crops Prod"], "year": ["2015"], "volume": ["67"], "fpage": ["448"], "lpage": ["56"], "pub-id": ["10.1016/j.indcrop.2015.01.080"]}, {"label": ["6."], "surname": ["Fageria", "Baligar"], "given-names": ["NK", "VC"], "article-title": ["Enhancing nitrogen use efficiency in crop plants"], "source": ["Adv Agron"], "year": ["2005"], "volume": ["88"], "fpage": ["97"], "lpage": ["185"], "pub-id": ["10.1016/S0065-2113(05)88004-6"]}, {"label": ["7."], "surname": ["Maaz", "Sapkota", "Eagle", "Kantar", "Bruulsema", "Majumdar"], "given-names": ["TM", "TB", "AJ", "MB", "TW", "K"], "article-title": ["Meta-analysis of yield and nitrous oxide outcomes for nitrogen management in agriculture"], "source": ["Glob ChangeBiol"], "year": ["2021"], "volume": ["27"], "fpage": ["2343"], "lpage": ["60"], "pub-id": ["10.1111/gcb.15588"]}, {"label": ["8."], "surname": ["Michalczyk", "Kersebaum", "Dauck", "Roelcke", "Yue", "Chen", "Zhang"], "given-names": ["A", "KC", "HP", "M", "SC", "XP", "FS"], "article-title": ["Quantifying nitrogen loss and wateruse via regionalization and multiple-year scenario simulations in the North China Plain. J. Plant Nutr"], "source": ["Soil Sci"], "year": ["2020"], "volume": ["183"], "fpage": ["718"], "lpage": ["33"]}, {"label": ["9."], "surname": ["Palmero", "Fernandez", "Garcia", "Haro", "Salvagiotti", "Ciampitti"], "given-names": ["F", "JA", "FO", "RJPPVV", "F", "IA"], "article-title": ["A quantitative review into the contributions of biological nitrogen fixationto agricultural systems by grain legumes"], "source": ["Europ J Agr"], "year": ["2022"], "volume": ["136"], "fpage": ["126514"], "pub-id": ["10.1016/j.eja.2022.126514"]}, {"label": ["10."], "surname": ["Mondal", "Skalicky", "Garai", "Hossain", "Sarkar", "Banerjee"], "given-names": ["M", "M", "S", "A", "S", "H"], "article-title": ["Supplementing nitrogen in combination with rhizobium inoculation and soil mulch in peanut ("], "italic": ["Arachis hypogaea"], "source": ["Agronomy"], "year": ["2020"], "volume": ["10"], "fpage": ["1582"], "pub-id": ["10.3390/agronomy10101582"]}, {"label": ["11."], "surname": ["Peoples", "Giller", "Jensen", "Herridge"], "given-names": ["MB", "KE", "ES", "DF"], "article-title": ["Quantifying countrytoglobal scale nitrogen fixation for grain legumes: I. Reliance on nitrogen fixation of soybean, groundnut and pulses"], "source": ["Plant Soil"], "year": ["2021"], "volume": ["469"], "fpage": ["1"], "lpage": ["14"], "pub-id": ["10.1007/s11104-021-05167-6"]}, {"label": ["12."], "surname": ["Tubbs", "Harris", "Beasley", "Smith", "Smith"], "given-names": ["RS", "GH", "JP", "AR", "NB"], "article-title": ["Effect of inoculant and nitrogen application at planting on peanut production in Georgia"], "source": ["Crop Manag"], "year": ["2012"], "volume": ["11"], "fpage": ["0"]}, {"label": ["13."], "surname": ["Huang", "Yang", "Zheng", "Olesen", "Rees", "Zou"], "given-names": ["WH", "YH", "HY", "JE", "RM", "J"], "article-title": ["Excessive N applications reduces yield and biological N fixation ofsummer-peanut in the North China Plain"], "source": ["Field Crops Res"], "year": ["2023"], "volume": ["302"], "fpage": ["109021"], "pub-id": ["10.1016/j.fcr.2023.109021"]}, {"label": ["14."], "surname": ["Giller", "Nambiar", "Rao", "Dart", "Day"], "given-names": ["KE", "PTC", "BS", "PJ", "JM"], "article-title": ["A comparison of nitrogen fixation in genotypes of groundnut "], "italic": ["(Arachis hypogaea"], "sup": ["15"], "source": ["Biol Fertil Soils"], "year": ["1987"], "volume": ["5"], "fpage": ["23"], "lpage": ["5"], "pub-id": ["10.1007/BF00264341"]}, {"label": ["15."], "surname": ["Mondal", "Skalicky", "Garai", "Hossain", "Sarkar", "Banerjee"], "given-names": ["M", "M", "S", "A", "S", "H"], "article-title": ["Supplementing nitrogen in combination with rhizobium inoculation and soil mulch in peanut ("], "italic": ["Arachis hypogaea"], "source": ["Agronomy"], "year": ["2020"], "volume": ["10"], "fpage": ["1513"], "pub-id": ["10.3390/agronomy10101513"]}, {"label": ["16."], "surname": ["Tang", "Ye", "Yao", "Zhao", "Xuan", "Tian", "Zhang", "Xu"], "given-names": ["WJ", "J", "XM", "PZ", "W", "YL", "YY", "S"], "article-title": ["Genome-wide associated study identifies NAC42-activated nitrate transporter conferring high nitrogen use efficiency in rice"], "source": ["Nat Commu"], "year": ["2019"], "volume": ["10"], "fpage": ["5279"], "pub-id": ["10.1038/s41467-019-13187-1"]}, {"label": ["17."], "surname": ["Delfini", "Belgoff", "Fern\u00e1ndez", "Fabra", "Castro"], "given-names": ["R", "C", "E", "A", "S"], "article-title": ["Symbiotic nitrogen fixation and nitrate reduction in two peanut cultivars with different growth habit and branching pattern structures"], "source": ["Plant Growth Regu"], "year": ["2010"], "volume": ["61"], "fpage": ["153"], "lpage": ["9"], "pub-id": ["10.1007/s10725-010-9461-1"]}, {"label": ["18."], "surname": ["Xia", "Zhao", "Hou", "Li", "Zhao", "Bi"], "given-names": ["H", "CZ", "L", "AQ", "SZ", "YP"], "article-title": ["Transcriptome profiling of peanut gynophores revealed global reprogramming of gene expression during early pod development in darkness"], "source": ["BMC Genom"], "year": ["2013"], "volume": ["14"], "fpage": ["517"], "pub-id": ["10.1186/1471-2164-14-517"]}, {"label": ["19."], "surname": ["Beringer", "Taha"], "given-names": ["H", "MA"], "sup": ["45"], "italic": ["Arachis hypogea"], "source": ["Exper Agr"], "year": ["1976"], "volume": ["12"], "fpage": ["1"], "lpage": ["7"], "pub-id": ["10.1017/S0014479700006992"]}, {"label": ["20."], "surname": ["Chahal", "Singh", "Shukla"], "given-names": ["RS", "SP", "UC"], "article-title": ["Uptake and translocation of zinc absorbed through roots and fruiting organs in peanuts"], "source": ["Plant Soil"], "year": ["1979"], "volume": ["51"], "fpage": ["109"], "lpage": ["15"], "pub-id": ["10.1007/BF02205930"]}, {"label": ["22."], "surname": ["Zhang", "Wang", "Wan", "Liu", "Zhang"], "given-names": ["M", "LF", "YS", "FZ", "K"], "article-title": ["Rational nitrogen strategies can improve peanut source supply capacity and pod yield"], "source": ["Agron J"], "year": ["2017"], "volume": ["109"], "fpage": ["2927"], "pub-id": ["10.2134/agronj2017.03.0190"]}, {"label": ["23."], "surname": ["Inanaga", "Utunomiya", "Horiguchi", "Nishihara"], "given-names": ["S", "M", "T", "T"], "article-title": ["Behaviour of fertilizer-N absorbed through root and fruit in peanut"], "source": ["Plant Soil"], "year": ["1990"], "volume": ["122"], "fpage": ["85"], "lpage": ["9"], "pub-id": ["10.1007/BF02851913"]}, {"label": ["24."], "surname": ["Hou", "Lin", "Wang", "Li", "Zhao", "Zhu", "Li", "Li"], "given-names": ["L", "RX", "XJ", "H", "CZ", "XJ", "CS", "GH"], "article-title": ["The mechanisms of pod zone nitrogen application on peanut pod yield"], "source": ["Rus J of Plant Physiol"], "year": ["2022"], "volume": ["69"], "fpage": ["51"], "pub-id": ["10.1134/S1021443722030050"]}, {"label": ["25."], "surname": ["Sun", "Li", "Wang", "Wang"], "given-names": ["H", "SX", "YF", "ML"], "article-title": ["Effects of nitrogen application on source of nitrogen accumulation and yields of different peanut cultivars"], "source": ["J Plant Nutri Fertil"], "year": ["2010"], "volume": ["16"], "fpage": ["153"], "lpage": ["7"]}, {"label": ["26."], "surname": ["Khator", "Shekhawat"], "given-names": ["K", "GS"], "article-title": ["Cdand Cuinduced phytotoxicity on 2\u20133 leaf stage of Cyamopsis tetragonoloba and its regulation by nitrate reductase and ROS quenching enzyme"], "source": ["Acta PhysiolPlanta"], "year": ["2020"], "volume": ["42"], "fpage": ["120"]}, {"label": ["28."], "surname": ["Ojiewo", "Janila", "Bhatnagar-Mathur", "Pandey", "Desmae", "Okori", "Mwololo", "Ajeigbe"], "given-names": ["C", "P", "P", "MK", "H", "P", "J", "H"], "article-title": ["Advances in crop improvement and delivery research for nutritional quality and health benefits of groundnut ("], "italic": ["Arachis hypogaea"], "source": ["Plant Sci"], "year": ["2020"], "volume": ["11"], "fpage": ["1"], "lpage": ["15"]}, {"label": ["29."], "surname": ["Mohapatra", "Dixit"], "given-names": ["AKB", "L"], "article-title": ["Integrated nutrient management in rainy season groundnut ("], "italic": ["Arachis hypogaea"], "source": ["Indian J Agron"], "year": ["2010"], "volume": ["55"], "fpage": ["123"], "lpage": ["7"], "pub-id": ["10.59797/ija.v55i2.4740"]}, {"label": ["30."], "surname": ["Xia", "Wang", "Hu", "Wang", "Zhang", "Wu", "Chi"], "given-names": ["GM", "YJ", "JQ", "SJ", "Y", "Q", "DC"], "article-title": ["Effects of supplemental irrigation on water and nitrogen use, yield, and kernel quality of peanut under nitrogen-supplied conditions"], "source": ["Agric Water Manag"], "year": ["2021"], "volume": ["243"], "fpage": ["106518"], "pub-id": ["10.1016/j.agwat.2020.106518"]}, {"label": ["31."], "surname": ["Gheysari", "Mirlatifi", "Bannayan", "Homaee", "Hoogenboom"], "given-names": ["M", "SM", "M", "M", "G"], "article-title": ["Interaction of water and nitrogen on maize grown for silage"], "source": ["Agric Water Manag"], "year": ["2009"], "volume": ["96"], "fpage": ["809"], "lpage": ["21"], "pub-id": ["10.1016/j.agwat.2008.11.003"]}, {"label": ["32."], "surname": ["Liu", "Gao", "Liu", "Yang", "Zhen", "Li", "Li", "Zhao"], "given-names": ["ZX", "F", "Y", "JQ", "XY", "XX", "Y", "JH"], "article-title": ["Timing and splitting of nitrogen fertilizer supply to increase crop yield and efficiency of nitrogen utilization in a wheat-peanut relay intercropping system in China"], "source": ["The Crop J"], "year": ["2018"], "volume": ["8"], "fpage": ["6"]}, {"label": ["33."], "surname": ["Effah", "Li", "Xie", "Liu", "Xu", "Karikari", "Anwar", "Zeng"], "given-names": ["Z", "LL", "JH", "C", "AX", "B", "S", "M"], "article-title": ["Regulation of nitrogen metabolism, photosynthetic activity, and yield attributes of spring wheat by nitrogen fertilizer in the semi-arid loess plateau region"], "source": ["J Plant Growth Regu"], "year": ["2022"], "volume": ["42"], "fpage": ["11201133"]}, {"label": ["34."], "surname": ["Jiang", "Du", "Tian", "Wang", "Xiong", "Xu", "Yan", "Ding"], "given-names": ["Q", "YL", "XY", "QS", "RH", "GC", "C", "YF"], "article-title": ["Effect of panicle nitrogen on grain filling characteristics of high-yielding rice cultivars"], "source": ["Europ J Agr"], "year": ["2016"], "volume": ["74"], "fpage": ["185"], "lpage": ["92"], "pub-id": ["10.1016/j.eja.2015.11.006"]}, {"label": ["35."], "surname": ["Inanaga", "Yoshida", "Hoshino", "Nishihara"], "given-names": ["S", "T", "T", "T"], "article-title": ["The effect of mineral elements on the maturity of peanut seed"], "source": ["Plant Soil"], "year": ["1988"], "volume": ["106"], "fpage": ["263"], "lpage": ["8"], "pub-id": ["10.1007/BF02371222"]}, {"label": ["36."], "surname": ["Luo", "Liu", "Wan", "Zhang", "Zhao"], "given-names": ["B", "FZ", "YS", "K", "WX"], "article-title": ["Dynamic changes of endogenous hormones content and dry matter accumulation of pods and kernels in different varieties (lines) of peanut ("], "italic": ["Arachis hypogaea"], "source": ["Acta Agron Sin"], "year": ["2013"], "volume": ["39"], "fpage": ["2083"], "lpage": ["93"], "pub-id": ["10.3724/SP.J.1006.2013.02083"]}, {"label": ["37."], "surname": ["Main", "Barber", "Boman", "Chapman", "Dodds", "Duncan"], "given-names": ["CL", "LT", "RK", "K", "DM", "S"], "article-title": ["Effects of nitrogen and planting seed size on cotton growth, development, and yield"], "source": ["Agron J"], "year": ["2013"], "volume": ["105"], "fpage": ["1853"], "pub-id": ["10.2134/agronj2013.0154"]}, {"label": ["39."], "surname": ["Miflin", "Habash"], "given-names": ["BJ", "DZ"], "article-title": ["The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops"], "source": ["J Exper Bot"], "year": ["2022"], "volume": ["53"], "fpage": ["979"], "lpage": ["87"], "pub-id": ["10.1093/jexbot/53.370.979"]}, {"label": ["40."], "surname": ["Liu", "Zhu", "Dong", "Tang", "Zheng", "Xiao"], "given-names": ["ZY", "YA", "Y", "L", "Y", "JX"], "article-title": ["Interspecies interaction for nitrogen use efficiency via up-regulated glutamine and glutamate synthase under wheat-faba bean intercropping"], "source": ["Field Crops Res"], "year": ["2021"], "volume": ["274"], "fpage": ["108324"], "pub-id": ["10.1016/j.fcr.2021.108324"]}, {"label": ["41."], "surname": ["Balotf", "Kavoosi", "Kholdebarin"], "given-names": ["S", "G", "B"], "article-title": ["Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings"], "source": ["Biotech Appl Biochem"], "year": ["2015"], "volume": ["63"], "fpage": ["220"], "lpage": ["9"], "pub-id": ["10.1002/bab.1362"]}, {"label": ["42."], "surname": ["Li", "Cheng", "Lu", "Lu"], "given-names": ["GH", "GG", "WP", "DL"], "article-title": ["Differences of yield and nitrogen use efficiency under different applications of slow release fertilizer in spring maize"], "source": ["J Integri Agr"], "year": ["2021"], "volume": ["20"], "fpage": ["554"], "lpage": ["64"], "pub-id": ["10.1016/S2095-3119(20)63315-9"]}, {"label": ["43."], "surname": ["Sun", "Sun", "Li", "Guo", "Ma"], "given-names": ["YJ", "YY", "XY", "X", "J"], "article-title": ["Relationship of nitrogen utilization and activities of key enzymes involved in nitrogen metabolism in rice under water-nitrogen interaction"], "source": ["Acta Agron Sin"], "year": ["2009"], "volume": ["35"], "fpage": ["2055"], "lpage": ["63"]}, {"label": ["45."], "surname": ["Sung", "Sun"], "given-names": ["FJM", "YW"], "article-title": ["Seasonal patterns of nitrate reductase and nitrogease activities in "], "italic": ["Arachis hypogaea"], "source": ["Field Crops Res"], "year": ["1990"], "volume": ["25"], "fpage": ["215"], "lpage": ["22"], "pub-id": ["10.1016/0378-4290(90)90004-U"]}, {"label": ["46."], "surname": ["Zhang", "Wan", "Dai", "Ning", "Song"], "given-names": ["ZM", "SB", "LX", "TY", "WW"], "article-title": ["Effects of nitrogen application rates on nitrogen metabolism and related enzyme activities of two different peanut cultivars"], "source": ["Scien Agricultura Sin"], "year": ["2011"], "volume": ["44"], "fpage": ["280"], "lpage": ["90"]}, {"label": ["47."], "surname": ["Nacry", "Bouguyon", "Gojon"], "given-names": ["P", "E", "A"], "article-title": ["Nitrogen acquisition by roots: physiological and developmental mechanisms ensuring plant adaptation to a fluctuating resource"], "source": ["Plant Soil"], "year": ["2013"], "volume": ["370"], "fpage": ["1"], "lpage": ["29"], "pub-id": ["10.1007/s11104-013-1645-9"]}, {"label": ["48."], "mixed-citation": ["Li J, Yang P, Sohail H, Du HB, Li J. The impact of short-term nitrogen starvation and replenishment on the nitrate metabolism of hydroponically grown spinach. Sci Hortic.2023;309."]}, {"label": ["49."], "surname": ["Bucher", "Santos", "Nogueira", "Rangel", "Souza", "Fernandes"], "given-names": ["CA", "LA", "EM", "RP", "SR", "MS"], "article-title": ["The transcription of nitrate transporters in upland rice varieties with contrasting nitrate-uptake kinetics"], "source": ["J Plant Nutri Soil Sci"], "year": ["2014"], "volume": ["177"], "fpage": ["395"], "lpage": ["403"], "pub-id": ["10.1002/jpln.201300086"]}, {"label": ["50."], "surname": ["Chen", "Guo", "Wang", "Liang", "Li", "He", "Zhen"], "given-names": ["XL", "PJ", "ZY", "JY", "GH", "WW", "A"], "article-title": ["Grafting improves growth and nitrogen-use efficiency by enhancing NO"], "sup": ["3"], "source": ["Plant Soil"], "year": ["2022"], "volume": ["480"], "fpage": ["305"], "lpage": ["27"], "pub-id": ["10.1007/s11104-022-05583-2"]}]
{ "acronym": [], "definition": [] }
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2024-01-14 23:43:47
BMC Plant Biol. 2024 Jan 13; 24:48
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PMC10787502
38216992
[ "<title>Introduction</title>", "<p id=\"Par15\">Low-dose computed tomography (CT) is the gold standard for early lung cancer detection [##REF##10682770##1##–##REF##21714641##3##]. These scans are designed to detect lung nodules, which are then determined by radiologists to be either benign or malignant. Currently, lung nodules are characterized according to the Lung CT Screening Reporting and Data System (Lung-RADS) developed by the American College of Radiology (ACR) [##REF##29053407##4##]. This system focuses on information derived from the nodules alone (e.g. solidity, shape, growth rate, and texture) with little consideration given to the nodule microenvironment. Studies, however, have shown that nodule microenvironments such as fibrosis may play a role in lung cancer malignancy. Karampitsakos et al<italic>.</italic> and Li et al<italic>.</italic> found that pulmonary fibrosis increases a patient’s risk of developing lung cancer. Salvatore et al<italic>.</italic> revealed that pulmonary fibrosis is observed in 6.6% of lung cancer screening participants compared to 0.03% among men and 0.02% among women of the general public [##REF##28377145##5##–##REF##26700157##7##]. Many risk factors are shared between pulmonary fibrosis and lung cancer and CT imaging has played a crucial role in the early detection of both diseases [##REF##29154106##8##, ##REF##31412851##9##]</p>", "<p id=\"Par16\">Deep learning (DL) algorithms, specifically convolutional neural networks (CNNs), have been used in lung nodule detection, segmentation, and classification tasks with high accuracy and efficiency [##UREF##0##10##–##UREF##3##17##]. An explainable model can be achieved with visualization maps attached to the model, specific features and structures can be highlighted in the images. This could help researchers to better understand how a model reaches decisions, and identify features associated with it [##UREF##4##18##, ##UREF##5##19##]. Zhu et al<italic>.</italic> [##UREF##1##11##] retain the segmented nodules by discarding the background information when performing nodule classification, and Xiao et al<italic>.</italic> [##REF##32537025##12##] isolate nodules from the encompassing tissues based on the presupposition that the presence of surrounding tissue might negatively impact the classification outcome. Other studies [##REF##31296975##13##–##UREF##3##17##] encompassed adjacent soft tissue structures due to the utilization of 2D or 3D bounding boxes as training data. However, these studies did not conduct a performance comparison between solitary nodules and nodules situated within diverse pulmonary contexts, therefore, the potential impact on the microenvironment still remains unclear. To the best of our knowledge, no DL-based studies have investigated the impact of the fibrotic microenvironment on the lung nodule classification task.</p>", "<p id=\"Par17\">The purpose of this study is to assess the influence of integrating a fibrotic microenvironment aspect into the classification of nodule malignancies through the utilization of DL algorithms. We developed a novel 3D classification network featuring attention gates, specifically designed to mitigate the loss of model efficiency in the processing of nodules and their associated microenvironment. This innovation incorporates several novel technical contributions: (1) multiple attention gates emanate from distinct network depths, serving the purpose of accentuating salient features at shallower network depths. This collective input significantly influences the final classification outcome; and (2) predictive vectors derived from these attention gates can be concatenated with additional clinical data. Subsequently, this combined information is passed through a fully connected layer, culminating in the generation of a definitive classification result. By iteratively incorporating and excluding microenvironmental data in experiments, our investigation aims to elucidate any conceivable associations between fibrotic tissue presence and the categorization of nodule malignancy.</p>" ]
[ "<title>Materials and methods</title>", "<p id=\"Par18\">Data were retrospectively collected in compliance with the Health Insurance Portability and Accountability Act, institutional review board approval, and waivers of informed consent.</p>", "<title>Study design</title>", "<p id=\"Par19\">4500 patients with chest CT scans containing fibrosis were identified. Patients less than 21 years old at the time of the initial CT scan were excluded. Lung nodules were identified and labeled with a single point marked approximately in the center of the nodule by a senior radiologist with over 20 years of experience via the VGG image annotator (VIA) web-based interface [##UREF##6##20##]. Two 3D DL models, one for nodule segmentation and one for nodule classification were used in this study (Fig. ##FIG##0##1##). The segmentation model was trained to delineate nodules. The classification model was trained with the objective of distinguishing between cancerous and non-cancerous nodules, while also determining the presence of fibrotic tissues in the surrounding region. Biopsy results and radiologist reports were used as the ground truth labels. The classification accuracy was compared to the nodule volume doubling time (VDT).</p>", "<title>Datasets</title>", "<p id=\"Par20\">The two datasets used in this study were the publicly available LIDC-IDRI with thoracic CT scans (1018 cases) and the in-house dataset (1088 cases). Information about the in-house dataset can be found in Table ##TAB##0##1##. The LIDC-IDRI dataset was used for the nodule segmentation task and pre-training of the nodule classification model [##REF##21452728##21##]. We leveraged the high-quality consensus nodule annotations provided in the LIDC dataset to train the segmentation model and the pretraining of the classification model. For the in-house dataset, screening and diagnostic CT examinations were collected from 4500 patients. A pool of 1088 cases met the inclusion and exclusion criteria. Among 1088 cases, 144 cases (13.23%) were confirmed to be malignant by pathological biopsy, and 57 cases (5.24%) were deemed “highly suspicious” by radiologists. For the nodule malignancy classification, we considered nodules in the two scans independently. A dataset containing 345 malignant (labeled 1) and 743 benign (labeled 0) nodules was constructed. Three separate data subsets were used in the training: (1) nodules only (microenvironment removed), (2) nodules with surrounding soft tissue microenvironment, and (3) nodules and microenvironment with semantic fibrosis metadata. For the pulmonary fibrosis classification task, 489 cases out of 1,088 cases were labeled by radiologists as fibrosis. Fibrosis is defined as fibrotic tissue presented adjacent to the nodule or presented in the nodule-growing microenvironment.</p>", "<title>Segmentation model</title>", "<p id=\"Par21\">The segmentation model within our pipeline served the purpose of preparing inputs for the nodule classification. In this stage, the model generated two distinct datasets: one that excluded background information and another that exclusively included background information. The underlying assumption was that by eliminating the background, any lung fibrosis-related details were either unavailable or significantly minimized. Consequently, the classification model was tasked with making decisions solely based on the attributes of the nodules themselves.</p>", "<p id=\"Par22\">The architecture of our 3D segmentation model was based upon the U-Net framework (3D UNet, Monai [##UREF##7##22##]), featuring an analysis path (encoder) and a synthesis path (decoder). Within this framework, we incorporated 3D convolutional layers and 3D pooling layers to extract intricate features from the input volume. In the decoding phase, 3D deconvolution was employed to reinstate the original feature map dimensions. Notably, the inclusion of residual units played a pivotal role in enabling the model to encompass contextual information surrounding the lung nodules. By incorporating these residual units, the entire network exhibited heightened precision, consequently ameliorating the segmentation of lung nodules within medical images.</p>", "<title>Nodule malignancy classification</title>", "<title>Volume doubling time (VDT)</title>", "<p id=\"Par23\">For cases with follow-up CT scans, the nodule volume doubling time (VDT) was calculated. We used our segmentation model to estimate nodule volume. We then used the modified Schwartz formula to calculate VDT: [##REF##28825886##23##]. This formula assumes an exponential growth rate for lung cancer. It assumes that malignant cells divide at a constant rate, and thus lesions grow exponentially with time. It is worth noting that a shrinking nodule will have a negative VDT value. Shrinking nodules were classified as benign in this study. According to most studies, a volume doubling time below 400 days represents a high likelihood of malignancy, and a VDT above 500 days is characteristic of a benign nodule [##REF##22970048##24##]. To prevent high false-positive rates, we set the threshold of malignancy to 500 days.</p>", "<title>DL-based classification model</title>", "<p id=\"Par24\">The proposed 3D Attention-gated Network (3D AG-Net) classification model was adopted from the 2D Attention-Gated Sononet [##REF##30802813##25##]. The 3D AG-Net consists of 17 3D convolution layers (3 × 3 × 3 convolution) to extract features from volumetric CT images input. Two soft attention gates, AG-1 and AG-2, were placed at the 11th and 14th layers, respectively (Fig. ##FIG##1##2##). Deviating from the main network flow, AG-1 and AG-2 can intercept salient features they deem necessary and filter out those features at their specific depths to make independent predictions. The attention maps generated by the AGs provide visualizations of the network behavior as well. Examples of the given nodules are shown in Fig. ##FIG##2##3##<bold>.</bold></p>", "<p id=\"Par25\">Hu et al<italic>. </italic>[##UREF##2##15##] demonstrated that network depths could affect prediction performance on the LIDC dataset, a trade-off between sensitivity and specificity was observed. Higher sensitivity was achieved at a deeper network, and higher specificity was achieved at an intermediate network. Hence, two attention-gated feature extraction modules were branched out at the 11th layer and 14th layer (intermediate depth) from the leading architecture (green lines with attention gates in Fig. ##FIG##1##2##) to optimize specificity performance. Meanwhile, the leading architecture has 17 layers to provide sufficient depth for improving sensitivity performance. At the final stage of the network, as indicated by red dashed lines in Fig. ##FIG##1##2##, predictive vectors generated by AG-1, AG-2, the final layer of the leading network, and semantic clinical metadata (e.g., lung fibrosis) can all be concatenated, followed by a fully connected layer to fine-tune the vectors and yield the optimized final prediction results.</p>", "<p id=\"Par26\">All experiments including preprocessing, developing, and evaluation of the model, were performed using Python version 3.6 and PyTorch 1.5 on NVIDIA GP102, GTX 1080 Ti.</p>", "<title>Evaluations and statistical analysis</title>", "<p id=\"Par27\">A tenfold cross-validation strategy was used to evaluate network performance and generalizability. True positive cases, true negative cases, false positive cases, and false negative cases are denoted as TP, TN, FP, and FN respectively. The quantitative evaluation metrics used are listed below..</p>", "<p id=\"Par28\">Qualitative evaluations were conducted with attention maps and class activation maps (CAM). A detailed description of CAM can be found in Additional file ##SUPPL##0##1##: Appendix SB. The one-way ANOVA pairwise t-test was performed with a confidence level set at 95%. In the process of evaluating multiple pairwise performances, a heightened susceptibility to committing a Type I error is observed. In order to mitigate this, a corrective measure known as the Bonferroni correction Eq. (##FORMU##5##5##) was applied on the α level restricting a more confined threshold for the adjusted p-value to reject the null hypothesis.</p>" ]
[ "<title>Results</title>", "<title>Nodule segmentation</title>", "<p id=\"Par29\">We observed a smooth decrease in the training loss with a fluctuated mean dice score in the validation loss due to the overfitting of the model. Therefore, we include a dropout rate of 20% during each epoch. Example cases of the segmentation results are shown in Fig. ##FIG##2##3## for the LIDC dataset and the in-house dataset. We were able to see the similarity between the ground truth and the generated mask. We achieved an average dice score of 0.761 on the validation dataset after 50 epochs of training. The ADAM optimizer was used with the learning rate and dropout rate as 0.0001 and 0.2, respectively. Data augmentation, including random flips, intensity scaling, and intensity shifting, were used to improve model performance. The dice loss function was used as a metric to access the model performance and evaluate the testing dataset. The sliding window inference method which uses a rectangular or cube region of fixed dimension that \"slides\" across an image was applied to create binary classifications on whether or not each voxel belongs to a nodule. Although computationally expensive, this method determines if the window has an object that interests us. Accelerated methods such as cache IO and transform function featured by MONAI were also used to expedite the training process.</p>", "<p id=\"Par30\">All experiments including preprocessing, developing, and evaluation of the model, were performed using Python version 3.6 and PyTorch 1.5 on NVIDIA GP102, GTX 1080 Ti.</p>", "<title>3D AG-Net Pre-training on LIDC-IDRI</title>", "<p id=\"Par31\">To ensure the effectiveness of the 3D AG-Net for the nodule malignancy classification task, we validated the classification model using the LIDC dataset, which has public benchmarks. The model was trained with an early stopping strategy with patience of 50 epochs based on the highest validation accuracy. The network used binary cross-entropy (BCE) loss for nodule malignancy classification. The input data is a 64 × 64 × 64 voxel CT nodule image, Adam optimizer with an initial learning rate set at 0.0002, batch size 128. All experiments were conducted on an Nvidia GTX 1080 Ti GPU with PyTorch library. We achieved 91.57%, 83.34%, 90.46%, and 0.95 for accuracy, sensitivity, precision, and AUC, respectively, with the S1 and S2 (benign) versus S4 and 5 (malignant), which is compatible with the state-of-the-art models' performances (Additional file ##SUPPL##0##1##: Appendix SC). The result trained with the complete LIDC dataset, S1, S2, S3 (benign) versus S4 and S5 (malignant), also reported here as 85.11%, 77.78%, 88.54%, and 0.90 ± 0.04 for accuracy, sensitivity, specificity, and AUC, respectively (Table ##TAB##1##2##). Although the model trained with the complete LIDC dataset achieved lower metrics scores, it serves as a better initializer for the training of the in-house dataset since the complete dataset better reflects the real-world conditions. Therefore the model is more generalized for later applications.</p>", "<title>3D AG-Net training on the in-house dataset</title>", "<p id=\"Par32\">For models trained with the in-house dataset with tenfold cross-validation, the experiment results are summarized in Table ##TAB##1##2## and Fig. ##FIG##3##4##. The performance of the 3D AG-Net without pretraining (trained from scratch) achieved 78.84 ± 5.88%, 62.00 ± 13.65%, 87.29 ± 5.98%, 0.83 ± 0.03 for accuracy, sensitivity, specificity, and AUC, respectively. The model with pretraining on LIDC dataset achieved results of 79.03 ± 2.97%, 65.46 ± 18.64%, 85.86 ± 6.29%, 0.84 ± 0.06 for accuracy, sensitivity, specificity, and AUC, respectively. When the background information was removed, it performed slightly worse in most of the metrics except for specificity, achieving 75.61 ± 7.02%, 50.00 ± 25.46%, 88.46 ± 7.88%, 0.78 ± 0.08 for accuracy, sensitivity, specificity, and AUC, respectively. When additional semantic fibrosis metadata was provided, the 3D AG-Net yielded the best AUC when comparing all other types of datasets. The result was 80.84 ± 3.31%, 74.67 ± 14.78%, 84.95 ± 5.43%, 0.89 ± 0.05 for accuracy, sensitivity, specificity, and AUC, respectively.</p>", "<p id=\"Par33\">We used 3D AG-Net without pretraining as the baseline to compare with the model pretrained on LIDC dataset (AUC increase 1.2%), the model trained with background removed (AUC decrease 6%, p-value &lt; 0.01), and model trained with additional semantic fibrosis metadata (AUC increase 7.2%, p-value &lt; 0.01). We found statistical differences (p-value &lt; 0.01) in AUC between adding (model trained with additional semantic fibrosis metadata) and removing (model trained with nodule background removed) fibrosis information. When semantic fibrosis information is available to the network, the nodule malignancy classification accuracy increases 5.23%. Nodule volume doubling time (VDT) as the current clinical guideline to predict nodule malignancy is also included in the experiment for comparison. The VDT method achieved 62.63%, 56.52%, and 65.48% of accuracy, sensitivity, and specificity respectively. Note that CNN trained with nodule-only images still outperformed the VDT method.</p>", "<p id=\"Par34\">The 3D AG-Net was also trained with the in-house dataset to predict lung fibrosis using the ground-truth fibrosis diagnosis provided by radiologists (fibrosis as 1, non-fibrosis as 0). The model achieved 73.01 ± 5.84%, 75.18 ± 13.32%, 70.83 ± 6.22%, and 0.75 ± 0.07 for accuracy, sensitivity, specificity, and AUC, respectively.</p>", "<title>Visualization</title>", "<p id=\"Par35\">Figure ##FIG##4##5## depicts network visualizations for nodule and fibrosis classification. Notably, distinct attention patterns emerge within the same case when different objectives, such as nodule or fibrosis classification, are employed.</p>", "<p id=\"Par36\">For nodule classification, the network exhibits a similar attention pattern for true positive cases (Fig. ##FIG##5##6##a) and false positive cases (Fig. ##FIG##5##6##b). AG-1 surveys the nodule and the microenvironment, AG-2 removes the attention on the nodule and surveys the surrounding microenvironment, and CAM at the last layer shifts the attention back to the nodule parenchyma. For true negative cases (Fig. ##FIG##5##6##c) and false negative cases (Fig. ##FIG##5##6##d), another attention pattern is observed. The network attention starts with AG-1 the nodule and the microenvironment. Sequentially at the AG-2 layer, the network removes the attention from the majority of the nodule parenchyma with a focus on the microenvironment. At the final stage, the network completely removes the attention from the nodule.</p>", "<p id=\"Par37\">In summary, the network conducts similar search patterns at AG-1 and AG-2 stages for all the cases, then if the network attention shifts back to the nodule at the final stage, the classification will be malignant, while the attention remains at the microenvironment, the classification will be benign. In Fig. ##FIG##5##6##<bold>.</bold>, We demonstrate cases of four possibilities including true positive (a), false positive (b), true negative (c), and false negative (d). It also presents the attention-gated maps and CAM of each case from left to right, respectively. We found that false positive cases usually occurred with larger nodule sizes, especially when the nodule size exceeds our per-defined 64 × 64 × 64 ROI, therefore, there is limited nodule shape, boundary, and microenvironment information presented to the network. For false negative cases, nodules with less diffuse characteristics and more well-defined boundaries are more likely to be classified as benign by the network.</p>", "<p id=\"Par38\">As for fibrosis classification, the network initially searches the surrounding soft tissue area close to the nodule and gradually enlarges the survey area in lung parenchyma to detect fibrotic tissues.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par39\">To evaluate the role of the microenvironment in nodule malignancy classification, we compared the model trained with nodule and microenvironment against the model trained with nodule alone (microenvironment removed). The statistical analysis showed a statistical significance (CI: 95%) that the performance of the method using not only the background information but also the semantic metadata was superior in accuracy (p-value = 0.0011) and sensitivity (p-value = 0.0013). The Bonferroni adjusted alpha coefficient was 0.01667. In addition, we found that although sensitivity decreased vastly by 15.46% after removing the nodule’s microenvironment, the specificity slightly increased by 2.6%, indicating that the detection ability of malignant nodules decreased when the microenvironment was removed. While benign nodule detection remained unaffected. Thus, the factors that contribute to a nodule's malignancy may be dependent on the nodule and its microenvironment.</p>", "<p id=\"Par40\">An intriguing observation pertains to comparing fibrosis incidence rates in malignant and benign nodules. Malignant nodules exhibit a fibrosis incidence rate of 65%, while benign nodules demonstrate a rate of 35%, representing a 30% higher fibrosis rate in the former. To examine the relationship between fibrosis and malignancy, we purposely added semantic fibrosis data into the classifier right before the final fully connected layer of the classification model. The classifier is, therefore, forced to consider the fibrosis information when making the final prediction. When comparing the model trained with and without the fibrosis metadata, we found that sensitivity increases by 9.21% when semantic fibrosis information is available, and specificity slightly decreases by 0.91%. In other words, the ability to detect malignant nodules was enhanced when fibrosis data was presented to the network during training.</p>", "<p id=\"Par41\">In this study, we utilized two distinct approaches to assess nodule malignancy: VDT and deep learning models (3D AG-Net). VDT serves as a well-established method for evaluating nodule behavior predicated upon growth rate analysis. This approach offers valuable insights into the pace of nodule size alteration. However, the precision of VDT is susceptible to fluctuations due to factors such as measurement techniques, nodule characteristics, and the threshold values employed to classify nodules as either rapidly expanding or stable. VDT, rooted in quantitative measurements, exhibits relative independence from extensive datasets for training, yet its simplicity constrains its ability to encapsulate intricate nodule attributes.</p>", "<p id=\"Par42\">Conversely, deep learning models have exhibited the capacity to uncover subtle features that may elude conventional methodologies. The accuracy of these models hinges on the quality and diversity of the training data, the architectural design of the model, and the criteria used for performance assessment. Consequently, deep learning models offer a more advanced and comprehensive evaluation of lung nodules.</p>", "<p id=\"Par43\">In our experimental approach, the VDT method yielded an accuracy of 62.63%, with a sensitivity of 56.52% and specificity of 65.48%. Notably, 3D AG-Net trained on nodule-only images, devoid of microenvironment information, achieved an accuracy of 75.61%, sensitivity of 50.00%, and specificity of 88.46%. It is of significance that this CNN, trained exclusively with nodule images, outperformed the VDT method by 12.98% in terms of accuracy.</p>", "<p id=\"Par44\">Note that there are some limitations in the study. First, we labeled all shrinking-size nodules as benign (negative VDT values based on the modified Schwartz formula). This assumption could lead to underestimating the VDT prediction accuracy and sensitivity because nodules with a decreased size could still be cancer. Second, we used a segmentation model to segment lung nodules and then remove the background. Errors such as cases with partial surrounding tissue leaking into the dataset, or nodules partially removed by the segmentation algorithm, could propagate from the segmentation stage and affect the classification performance. Therefore, the performance without microenvironment could be over- or under-estimated due to the limitation of the segmentation model. It is worth considering the exploration of advanced segmentation models such as AMSUnet [##REF##37276753##26##] and Dual-Branch-UNet [##UREF##8##27##] in future research endeavors.</p>" ]
[ "<title>Conclusion</title>", "<p id=\"Par45\">We have developed a DL-based pipeline containing an auto-segmentation model (3D U-net) and an attention-gated classification model (3D AG-Net) to classify lung nodule malignancy and pulmonary fibrosis. The model’s attention can be visualized at different depths thus making the model behavior interpretable. We have successfully demonstrated that the nodule microenvironment, especially fibrosis, contributes to the performance of the nodule malignancy classification model. Microenvironment data increases nodule malignancy classification accuracy, sensitivity, and AUC. Model Performance is further increased when semantic lung fibrosis information becomes accessible.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Chest Computed tomography (CT) scans detect lung nodules and assess pulmonary fibrosis. While pulmonary fibrosis indicates increased lung cancer risk, current clinical practice characterizes nodule risk of malignancy based on nodule size and smoking history; little consideration is given to the fibrotic microenvironment.</p>", "<title>Purpose</title>", "<p id=\"Par2\">To evaluate the effect of incorporating fibrotic microenvironment into classifying malignancy of lung nodules in chest CT images using deep learning techniques.</p>", "<title>Materials and methods</title>", "<p id=\"Par3\">We developed a visualizable 3D classification model trained with in-house CT dataset for the nodule malignancy classification task. Three slightly-modified datasets were created: (1) nodule alone (microenvironment removed); (2) nodule with surrounding lung microenvironment; and (3) nodule in microenvironment with semantic fibrosis metadata. For each of the models, tenfold cross-validation was performed. Results were evaluated using quantitative measures, such as accuracy, sensitivity, specificity, and area-under-curve (AUC), as well as qualitative assessments, such as attention maps and class activation maps (CAM).</p>", "<title>Results</title>", "<p id=\"Par4\">The classification model trained with nodule alone achieved 75.61% accuracy, 50.00% sensitivity, 88.46% specificity, and 0.78 AUC; the model trained with nodule and microenvironment achieved 79.03% accuracy, 65.46% sensitivity, 85.86% specificity, and 0.84 AUC. The model trained with additional semantic fibrosis metadata achieved 80.84% accuracy, 74.67% sensitivity, 84.95% specificity, and 0.89 AUC. Our visual evaluation of attention maps and CAM suggested that both the nodules and the microenvironment contributed to the task.</p>", "<title>Conclusion</title>", "<p id=\"Par5\">The nodule malignancy classification performance was found to be improving with microenvironment data. Further improvement was found when incorporating semantic fibrosis information.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s12967-023-04798-w.</p>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>Not applicable.</p>", "<title>Author contributions</title>", "<p>YL and SJ designed and experimented the classification model, HYH and SJ designed and experimented the segmentation model, AD and MS analyzed and interpreted the patient data regarding the lung CT images and the histological examinations. BP and TL reviewed the low dose lung CT screening protocols and parameters, and was a major contributor in writing the manuscript. All authors collectively designed the method and experiments, read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This was an independent, investigator-initiated study supported by Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI). BIPI had no role in the design, analysis, or interpretation of the results in this study; BIPI was given the opportunity to review the manuscript for medical and scientific accuracy as it relates to BIPI substances, as well as intellectual property considerations.</p>", "<title>Availability of data and materials</title>", "<p>The data that support the findings of this study are available from the corresponding author upon reasonable request.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par46\">This retrospective study was approved by the IRB at Columbia University (IRB No. AAAS1829).</p>", "<title>Consent for publication</title>", "<p id=\"Par47\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par48\">Mary M. Salvatore—Grant support, lecture, and advisory board Genentech and Boehringer Ingelheim. Image analysis for AbbVie, Bioclinica, LungLife AI. The remaining author have no conflicts to disclose.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>The workflow. Models to segment and classify nodules based on their central point coordinates provided by radiologists. CT images were cropped into 64 × 64 × 64 voxel volumes according to the center coordinates and fed into the models. Nodule volumes are passing (1) the 3D UNet for nodule segmentation. With the segmentation mask, surrounding soft tissue (background) can be removed; hence the nodule volume estimation can be performed. Nodule volumes then go through (2) the classification model (3D Attention Net) to predict nodule malignancy and pulmonary fibrosis. Separate datasets (with or without semantic fibrosis information) can be selected as the input to the classifier. The model’s attention at different layers can be visualized and interpreted via attention coefficient maps and CAMs</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>The schematics of the 3D Attention Network. The light-yellow blocks and the dark-yellow blocks indicate the convolutional layers and the ReLU, respectively. The orange blocks indicate the max-pooling operation resulting in a 1/2 image size. The purple blocks indicate the fully connected layer, and the green blocks indicate additional clinical metadata available. The green arrow-line and the red dashed-arrow-line are connecting operations and concatenating vectors, respectively</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Example cases for nodule segmentation. Four cases from the LIDC dataset and four In-house datasets were randomly selected and displayed here. The LIDC data with radiologist hand-drawn annotations for training and testing and the In-house data were used for inference only. For the LIDC dataset, columns (<bold>a</bold>), (<bold>b</bold>), and (<bold>c</bold>) are the original CT images, ground-truth segmentation provided by radiologists, and the generated masks by segmentation model, respectively. For the In-house dataset, columns (<bold>d</bold>), (<bold>e</bold>), and (<bold>f</bold>) are the original CT images, background removed (nodule only) images via auto-segmentation, and the background only (soft lung tissue) images, respectively</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Receiver operating characteristic (ROC) curves and area under the curves (AUC) of experiments with different datasets and methods. The ROC curves demonstrated here are the averaged ROC based on the tenfold cross-validation. The averaged AUCs with one standard deviation are computed and listed in the legend area. The blue line, red line, green line, cyan line, magenta line, and the red dashed-line are indicating the nodule malignancy prediction results on the LIDC dataset, In-house dataset with metadata, In-house dataset (pretrained with LIDC), In-house dataset, and In-house dataset (background removed), respectively. Statistical differences were found in <bold>1</bold> LIDC dataset, trained from sketch v.s. In-house dataset, trained from sketch (p-value: 0.0319); <bold>2</bold> LIDC dataset, trained from sketch v.s. In-house dataset, background removed (p-value: 0.0001); <bold>3</bold> In-house dataset, adding fibrosis metadata v.s. In-house dataset, background removed (p-value: 0.0002)</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Network attention gates (AGs) and class activation maps (CAMs) visualizations. An example is network visualization for nodule prediction (first row) and lung fibrosis prediction (second row) tasks. The first, second, third, and fourth columns indicate the ground-truth (GT) CT image, first attention gate (AG-1) at No. 11 layer depth, second attention gate (AG-2) at No. 14 layer depth, and the class activation map (CAM) at the final layer, respectively. The fifth column indicates the nodule ground-truth mask (GT Mask), which is not available when the model was trained. The case demonstrated here is a benign nodule in the non-fibrotic lung, where both nodule malignancy and fibrosis models made the correct inferences. From the AGs and CAMs, we can observe the nodule network focuses on nodule parenchyma and its surrounding tissues, while the fibrosis network focuses on other lung tissue with the nodule parenchyma excluded</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Results of the 3D AG-Net on the in-house dataset. A true positive case (<bold>a</bold>), a false positive case (<bold>b</bold>), a true negative case (<bold>c</bold>), and a false negative case (<bold>d</bold>) were shown. In each case, it showed the center slice of the 64 × 64 × 64 volume, the slice with the AG-1 heatmap, the slice with the AG-2 heatmap, and the slice with CAM on top of it from left to right, respectively</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Dataset information</p></caption></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Quantitative results</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Dataset</th><th align=\"left\">LIDC-IDRI</th><th align=\"left\">In-house</th><th align=\"left\" colspan=\"3\">In-house</th></tr></thead><tbody><tr><td align=\"left\">Training strategy/data processing</td><td align=\"left\">NA</td><td align=\"left\">NA</td><td align=\"left\">Pretrained on LIDC-IDRI data</td><td align=\"left\">Pretrained on LIDC-IDRI data/Nodule surrounding tissues removed</td><td align=\"left\">Pretrained on LIDC-IDRI data/semantic fibrosis data added</td></tr><tr><td align=\"left\">Micro-environment information</td><td align=\"left\">Available</td><td align=\"left\">Available</td><td align=\"left\">Available</td><td align=\"left\">Not available</td><td align=\"left\">Available</td></tr><tr><td align=\"left\">Accuracy (%)<sup>*</sup></td><td align=\"left\">85.11 (3.19)</td><td align=\"left\">78.84 (5.88)</td><td align=\"left\">79.03 (2.97)<sup>*</sup></td><td align=\"left\">75.61 (7.02)<sup>*</sup></td><td align=\"left\">80.84 (3.31)<sup>*</sup></td></tr><tr><td align=\"left\">Sensitivity (%)<sup>†</sup></td><td align=\"left\">77.78 (12.24)</td><td align=\"left\">62.00 (13.65)</td><td align=\"left\">65.46 (18.64)<sup>†</sup></td><td align=\"left\">50.00 (25.46)<sup>†</sup></td><td align=\"left\">74.67 (14.78)<sup>†</sup></td></tr><tr><td align=\"left\">Specificity (%)<sup>§</sup></td><td align=\"left\">88.54 (5.87)</td><td align=\"left\">87.29 (5.98)</td><td align=\"left\">85.86 (6.29)<sup>§</sup></td><td align=\"left\">88.46 (7.88)<sup>§</sup></td><td align=\"left\">84.95 (5.43)<sup>§</sup></td></tr><tr><td align=\"left\">AUC</td><td align=\"left\">0.90 (0.04)</td><td align=\"left\">0.83 (0.03)</td><td align=\"left\">0.84 (0.06)</td><td align=\"left\">0.78 (0.08)</td><td align=\"left\">0.89 (0.05)</td></tr></tbody></table></table-wrap>" ]
[ "<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{VDT}} = \\frac{{Ln\\left( 2 \\right)*{\\text{Days Between Scans}}}}{{Ln\\left( {Volume\\, on\\, {\\text{Second Scan}}/ {\\text{Volume on Baseline Scan}}} \\right)}}$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mrow><mml:mtext>VDT</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>L</mml:mi><mml:mi>n</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mn>2</mml:mn></mml:mfenced><mml:mrow/><mml:mo>∗</mml:mo><mml:mrow><mml:mtext>Days Between Scans</mml:mtext></mml:mrow></mml:mrow><mml:mrow><mml:mi>L</mml:mi><mml:mi>n</mml:mi><mml:mfenced close=\")\" 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{\\text{FP}} + {\\text{FN}}}}$$\\end{document}</tex-math><mml:math id=\"M4\" display=\"block\"><mml:mrow><mml:mtext>Accuracy</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>TP</mml:mtext><mml:mo>+</mml:mo><mml:mtext>TN</mml:mtext></mml:mrow><mml:mrow><mml:mtext>TP</mml:mtext><mml:mo>+</mml:mo><mml:mtext>TN</mml:mtext><mml:mo>+</mml:mo><mml:mtext>FP</mml:mtext><mml:mo>+</mml:mo><mml:mtext>FN</mml:mtext></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></disp-formula>", "<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{Sensitivity}} = \\frac{{{\\text{TP}} }}{{{\\text{TP}} + {\\text{FN}}}}$$\\end{document}</tex-math><mml:math id=\"M6\" display=\"block\"><mml:mrow><mml:mtext>Sensitivity</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mtext>TP</mml:mtext><mml:mrow><mml:mtext>TP</mml:mtext><mml:mo>+</mml:mo><mml:mtext>FN</mml:mtext></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></disp-formula>", "<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{Specificity}} = \\frac{{{\\text{TN}}}}{{{\\text{TN}} + {\\text{FP}}}}$$\\end{document}</tex-math><mml:math id=\"M8\" display=\"block\"><mml:mrow><mml:mtext>Specificity</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mtext>TN</mml:mtext><mml:mrow><mml:mtext>TN</mml:mtext><mml:mo>+</mml:mo><mml:mtext>FP</mml:mtext></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></disp-formula>", "<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{Dice Score}} = \\frac{{{2 } \\times {\\text{the Area of Overlap}}}}{{\\text{the total number of pixels in both images}}}$$\\end{document}</tex-math><mml:math id=\"M10\" display=\"block\"><mml:mrow><mml:mrow><mml:mtext>Dice Score</mml:mtext></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:mrow><mml:mtext>the Area of Overlap</mml:mtext></mml:mrow></mml:mrow><mml:mrow><mml:mtext>the total number of pixels in both images</mml:mtext></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></disp-formula>", "<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{Bonferroni-corrected p value}} = \\frac{{\\text{The original p value}}}{{\\text{The number of tests performed}}}$$\\end{document}</tex-math><mml:math id=\"M12\" display=\"block\"><mml:mrow><mml:mrow><mml:mtext>Bonferroni-corrected p value</mml:mtext></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>The original p value</mml:mtext></mml:mrow><mml:mrow><mml:mtext>The number of tests performed</mml:mtext></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></disp-formula>" ]
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[ "<table-wrap-foot><p>Data are presented in the format of Mean (Standard deviation), <italic>AUC</italic> area under the receiver operating characteristic</p><p>*One-way ANOVA analysis performed with Bonferroni correction on accuracy: p-value = 0.0011 (statistical significance)</p><p><sup>†</sup>One-way ANOVA analysis performed with Bonferroni correction on sensitivity: p-value = 0.0013 (statistical significance)</p><p><sup>§</sup>One-way ANOVA analysis performed with Bonferroni correction on specificity p-value = 0.35556 (statistical non-significance)</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"12967_2023_4798_MOESM1_ESM.docx\"><caption><p><bold>Additional file 1</bold>: <bold>Appendix SA.</bold> How the nodule segmentation model is implemented on LIDC-IRDI and in-house dataset. <bold>Appendix SB.</bold> How Class Activation Maps (CAM) assist the prediction of our model. <bold>Appendix SC.</bold> Performance comparison with state-of-the-art methods with LIDC dataset.</p></caption></media>" ]
[{"label": ["10."], "surname": ["Jogin", "Mohana", "Madhulika", "Divya", "Meghana", "Apoorva", "Jogin"], "given-names": ["M", "MS", "GD", "RK", "S", "M"], "article-title": ["Feature extraction using convolution neural networks (CNN) and deep learning"], "source": ["2018 3rd IEEE International conference on recent trends in electronics, information communication technology (RTEICT)"], "year": ["2018"], "publisher-loc": ["Bangalore"], "publisher-name": ["IEEE"], "fpage": ["2319"], "lpage": ["2323"]}, {"label": ["11."], "surname": ["Zhu", "Liu", "Fan", "Xie"], "given-names": ["W", "C", "W", "X"], "article-title": ["DeepLung: 3D deep convolutional nets for automated pulmonary nodule detection and classification"], "source": ["bioRxiv"], "year": ["2017"], "pub-id": ["10.1101/189928v1"]}, {"label": ["15."], "surname": ["Hu", "Liu", "Zhang", "Jiang", "Liu", "Zhang"], "given-names": ["L", "Q", "J", "F", "Y", "S"], "article-title": ["A-DARTS: attention-guided differentiable architecture search for lung nodule classification"], "source": ["JEI"], "year": ["2021"], "volume": ["30"], "fpage": ["013012"]}, {"label": ["17."], "surname": ["Dutande", "Baid", "Talbar"], "given-names": ["P", "U", "S"], "article-title": ["LNCDS: A 2D\u20133D cascaded CNN approach for lung nodule classification, detection and segmentation"], "source": ["Biomed Signal Process Control"], "year": ["2021"], "volume": ["67"], "fpage": ["102527"], "pub-id": ["10.1016/j.bspc.2021.102527"]}, {"label": ["18."], "surname": ["Singh", "Sengupta", "Lakshminarayanan"], "given-names": ["A", "S", "V"], "article-title": ["Explainable deep learning models in medical image analysis"], "source": ["J Imaging Sci Technol"], "year": ["2020"], "pub-id": ["10.3390/jimaging6060052"]}, {"label": ["19."], "surname": ["Suryani", "Chang", "Feng", "Lin", "Lin", "Cheng"], "given-names": ["AI", "C-W", "Y-F", "T-K", "C-W", "J-C"], "article-title": ["Lung tumor localization and visualization in chest X-ray images using deep fusion network and class activation mapping"], "source": ["IEEE Access"], "year": ["2022"], "volume": ["10"], "fpage": ["124448"], "lpage": ["124463"], "pub-id": ["10.1109/ACCESS.2022.3224486"]}, {"label": ["20."], "mixed-citation": ["Dutta A, Zisserman A. The VIA annotation software for images, audio and video. arXiv [cs.CV]. 2019. "], "ext-link": ["http://arxiv.org/abs/1904.10699"]}, {"label": ["22."], "mixed-citation": ["Jorge Cardoso M, Li W, Brown R, Ma N, Kerfoot E, Wang Y, et al. MONAI: an open-source framework for deep learning in healthcare. arXiv. 2022. "], "ext-link": ["http://arxiv.org/abs/2211.02701"]}, {"label": ["27."], "surname": ["Jian", "Wu", "Chen", "Fu", "Yang"], "given-names": ["M", "R", "H", "L", "C"], "article-title": ["Dual-branch-UNet: a dual-branch convolutional neural network for medical image segmentation"], "source": ["Comput Model Eng Sci"], "year": ["2023"], "volume": ["137"], "fpage": ["705"], "lpage": ["716"]}]
{ "acronym": [ "3D AG-Net", "AUC", "CAM", "CNNs", "CT", "DL", "ROC", "VDT", "VIA" ], "definition": [ "3D Attention-gated Network", "Area-under-curve", "Class activation map", "Convolutional neural networks", "Computed tomography", "Deep learning", "Receiver operating characteristic", "Volume doubling time", "VGG image annotator" ] }
27
CC BY
no
2024-01-14 23:43:47
J Transl Med. 2024 Jan 13; 22:51
oa_package/a3/5c/PMC10787502.tar.gz
PMC10787503
38217008
[ "<title>Introduction</title>", "<p id=\"Par25\">Between a third and half of patients with liver cirrhosis develop minimal hepatic encephalopathy (MHE), which is defined as the preclinical state of hepatic encephalopathy (HE) [##REF##35724930##1##–##REF##15554421##3##], and is characterized by mild cognitive impairment including alterations in attention, motor performance and balance [##REF##11726083##4##–##REF##27988985##7##]. These alterations are associated with a higher risk of falls, impaired driving ability, and a general deterioration in quality of life of patients [##REF##20978484##8##–##REF##30622374##11##]. Moreover, patients with MHE have an increased risk of developing HE [##REF##11569701##12##]. Early detection of MHE and treatment can help reduce hospitalization costs, prolong life expectancy in patients, and improve overall quality of life [##REF##17295846##13##, ##REF##26164219##14##].</p>", "<p id=\"Par26\">Advances in functional magnetic resonance imaging (fMRI) technology over recent years has made it possible to study functional changes in the brain and cerebellum of people with pathologies affecting neurological function such as MHE, and associate these changes with cognitive alterations specific to each patient. Of particular interest is the emergence of resting-state fMRI, a technique for studying the function of various brain networks and the connections between them without the need for patients to perform any tasks during analysis. Recent studies in MHE have described structural changes such as volume reduction in the hippocampus, focal damage in the precuneus, and microstructural alterations in white matter [##REF##29023586##15##–##REF##24218171##17##]. Brain networks such as the default mode network, the attention network, the visual network, the hippocampus and the thalamus have also shown altered function in MHE patients [##REF##26321490##18##–##REF##23332976##23##]. Some of these alterations correlate with cognitive impairment and memory performance and also with plasma levels of pro-inflammatory cytokines [##REF##29023586##15##, ##REF##29941971##22##]. These studies were performed in patients with liver cirrhosis due to several aetiologies, mainly alcohol-, HCV- and HBV-related cirrhosis [##REF##26321490##18##–##REF##23332976##23##]. Ahluwalia et al. [##REF##25939692##24##] found that brain reserve as shown using the MRI neurometabolic and neurostructural profile is significantly impaired in abstinent alcoholic patients with cirrhosis compared to nonalcoholic patients with cirrhosis. MRI results in that study showed a greater effect of hyperammonemia, brain edema, and significantly higher cortical damage in alcoholic-related cirrhosis as compared to nonalcoholic patients. Studies in patients with HBV-related cirrhosis show abnormalities in subcortical and cortical functional networks, which correlate with disease duration and psychometric tests [##REF##29988437##25##]. HCV-related cognitive decline is associated with neuroinflammation and structural disintegrity in basal ganglia, frontal and occipital white matter [##REF##31405320##26##], but functional MRI studies in HCV patients with MHE are scant.</p>", "<p id=\"Par27\">The underlying mechanisms of alterations in neuronal connectivity associated to MHE are not known. Alterations in the gut-liver-brain axis seem to play a relevant role in the induction of MHE [##REF##24690956##27##]. Gut microbiome is altered in patients with liver cirrhosis which may contribute to alterations in the immune system and cognition [##REF##24690956##27##]. Peripheral inflammation and hyperammonemia play synergistic roles in inducing MHE [##REF##14739095##28##, ##REF##22072427##29##], and we previously showed that MHE appearance is associated with specific changes in immune system and peripheral inflammation [##REF##28751644##30##]. In animal models of hyperammonemia and MHE, it was shown that peripheral alterations are transmitted to brain inducing neuroinflammation, which alters neurotransmission, leading to cognitive and motor impairment [##REF##30830722##31##]. A similar process would occur in MHE patients. Patients died with liver disease show neuroinflammation in cerebellum, with activation of microglia and astrocytes and loss of Purkinje and granular neurons [##REF##29445232##32##]. Based on these studies, the sequence of events that would induce MHE could be the following: alterations in immunological system and in inflammatory parameters associated with MHE would be transmitted to brain, leading to alterations in neurotransmission and functional connectivity, which would trigger cognitive and motor alterations.</p>", "<p id=\"Par28\">Together with the early detection provided by the Psychometric Hepatic Encephalopathy Score (PHES) as the “gold standard” for the diagnosis of MHE [##REF##11434627##33##, ##REF##11870389##34##], some treatments have shown an ability to prevent MHE progression towards HE. According to clinical practice guidelines [##REF##35724930##1##], treatment can be initiated with lactulose and/or rifaximin on suspicion of MHE. Both treatments exert their effects mainly by regulating the activity of the gut microbiota, and have proven effective for reversal of MHE [##REF##31476436##35##–##REF##23565181##37##]. No remarkable differences in the effectivity of one over the other can be found in the literature [##REF##37467180##38##], although rifaximin is better tolerated than lactulose [##REF##26201713##39##].</p>", "<p id=\"Par29\">Some clinical trials studied the effect of rifaximin in patients with MHE. They reported an improvement in driving and cognitive skills, quality of life and reduction of endotoxins [##REF##23565181##37##, ##UREF##0##40##]. Other studies showed that rifaximin prevented HE episodes and relapses [##REF##25339518##36##, ##REF##24365449##41##]. We previously showed that rifaximin treatment reverses immunophenotype and inflammatory alterations and improves cognitive function in some MHE patients but not in others [##REF##31462286##42##], and that patients with clinical signs of metabolic syndrome have a poor response to rifaximin for MHE [##REF##35165326##43##]. Moreover, after rifaximin treatment, there was a decrease in a parameter of axonal injury in patients who responded to treatment [##REF##37834174##44##].</p>", "<p id=\"Par30\">The present study investigates the effects of rifaximin treatment on brain function in MHE patients using fMRI techniques. We analysed whether improvement by rifaximin of cognitive function and peripheral inflammation is associated with changes in brain functional connectivity. We analysed both independent brain network function and the functional connections between these networks, the latter representing a new approach to study of the effects of MHE and rifaximin. We also examined both pre- and post-treatment differences between patients who respond favourably or not to rifaximin treatment. Finally, we ascertained the correlation between these changes and improvements in psychometric performance and inflammatory parameters after treatment.</p>" ]
[ "<title>Patients and methods</title>", "<title>Participants</title>", "<p id=\"Par31\">A total of 53 patients with liver cirrhosis and 23 healthy controls without liver disease were enrolled as volunteers onto the study after written informed consent. Patients were recruited between July 2015 and January 2019 from the outpatient clinics of Hospital Clinico Universitario and Hospital Arnau de Vilanova, in Valencia, Spain. Inclusion criteria were clinical, biochemical, and histological evidence of liver cirrhosis. For healthy controls, liver disease was discarded via clinical, analytical, and serologic analysis. Exclusion criteria included HE or history of HE, alcohol intake during the 6 months prior to recruitment, infections, antibiotic use or gastrointestinal bleeding during the 6 weeks prior to recruitment, history of shunt surgery or transjugular intrahepatic portosystemic shunt for portal hypertension, use of drugs that affect cognitive function, hepatocellular carcinoma, and neurological or psychiatric disorders. Patients included in the study before and after rifaximin treatment, did not show fever or any clinical or biological sign of recent infection. Psychometric, attention and coordination tests, and blood collection were performed on the same day. Twenty-two patients were classified as without MHE (nMHE) and 31 as with MHE using the PHES battery (see below) [##REF##11434627##33##, ##REF##11870389##34##]. fMRI acquisition was performed on healthy controls and MHE patients in the week following neuropsychological assessment. After fMRI acquisition, four subjects (one healthy control and three MHE patients) were excluded from the study due to poor acquisition caused by excessive head movement during the process (translation &gt; 2.5 mm or rotation &gt; 2.5˚). After this reduction, 22 healthy controls and 28 patients remained (Fig. ##FIG##0##1##).</p>", "<p id=\"Par32\">Study protocols were approved by Scientific and Research Ethics Committees of Hospitals Clinico and Arnau Vilanova, Valencia, Spain, (F-CE-GEva-15; 2018.51) and classified by the Spanish Agency of Medicines and Medical Devices (CMF-NRT-2017). The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. The demographic characteristics and disease aetiology of each group are shown in Table ##TAB##0##1##.</p>", "<title>Diagnosis of MHE</title>", "<p id=\"Par33\">MHE was diagnosed using the PHES battery of tests [##REF##11434627##33##, ##REF##11870389##34##]. Scores were adjusted for age and education level using Spanish normality tables (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.redeh.org/TEST_phes.htm\">https://www.redeh.org/TEST_phes.htm</ext-link>. Accessed on 14 July 2023). Patients were classified as MHE when PHES score was ≤  − 4 points.</p>", "<p id=\"Par34\">Additional psychometric tests performed were focused on different cognitive functions: cognitive flexibility and inhibitory control (Stroop test); selective, sustained attention and mental concentration (d2 test); mental processing speed (Oral Symbol Modalities test, SDMT); working memory (digit span and letter-number sequencing test, from Wechsler Adults Intelligence Scale), and bimanual and visuomotor coordination tests. All tests were performed as previously described [##REF##28146589##45##].</p>", "<title>Ammonia and pro-inflammatory cytokines level measurement</title>", "<p id=\"Par35\">Blood ammonia levels were measured immediately after blood collection using the Ammonia Test Kit II for the PocketChemBA system (Arkay, Inc., Kyoto, Japan). Plasma concentrations of IL-6, IL-18, IL-22 (Affymetrix eBioscience, Vienna, Austria), IL-15, CCL20, CXCL13 and CX3CL1 (R&amp;D Systems, Minneapolis, MN, USA) were measured by ELISA according to the manufacturer’s instructions.</p>", "<title>Rifaximin treatment</title>", "<p id=\"Par36\">Of the 28 MHE patients, 22 were prescribed rifaximin treatment (1.2 g/day, in three doses of 400 mg every 8 h) after the first fMRI acquisition session. The remaining 6 subjects underwent the prior acquisition of MRI, but they subsequently refused to continue in the study, so these patients could not be followed up, regardless of whether or not they had been prescribed treatment. After 6 months of treatment, patients underwent a second psychometric evaluation and fMRI acquisition session, during which four patients dropped out of the study: one due to adverse effects, two died and one declined to undergo a second interview and acquisition session. This reduced the study group to a total of 18 patients with known treatment response and follow-up fMRI acquisition, of which 13 responded favourably to rifaximin (responders), while the other five showed a lack of response (non-responders). Patients whose PHES score results were consistent with nMHE patients (PHES score &gt; − 4) were classified as responders, while those not matching these criteria were classified as non-responders (Fig. ##FIG##0##1##). The demographic characteristics and disease aetiology of response groups are in Table ##TAB##0##1##.</p>", "<p id=\"Par37\">No HE episodes occurred during the 6 months of treatment with rifaximin, and there were few cases of other decompensations, such as ascites (2 patients) or portal thrombosis (1 patient), all occurring in the responder group, which indicates that most patients were clinically stable.</p>", "<title>Image acquisition</title>", "<p id=\"Par38\">All subjects underwent an MRI scan using a 3 T Philips Achieva scanner (Philips Medical Systems, Netherlands). Sagittal high-resolution three-dimensional 3D MPRAGE T1 images were acquired (TR = 8.42 ms, TE = 3.8 ms, matrix = 320 × 320 × 250, voxel size = 1 × 1 × 1 mm, flip angle = 8˚). In addition, functional MRI resting-state data was acquired using a gradient-echo T2-weighted echo-planar imaging (EPI) sequence (5 min, 150 volumes, TR = 2000 ms, TE = 30 ms, matrix = 80 × 80 × 31, voxel size = 3 × 3 × 3 mm, flip angle = 85˚). During the resting sequence, participants were instructed to remain motionless and relax with their eyes open, not fall asleep and think of nothing in particular.</p>", "<title>Image pre-processing</title>", "<p id=\"Par39\">All processing and data analysis of fMRI data were conducted using the Oxford Centre for Functional MRI of the Brain (FMRIB) Software Library (FSL) version 6.0.1, and third party tools specially developed for this software [##REF##21979382##46##].</p>", "<p id=\"Par40\">The first ten images of the fMRI time series were discarded to account for magnetic saturation effects. Remaining volumes were motion-corrected using MCFLIRT [##REF##12377157##47##]. Brain extraction, or cropping, was then performed on motion-corrected fMRI volumes and structural images using FMRIB’s Brain Extraction Tool [##REF##12391568##48##] and interleaved slice timing correction was conducted. Volumes were spatially smoothed with a 4 mm full width at half maximum Gaussian kernel, and high-pass filtered with a cut-off of 100 s. For their registration to standard space, functional images were linearly registered to their corresponding structural images. Afterwards, non-linear registration of the structural images to MNI152 standard space was performed. Finally, functional images were non-linearly registered to MNI152 standard space using the previous registration of their corresponding structural images. All steps described were applied as part of the FSL Multivariate Exploratory Linear Optimized Decomposition into Independent Components (MELODIC) tool process, which applied single-subject independent component analysis (ICA) on the images resulting from the previously described steps. Automatic estimation of dimensionality was used in this analysis.</p>", "<p id=\"Par41\">The resulting images from MELODIC were denoised using ICA-AROMA [##REF##25770991##49##], which performs another single-subject ICA to remove motion-related components.</p>", "<title>Analysis of fMRI data</title>", "<p id=\"Par42\">Group-level ICA was applied using MELODIC. Connectivity networks were obtained from the images of a random selection of subjects in which the main study groups were equally represented, as per standard protocol in this methodology (10 healthy controls, 5 responder patients and the 5 non-responder patients). All images used were temporally concatenated, and then split into 20–80 independent components, close to the optimal recommended value for studying connectivity alterations in neuropsychiatric diseases [##REF##21687724##50##]. The model with 60 components yielded the most satisfactory results in terms of quantity and quality of detected resting-state networks (RSNs) upon visual inspection. Thirteen components were considered of biological interest (Fig. ##FIG##1##2##).</p>", "<p id=\"Par43\">Each of the 13 networks of interest was assigned a name according to its spatial distribution and how it overlapped with RSNs found in reference studies [##REF##16087444##51##, ##REF##19620724##52##]. In cases in which a spatial parallel with these references was not clear, a name was provided depending on the area were the signal of the network was most intense [##UREF##1##53##].</p>", "<p id=\"Par44\">We next performed FSL dual regression [##REF##28348512##54##]. The spatial maps of all components of biological interest were used as spatial regressors on each subject’s fully pre-processed functional images to obtain the time series and spatial map of each identified RSN of each subject. Time series were obtained using dual-threshold regression for their future use as part of the inter-network functional connectivity analysis. Resulting spatial representations were used for the intra-network functional connectivity analysis.</p>", "<p id=\"Par45\">Intra-network functional connectivity was analysed using FSL Randomise [##REF##24530839##55##]. We applied 5000 permutations, family-wise error correction for multiple comparisons and threshold-free cluster enhancement (TFCE) in every test. The analyses were restricted to voxels present in all subjects included in each specific comparison, using a binary mask previously generated by the program.</p>", "<p id=\"Par46\">Inter-network functional connectivity was analysed using graph theory and the FSLNets package available in MATLAB (<ext-link ext-link-type=\"uri\" xlink:href=\"https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSLNets\">https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSLNets</ext-link>). The normalised time series obtained during dual-threshold regression were used for this analysis. Partial correlations between each network pair in each subject were calculated and transformed to Fisher’s z-scores for further analyses. We applied 5000 permutations and family-wise error correction for multiple comparisons in all performed tests.</p>", "<p id=\"Par47\">Three main comparisons were performed both when analysing Intra and Inter-network functional connectivity. All comparisons were modelled using general linear models (GLM) created with the GLM function included in FSL. The aforementioned comparisons included:<list list-type=\"order\"><list-item><p id=\"Par48\">An analysis of the general effect of rifaximin by comparing all patients before and after treatment. In this case we used a GLM similar to a paired t test. The time point of each sample (before vs after treatment) and the identity of each patient, as a correction for the repeated measures analysis, were the factors considered in this case.</p></list-item><list-item><p id=\"Par49\">An analysis of the differences in the effects of rifaximin between responding and non-responding patients. In this case we used a GLM similar to a 2-way mixed effect ANOVA. The time point of each sample (before vs after treatment), the response group of each patient (responding vs non-responding), and the identity of each patient, as a correction for the repeated measures analysis, were the factors considered in this case.</p></list-item><list-item><p id=\"Par50\">An analysis of the pre-existing differences before treatment between responders and non-responders. In this case we used a GLM similar to a t test. The only factor considered in this case was the response group of each patient (responding vs non-responding).</p></list-item></list></p>", "<p id=\"Par51\">For resting-state networks with significant results in either of the first two analyses during the intra-network functional connectivity analysis, a posterior comparison of post-treatment patients and healthy controls was performed using a GLM similar to a t-test. This final analysis was performed to observe if the changes experimented after treatment were sufficient to overcome alterations previously observed by Garcia-Garcia et al. [##REF##29023586##15##]. In this case, the study group of the subjects (patients <italic>vs</italic>. controls) was the only factor we considered.</p>", "<p id=\"Par52\">In analyses 1 and 2 the images of all patients before and after treatment were used. In analysis 3 only the images obtained before treatment were included. For the posterior comparison of patients and controls we used the images of controls and the images of either all the patients after treatment (posterior comparisons related to analysis 1) or responding patients after treatment (posterior comparisons related to analysis 2). In all cases results were considered significant at p &lt; 0.05 after applying the aforementioned corrections.</p>", "<p id=\"Par53\">Statistically significant clusters were associated with functional regions of the brain cortex or cerebellum using Glasser’s functional parcellation atlas [##REF##27437579##56##], Yeo’s cerebellar atlas [##REF##33427596##57##], and chapters 2–9 of A Connectomic Atlas of the Human Cerebrum [##UREF##1##53##].</p>", "<title>Correlation analysis</title>", "<p id=\"Par54\">Correlation analyses were performed using the values obtained in fMRI data analysis. The analyses were limited to intra and inter-network connections experiencing significant changes during treatment or that showed significant between- response group differences before treatment, psychometric tests and biochemical determinations. Spearman’s correlation test was performed using R software (version 4.1.1). False discovery rate (FDR) correction was applied on the resulting correlations, and two-sided p values &lt; 0.05 after correction were considered significant.</p>" ]
[ "<title>Results</title>", "<title>Effects of rifaximin treatment on cognitive tests and inflammatory parameters</title>", "<p id=\"Par55\">MHE patients performed worse in almost all psychometric tests and showed altered levels of all biochemical measurements compared to healthy controls and nMHE patients (Additional file ##SUPPL##0##1##: Table S1). A significant increase in PHES score was observed after treatment (p &lt; 0.01), together with better performance of Oral SDMT (p &lt; 0.001) and Stroop test neutral and incongruent tasks (p &lt; 0.05). d2 and Digit Span and letter-number sequencing tests were not affected (Table ##TAB##1##2##). An improvement in all biochemical measurements (except for ammonia and CXCL13 levels) was observed after treatment (Table ##TAB##1##2##).</p>", "<p id=\"Par56\">All observed improvements were greater when the analyses were restricted to responders, but besides the PHES subtests, no new tests showed previously unobserved changes. A similar effect was observed in all biochemical measurements except fractalkine. None of these changes were observed in non-responders, except for a significant improvement in the number of correct answers in oral SDMT (p &lt; 0.05) (Table ##TAB##1##2##).</p>", "<p id=\"Par57\">We found preexisting differences between responders and non-responders in the performance of both bimanual (p &lt; 0.05) and visuomotor coordination tests (p &lt; 0.05) (Table ##TAB##1##2##).</p>", "<title>Intra-network connectivity</title>", "<p id=\"Par58\">All changes and alterations described in this section, as well as their size and location, are included in Table ##TAB##2##3##. An increased connectivity after rifaximin treatment was observed in cluster MSMN-1 of the medial sensorimotor network (MSMN) in all patients, regardless of response group (Fig. ##FIG##2##3##A). A similar, non-significant tendency was observed in MSMN-2. On the other hand, reduced connectivity was observed in clusters LFPN-1 and LFPN-2 of the left fronto-parietal network (LFPN) (Fig. ##FIG##2##3##B and C), and in cluster LSMN-1 of the lateral sensorimotor network (LSMN) (Fig. ##FIG##2##3##D). Additional non-significant tendencies towards reduction in connectivity were also observed in clusters RFPN-1, LFPN-3 and LFPN-4, in both the right and left fronto-parietal networks (Table ##TAB##2##3##).</p>", "<p id=\"Par59\">When restricting the sample to responding patients, decreased connectivity was observed in cluster LSMN-2 of the LSMN (Fig. ##FIG##2##3##E), as well as a similar non-significant tendency in clusters LFPN-5 and LFPN-6 of the LFPN, which was comparable to results from analysing the whole cohort.</p>", "<p id=\"Par60\">An interaction between treatment effect and response group was observed in cluster THN-1 of the thalamic network (THN) (Fig. ##FIG##2##3##F). A non-significant trend was observed also in cluster THN-2, as well as in cluster LVN-1 of the lateral visual network (LVN). In all cases the observed interaction pointed to a relative increase of connectivity in responders and a relative decrease in non-responders (Table ##TAB##2##3##).</p>", "<p id=\"Par61\">A pre-existing difference in connectivity between responders and non-responders was also found. Responders showed enhanced connectivity in cluster BGN-1, associated with their basal ganglia network (BGN) in comparison to non-responders (Fig. ##FIG##2##3##G). A similar, non-significant, tendency was observed in BGN-2.</p>", "<p id=\"Par62\">The activity of networks whose activity was affected following rifaximin treatment (MSMN, LFPN and LSMN in all patients, and THN in responding patients) was compared with the activity of those networks in healthy controls. With the exception of LFPN, no significant differences were observed between patients after treatment and controls in any network. The differences observed in LFPN were roughly similar to the alterations detected by García-García et al. [##REF##29023586##15##].</p>", "<title>Inter-network connectivity</title>", "<p id=\"Par63\">Our study of the effects of rifaximin independently of patient response revealed a significant change in THN-LSMN connectivity (Fig. ##FIG##3##4##B). The connection went from a slightly negative to a slightly positive value after treatment (p = 0.008). When considering each group separately, non-responders showed no significant change in this connection, but the change observed in responders remained significant (p = 0.027) (Fig. ##FIG##3##4##F).</p>", "<p id=\"Par64\">The connection between the medial visual network (MVN) and THN evolved from a negative value to a more neutral one after treatment (Fig. ##FIG##3##4##D). However, this change was significant only when the analysis was restricted to responders (p = 0.034) (Fig. ##FIG##3##4##H), rather than all patients together (p = 0.053), although the latter trend nonetheless approached significance.</p>", "<p id=\"Par65\">A significant interaction between treatment and response group was observed in the functional connectivity between the MSMN and the dorsal attention network (DAN) (p = 0.049) (Fig. ##FIG##3##4##G). In this case, responders showed an evolution from a positive towards a more neutral value, while non-responders connectivity progressed in the opposite direction. When considering the evolution of each group separately, only the change observed in responders remained significant (p = 0.026). Although the between-group differences in connectivity before the treatment were not significant (p = 0.063), they were nonetheless noteworthy (Fig. ##FIG##3##4##G).</p>", "<p id=\"Par66\">Another significant interaction was observed in the connection between the right fronto-parietal network (RFPN) and the supramarginal gyrus network (SPGN) (p = 0.019) (Fig. ##FIG##3##4##E). In this case responders progressed from a negative value towards a more neutral one, while non-responders showed an evolution in the opposite direction (Fig. ##FIG##3##4##E). Along the same line as the above described connections, when analysing the response groups separately, non-responding patients showed no level of significance, but the changes experienced by responders remained significant (p = 0.026).</p>", "<p id=\"Par67\">No connections between different networks showed significant or approaching significant differences when comparing the initial state of the two response groups.</p>", "<title>Correlation analysis</title>", "<p id=\"Par68\">Results of correlations between intra-network changes and psychometric tests showed that PHES score, DST and Oral SDMT correlated with both clusters related to the fronto-parietal network. DST and Oral SDMT showed a high correlation with LSMN-1 as well (Table ##TAB##3##4##). Clusters MSMN-1, LSMN-2, THN-1 and BGN-1 showed no significant correlations with any psychometric tests.</p>", "<p id=\"Par69\">Observing the correlations between intra-network alterations and biochemical parameters, all clusters related to the general effect of rifaximin were significantly correlated with at least one inflammatory parameter (Table ##TAB##3##4##). Ammonia levels showed a significant correlation with intra-network connectivity only in the MSMN-1 cluster. The presence of IL6 was particularly notable, as it was the only biochemical measurement that correlated with all the included clusters. IL15, IL18 and IL22 had a remarkable presence as well, with LFPN-1 being the only cluster that did not correlate with at least one of them. All significant correlations of clusters LFPN-1, LFPN-2, LSMN-1 and LSMN-2 with all biochemical parameters were positive, while those of MSMN-1 were negative.</p>", "<p id=\"Par70\">When we analysed the THN-1 correlations in the total group of treated MHE patients, no significant correlations were observed. Given that the evolution of this cluster is opposite in responder and non-responder patients (Table ##TAB##2##3##), we performed the correlations in the responder group to assess whether the change in THN-1 signal was accompanied by improvements in cognitive or biochemical parameters. As for the correlations with the psychometric tests, only the incongruent Stroop task and the SDMT scores tended to be significant (r = 0.5, p = 0.06 and r = 0.49, p = 0.07, respectively) but the small sample size and the correction of the p-values made them less significant. For biochemical parameters, significant correlations were observed with IL18 (r = − 0.61; p = 0.008) and IL15 (r = − 0.63; p = 0.007), which after correction for p-values became trends (p = 0.07 and p = 0.06, respectively) (Additional file ##SUPPL##0##1##: Table S2).</p>", "<p id=\"Par71\">There were no significant correlations between functional connectivity in BGN-1 cluster and psychometric or biochemical parameters.</p>", "<p id=\"Par72\">Several significant results also emerged from analysis of correlations between inter-network connections and psychometric tests (Table ##TAB##4##5##). In this case, however, the only connection that showed significant correlations was the MVN-THN connection. This connection correlated significantly with several scores in almost all psychometric tests performed, including tests assessing cognitive flexibility (Stroop test), mental processing speed (Oral SDMT, and DST from the PHES battery) and sustained concentration (d2 test). Subtest LTT from the PHES battery and the Bimanual coordination test, which evaluate motor coordination, were significantly correlated with the MVN-THN connection as well (Table ##TAB##4##5##).</p>", "<p id=\"Par73\">Significant correlations between inter-network connections and biochemical measurements were also observed, but quite limited. IL18 correlated significantly with both the MSMN-DAN and RFPN-SPGN connections (Table ##TAB##4##5##). CXCL13 showed a significant correlation with the MSMN-DAN connection as well, while IL6 and IL15 correlated with the RFPN-SPGN connection.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par74\">In this study, we assessed the effects of rifaximin on the activity of different resting-state networks in MHE patients, and on the functional connectivity between them. Besides this, we also investigated pre-existing differences between patients who did or did not respond to treatment. Finally, we analysed the relationship between these alterations and different psychometric and biochemical alterations also observed in patients with MHE.</p>", "<p id=\"Par75\">It is important to note that, given the non-absorbable nature of rifaximin, all the effects observed in this study would be an indirect result of its effects on the gut microbiota and the regulation of inflammatory imbalances present in patients with cirrhosis [##REF##23565181##37##, ##REF##31462286##42##].</p>", "<title>Effect on sensorimotor, fronto-parietal, and thalamic networks following rifaximin treatment</title>", "<p id=\"Par76\">Most intra-network effects were observed after rifaximin treatment on a general level, encompassing all treated patients. We observed generally increased sensorimotor network activity in the left caudate, and generally decreased left fronto-parietal network activity in the left Brodmann area 2. Both these abovementioned effects after rifaximin treatment have previously been reported in MHE patients performing N-back and inhibitory control tests with fMRI [##REF##24590688##58##]. Additionally, thalamic network activity in area a9-46v was relatively increased in responders compared to non-responders. Area a9-46v is part of the dorsolateral prefrontal cortex (DLPFC), which is involved in executive function. The effects observed in the thalamic network function after rifaximin treatment could be related to improved thalamus function and structure, which have been reported as deteriorated in MHE patients [##REF##23332976##23##, ##REF##33716679##59##]. A significant reduction of the connectivity of the lateral sensorimotor network was observed as well, but the clusters we found were very small, and of dubious biological significance.</p>", "<p id=\"Par77\">Studying inter-network connections, a general effect was observed after rifaximin treatment in the connection between the thalamic and lateral sensorimotor networks, which went from a negative to a positive z-score. Other inter-network connections also underwent significant changes, but in these cases among responding patients only. These included connections between medial sensorimotor and dorsal attention network (involved in visuospatial attention) the right fronto-parietal and supramarginal gyrus network (somatosensory perception) and the thalamic and medial visual network (visuospatial perception).</p>", "<p id=\"Par78\">Taken together, these results suggest that rifaximin indirectly helps improve communication between brain networks and areas mainly involved in executive function (i.e.: the fronto-parietal and dorsal attention networks, and the DLPFC), in areas that play a role mainly in processing different stimuli (i.e.: the thalamus, Brodmann area 2, supramarginal gyrus and visual network), which provide the sensorial information necessary for executive functions to be performed. Although seemingly less remarkable, changes in communication with areas involved in motor control (i.e.: sensorimotor network and caudate) were also observed.</p>", "<p id=\"Par79\">Several significant results were observed upon performing correlation analyses between the above changes and patient cognitive performance. Most tests were present to a certain level in these analyses, but the most prevalent were the Oral-SDMT and other tests that evaluate functions such as attention, and mental processing speed. Most of the significant correlations we observed involved clusters related to the activity of the left fronto-parietal network, which is directly involved in these and other executive functions.</p>", "<p id=\"Par80\">The correlation between the cognitive performance of the patients and the connection between the medial visual and the thalamic network was remarkable as well. This could be due to the fact that, even though this connection evolved in a similar direction both in responding and non-responding patients, the changes observed were significant only when limited to the responding patients; a similar pattern to that observed in most cognitive tests. These correlations would suggest a relationship between the improvement of the visuospatial perception and processing of the patients, and their cognitive performance, especially in attention related tests.</p>", "<title>Reduction of aberrant hyperconnectivity following rifaximin treatment</title>", "<p id=\"Par81\">In most cases, the connections between different networks seemed to evolve towards greater independence from each other after treatment. Similarly, global intra-network changes in connectivity of the fronto-parietal and lateral sensorimotor networks followed a similar tendency of decreased connectivity after treatment. These results suggest that the treatment improves brain function by ameliorating aberrant hyperconnectivity suffered by MHE patients. This pattern has already been observed in MHE and in other pathologies, such as multiple sclerosis and traumatic brain injury [##REF##27250065##19##, ##REF##22662152##21##, ##REF##24933491##60##].</p>", "<title>Rifaximin treatment does not recover a normal brain function</title>", "<p id=\"Par82\">Of the networks affected by rifaximin treatment, only the left fronto-parietal network has been previously identified as altered in MHE patients compared to healthy controls [##REF##29023586##15##]. The activity of the sensorimotor and thalamic networks, which were shown to be affected by rifaximin in this study, has not shown significant alterations in previous studies, so it was expected to neither observe significant differences between patients after treatment and controls when studying the activity of these networks.</p>", "<p id=\"Par83\">Despite the effects of rifaximin, the left fronto-parietal network remained similarly altered. These and other alterations in connectivity were largely still present in patients after treatment. This result is not unexpected, considering that the changes driven by rifaximin in left fronto-parietal network connectivity occurred at different locations from those caused by MHE, and that they further decreased connectivity, rather than increasing it to a state approaching that of a healthy individual [##REF##29023586##15##]. Besides this network, there seem to be no other common point between the networks altered in patients with MHE and those affected by rifaximin. Altogether, these results suggest that 6 months of rifaximin treatment improves cognitive function at a neurological level in responding patients yet the mechanisms involved do not necessarily include reversal of MHE-related alterations, thus pointing to a certain level of redundancy in the neural circuits involved in the process [##REF##34416675##61##]. On the other hand, the prevalence of the alterations caused by MHE suggest a high risk of relapse if patients abandon treatment [##REF##28051800##62##].</p>", "<title>Reduction of inflammation, but not ammonia levels after rifaximin treatment</title>", "<p id=\"Par84\">Comparing inflammatory cytokine levels before and after treatment, most decreased significantly in responders, but not in non-responders. Even taking a lack of significance due to the small patient sample size into account, IL6 and IL22 were the only cytokines that evolved in a similar way regardless of group response. In other cases, non-responders showed lesser reduction in levels, or directly increased levels, as observed with IL18 and CXCL13. Most of these cytokines show significant correlations with several significant changes revealed in brain function analysis. This points to the already observed effect of inflammation on the brain function in MHE patients, and how its decrease, promoted by rifaximin, helps restore these functions. Similar results have previously been reported, particularly regarding CCL20, CX3CL1, and IL15 levels, which are known to promote lymphocyte infiltration into the brain [##REF##31462286##42##]. Anti-inflammatory effects of rifaximin could be mediated by induction of the expression of pregnane-X-receptor (PXR) in intestinal epithelial cells [##REF##20816942##63##], promoting the transcription of genes for detoxification enzymes and cytokines, ultimately reducing inflammation and improving MHE.</p>", "<p id=\"Par85\">In contrast, no significant decrease in ammonia levels was observed in any patient group after treatment. Lower ammonia levels were reported in patients with overt HE grade I or II after rifaximin treatment [##REF##36555935##64##], but ammonia reduction with rifaximin was not statistically significant in MHE patients [##REF##31476436##35##]. It should be noted, however, that the blood ammonia levels of our study patients were not as high as those of patients with overt HE, which could explain why rifaximin did not alter them substantially.</p>", "<p id=\"Par86\">These results suggest that the improved inflammatory state of MHE patients, and the resulting reduction of lymphocyte infiltration, following rifaximin treatment is enough to favour the observed changes in brain function and enhanced cognitive performance.</p>", "<title>Responding and non-responding patients show pre-existing differences</title>", "<p id=\"Par87\">When considering intra-network connectivity, a difference was observed in the connectivity of the basal ganglia network. It was located in the right medial part of the Brodmann area 7, which is part of the precuneus, and was the largest cluster found in this study. Both the precuneus and basal ganglia play important roles in executive functions such as working memory and visuospatial attention, which have already been observed in this study to improve after treatment in responders. Alterations located in the precuneus and/or related to a decreased connectivity of the basal ganglia have been already reported in patients with MHE [##REF##29023586##15##, ##REF##22465844##16##]. These results indicate that patients responding positively to rifaximin have less deteriorated connectivity in this specific area than patients who do not respond to this treatment.</p>", "<p id=\"Par88\">The lack of significant correlation between pre-existing differences in basal ganglia activity and any neuropsychological test or biochemical parameter would suggest that this difference in connectivity is not related to a remarkable difference in inflammation, hyperammonemia or cognitive performance between groups before treatment.</p>", "<p id=\"Par89\">The main limitation of this study is the small study sample (subdivided even further in certain analyses), which particularly affected the non-responder group and reduced the statistical power of the results obtained. However, the conditions for results to be statistically significant were very strict, because we performed systematic corrections of p-values. Additionally, due to the limitation in sample size and computational power, it was not possible to include all the psychometric and inflammatory measurements as regressors in the general linear models of the performed analyses. In most cases, however, the changes observed in these variables were consistent with the separation of patients in responding and non-responding groups.</p>", "<p id=\"Par90\">Another limitation could be the lack of a placebo group and the open-label design. However, results of this study could be the basis of future randomised, double blind and placebo controlled clinical trials. This study is an exploratory study for characterizing brain functional connectivity modulation by rifaximin treatment that could be useful for future, placebo-controlled trials in MHE utilizing brain MR imaging.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"Par91\">In conclusion, although rifaximin does not directly correct most functional alterations caused by MHE, it favours subtle changes in brain function which frequently correct aberrant hyperconnectivity, and which are related to improvement in different executive functions as well as pro-inflammatory cytokine normalization in patients who respond favourably to treatment. This improvement, however, does not extend to blood ammonia levels in MHE patients. We also found pre-existing increased connectivity in the precuneus of patients who showed a favourable response to treatment. Finally, the results obtained in this study using the analysis of inter-network connections via FSLNets and graph theory show the potential of an approach rarely applied in the study of functional connectivity, and completely novel in the study of rifaximin effects on MHE.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Rifaximin is a non-reabsorbable antibiotic which acts at gut level, and improves cognition and inflammatory parameters in minimal hepatic encephalopathy (MHE) patients, but not all patients show the same level of response. This study aims to assess brain activity, both within and between brain networks, following rifaximin treatment, considering the differences between response groups as well.</p>", "<title>Methods</title>", "<p id=\"Par2\">Twenty-two healthy controls and 53 patients with cirrhosis (22 without and 31 with MHE, diagnosed by Psychometric Hepatic Encephalopathy Score, PHES) performed psychometric, attention and coordination tests, and blood inflammatory parameters were measured. Resting-state functional magnetic resonance imaging (fMRI) acquisitions were performed on controls and MHE patients. Eighteen MHE patients underwent a rifaximin treatment for 6 months, after which all measures were repeated. fMRI images were analysed and changes after treatment were assessed.</p>", "<title>Results</title>", "<p id=\"Par3\">After rifaximin treatment, 13 patients improved their PHES score (Responder patients) while 5 did not (Non-responder patients). No significant decrease in blood ammonia was observed after rifaximin treatment, but there was a decrease in plasma inflammatory cytokines in responder patients. A global effect of rifaximin was detected on the sensorimotor and fronto-parietal networks. Responder patients showed a relative increase of thalamic network connectivity in comparison to non-responder patients. Before treatment, responder and non-responder patients showed connectivity differences in basal ganglia network. The connection of the sensorimotor and thalamic networks between them and with other networks suffered changes after treatment. These connections between networks mostly decreased after treatment. All changes and differences showed a significant level of correlation with the performance of psychometric tests and the blood levels of inflammatory biomarkers.</p>", "<title>Conclusions</title>", "<p id=\"Par4\">There was an improvement of the communication between executive, motor and attention-related brain areas, and their functional independence following rifaximin treatment. Patients who respond also show a less deteriorated connection involved in these functions before treatment. Results suggest that the improved inflammatory state of MHE patients, following rifaximin treatment would favour the observed changes in brain function and enhanced cognitive performance.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s12967-023-04844-7.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>None.</p>", "<title>Author contributions</title>", "<p>Conceptualization: CM, FC, AU. Formal analysis: FC, JJG, AF, AU. Funding acquisition: CM, DEG, MPB, EK. Investigation: FC, JJG, AF, AU, MPB, MPR, JLL, DEG, EK, VB, CM. Methodology: FC, JJG, AF, MPB, JLL, VB. Project administration: CM, DEG, VB. Resources: CM, DEG, MPR, MPB, JLL, EK, VB. Software: FC, VB, JLL. Supervision: CM, DEG, VB. Visualization: FC, AU. Writing—original draft: FC, AU, CM. Writing—review and editing: all authors. All authors have read and agreed to the published version of the manuscript.</p>", "<title>Funding</title>", "<p>This work was supported by Agencia Valenciana de Innovación, Generalitat Valenciana (Consolidacio Cadena Valor) to C.M.; by Consellería Educación, Generalitat Valenciana (CIPROM2021/082 to C.M. and to D.E.G.); Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (PI23/00062 to CM and MPB; Contrato Joan Rodés, JR23/00029) co-funded with European Regional Development Funds (ERDF); F. Sarabia Donation (PRV00225) to C.M.; and Universidad de Valencia, Ayudas para Acciones Especiales (UV-INV_AE-2633839) to C.M. This study has been conducted in accordance with the theme 075–01025-23–01 ITEB RAS has been assigned to E.K. for the year 2023 and 2024–2025 planning period. A.F. and F.C.F have pre-doctoral contracts from Generalitat Valenciana (GRISOLIAP/2019/003; ACIF/2019/232). J.J.G. has a contract from Generalitat Valenciana (CIAPOT/2021/20). A.U. has a PROMETEO contract from Generalitat Valenciana (CIPROM2021/082). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.</p>", "<title>Availability of data and materials</title>", "<p>Data is contained within the article and Additional files.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par92\">Study protocols were approved by Scientific and Research Ethics Committees of Hospitals Clinico and Arnau Vilanova, Valencia, Spain, (F-CE-GEva-15; 2018.51) and classified by the Spanish Agency of Medicines and Medical Devices (CMF-NRT-2017). The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.</p>", "<title>Consent for publication</title>", "<p id=\"Par93\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par94\">The authors declare no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Flow chart of the steps followed in the selection of the study sample and follow-up that led to the final study groups</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Spatial maps of the 13 resting-state networks identified and analysed in the study. Maps are thresholded at 3 &lt; Z &lt; 10. Images are shown following MNI convention</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Spatial maps of all the clusters in which significant results were observed during the intra-network functional connectivity analysis. Clusters are shown as follows: <bold>a</bold> MSMN-1, <bold>b</bold> LFPN-1, <bold>c</bold> LFPN-2, <bold>d</bold> LSMN-1, <bold>e</bold> LSMN-2, <bold>f</bold> THN-1, <bold>g</bold> BGN-1. All clusters (green) are pointed (yellow arrows). The resting-state network (red) associated with each cluster is thresholded at 3 &lt; Z &lt; 10. All results were cluster-corrected for multiple comparisons using family-wise error (FWE), in combination with a threshold of p &lt; 0.001 at the uncorrected voxel level. Clusters were considered significant at p &lt; 0.05 after FWE correction. Images are shown following MNI convention</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Boxplots showing z scores of inter-networks connections with significant changes after rifaximin treatment. <bold>A</bold>–<bold>D</bold> Considering all patients as one group, before and after treatment. <bold>E</bold>–<bold>H</bold> Stratifying patients by response group (Resp: responding patients; NoResp: non-responding patients). Significant pre- and post-treatment differences were analysed using a general linear model similar to a paired t-test and are indicated by (*): *p &lt; 0.05, **p &lt; 0.01. Significant interactions between treatment and response group were analysed using a general linear model similar to a 2-way mixed effect ANOVA and are indicated by (α): α p &lt; 0.05. All p values were corrected for multiple comparisons using FDR, and differences were considered significant at p &lt; 0.05 after correction</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Demographic characteristics and liver disease aetiology by group</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" rowspan=\"2\">Controls (n = 22)</th><th align=\"left\" rowspan=\"2\">nMHE patients (n = 22)</th><th align=\"left\" rowspan=\"2\">MHE patients (n = 31)</th><th align=\"left\" colspan=\"2\">MHE patients following treatment (n = 18)</th></tr><tr><th align=\"left\">Response (n = 13)</th><th align=\"left\">No response (n = 5)</th></tr></thead><tbody><tr><td align=\"left\">Sex (M/F)</td><td align=\"left\">14/8</td><td align=\"left\">15/7</td><td align=\"left\">26/5</td><td align=\"left\">11/2</td><td align=\"left\">5/0</td></tr><tr><td align=\"left\">Age<sup>†</sup></td><td align=\"left\"><p>60 ± 6</p><p>(50–73)</p></td><td align=\"left\"><p>62 ± 8</p><p>(50–81)</p></td><td align=\"left\"><p>64 ± 9</p><p>(48–85)</p></td><td align=\"left\"><p>62 ± 7</p><p>(53–74)</p></td><td align=\"left\"><p>65 ± 10</p><p>(49–74)</p></td></tr><tr><td align=\"left\">Aetiology</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Alcohol</td><td align=\"left\"/><td align=\"left\">7</td><td align=\"left\">13</td><td align=\"left\">8</td><td align=\"left\">1</td></tr><tr><td align=\"left\"> Hepatitis (HCV/HBV)</td><td align=\"left\"/><td align=\"left\">11/0</td><td align=\"left\">9/1</td><td align=\"left\">4/0</td><td align=\"left\">0/1</td></tr><tr><td align=\"left\"> Metabolic</td><td align=\"left\"/><td align=\"left\">2</td><td align=\"left\">6</td><td align=\"left\">0</td><td align=\"left\">2</td></tr><tr><td align=\"left\"> Other</td><td align=\"left\"/><td align=\"left\">2</td><td align=\"left\">2</td><td align=\"left\">1</td><td align=\"left\">1</td></tr><tr><td align=\"left\">Child Pugh A/B/C<sup>‡</sup></td><td align=\"left\"/><td align=\"left\">19/3/0</td><td align=\"left\">17/10/4*</td><td align=\"left\">8/5/0</td><td align=\"left\">4/1/0</td></tr><tr><td align=\"left\">MELD<sup>†</sup><sup>,</sup><sup>§</sup></td><td align=\"left\"/><td align=\"left\">8 ± 2</td><td align=\"left\">10 ± 4*</td><td align=\"left\">9 ± 3</td><td align=\"left\">8 ± 2</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Psychometric and biochemical characteristics of responding and non-responding MHE patients before and after rifaximin treatment</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">General</th><th align=\"left\" colspan=\"2\">Responder</th><th align=\"left\" colspan=\"2\">Non-responder</th></tr><tr><th align=\"left\">Before treatment</th><th align=\"left\">After treatment</th><th align=\"left\">Before treatment</th><th align=\"left\">After treatment</th><th align=\"left\">Before treatment</th><th align=\"left\">After treatment</th></tr></thead><tbody><tr><td align=\"left\">PHES global score<sup>‡</sup></td><td char=\"±\" align=\"char\">− 7.3 ± 0.85</td><td char=\"±\" align=\"char\">− 4.6 ± 0.76**</td><td char=\"±\" align=\"char\">− 6.7 ± 0.94</td><td char=\"±\" align=\"char\">− 3.2 ± 0.66**</td><td char=\"±\" align=\"char\">− 8.8 ± 1.8</td><td char=\"±\" align=\"char\">− 8.4 ± 0.68</td></tr><tr><td align=\"left\"> DST (items completed)<sup>†</sup></td><td char=\"±\" align=\"char\">21 ± 2</td><td char=\"±\" align=\"char\">27 ± 1.9*</td><td char=\"±\" align=\"char\">22 ± 2.5</td><td char=\"±\" align=\"char\">30 ± 2*</td><td char=\"±\" align=\"char\">16 ± 2.8</td><td char=\"±\" align=\"char\">19 ± 1.9</td></tr><tr><td align=\"left\"> NCT-A (seconds)<sup>‡</sup></td><td char=\"±\" align=\"char\">74 ± 11</td><td char=\"±\" align=\"char\">57 ± 7*</td><td char=\"±\" align=\"char\">67 ± 12</td><td char=\"±\" align=\"char\">50 ± 8.2</td><td char=\"±\" align=\"char\">93 ± 26</td><td char=\"±\" align=\"char\">73 ± 11</td></tr><tr><td align=\"left\"> NCT-B (seconds)<sup>‡</sup></td><td char=\"±\" align=\"char\">228 ± 39</td><td char=\"±\" align=\"char\">159 ± 23</td><td char=\"±\" align=\"char\">212 ± 44</td><td char=\"±\" align=\"char\">127 ± 21*</td><td char=\"±\" align=\"char\">271 ± 85</td><td char=\"±\" align=\"char\">238 ± 49</td></tr><tr><td align=\"left\"> SD (seconds)<sup>‡</sup></td><td char=\"±\" align=\"char\">119 ± 8.2</td><td char=\"±\" align=\"char\">107 ± 9.3*</td><td char=\"±\" align=\"char\">116 ± 11</td><td char=\"±\" align=\"char\">92 ± 6.5*</td><td char=\"±\" align=\"char\">127 ± 12</td><td char=\"±\" align=\"char\">147 ± 21</td></tr><tr><td align=\"left\"> LTT (seconds + errors)<sup>‡</sup></td><td char=\"±\" align=\"char\">210 ± 14</td><td char=\"±\" align=\"char\">178 ± 16</td><td char=\"±\" align=\"char\">196 ± 13</td><td char=\"±\" align=\"char\">155 ± 13*</td><td char=\"±\" align=\"char\">247 ± 37</td><td char=\"±\" align=\"char\">237 ± 35</td></tr><tr><td align=\"left\">Stroop-congruent task<sup>‡</sup></td><td char=\"±\" align=\"char\">76 ± 4.2</td><td char=\"±\" align=\"char\">82 ± 4.2</td><td char=\"±\" align=\"char\">80 ± 4.9</td><td char=\"±\" align=\"char\">86 ± 4.8</td><td char=\"±\" align=\"char\">67 ± 6.9</td><td char=\"±\" align=\"char\">69 ± 5</td></tr><tr><td align=\"left\">Stroop-neutral task<sup>‡</sup></td><td char=\"±\" align=\"char\">56 ± 2.3</td><td char=\"±\" align=\"char\">62 ± 2.6*</td><td char=\"±\" align=\"char\">58 ± 2.4</td><td char=\"±\" align=\"char\">64 ± 3.1*</td><td char=\"±\" align=\"char\">50 ± 4.7</td><td char=\"±\" align=\"char\">56 ± 2.8</td></tr><tr><td align=\"left\">Stroop-incongruent task<sup>†</sup></td><td char=\"±\" align=\"char\">28 ± 2.1</td><td char=\"±\" align=\"char\">35 ± 2.2*</td><td char=\"±\" align=\"char\">29 ± 2.1</td><td char=\"±\" align=\"char\">35 ± 2.1*</td><td char=\"±\" align=\"char\">24 ± 5</td><td char=\"±\" align=\"char\">37 ± 7.1</td></tr><tr><td align=\"left\">Bimanual coordination (min)<sup>‡</sup></td><td char=\"±\" align=\"char\">3.3 ± 0.37</td><td char=\"±\" align=\"char\">3 ± 0.2</td><td char=\"±\" align=\"char\">2.8 ± 0.18</td><td char=\"±\" align=\"char\">2.7 ± 0.13</td><td char=\"±\" align=\"char\">4.5 ± 1.1<sup>α</sup></td><td char=\"±\" align=\"char\">3.8 ± 0.49</td></tr><tr><td align=\"left\">Visuo-motor coordination (min)<sup>‡</sup></td><td char=\"±\" align=\"char\">3.8 ± 0.21</td><td char=\"±\" align=\"char\">3.6 ± 0.27</td><td char=\"±\" align=\"char\">3.5 ± 0.24</td><td char=\"±\" align=\"char\">3.3 ± 0.31</td><td char=\"±\" align=\"char\">4.4 ± 0.3<sup>α</sup></td><td char=\"±\" align=\"char\">4.4 ± 0.41</td></tr><tr><td align=\"left\" colspan=\"7\">d2 test</td></tr><tr><td align=\"left\"> TR Values<sup>†</sup></td><td char=\"±\" align=\"char\">279 ± 19</td><td char=\"±\" align=\"char\">290 ± 20</td><td char=\"±\" align=\"char\">288 ± 23</td><td char=\"±\" align=\"char\">308 ± 23</td><td char=\"±\" align=\"char\">246 ± 23</td><td char=\"±\" align=\"char\">233 ± 32</td></tr><tr><td align=\"left\"> TA Values<sup>†</sup></td><td char=\"±\" align=\"char\">97 ± 7.9</td><td char=\"±\" align=\"char\">105 ± 9.5</td><td char=\"±\" align=\"char\">97 ± 10</td><td char=\"±\" align=\"char\">113 ± 11</td><td char=\"±\" align=\"char\">96 ± 5.8</td><td char=\"±\" align=\"char\">80 ± 16</td></tr><tr><td align=\"left\"> O Values<sup>‡</sup></td><td char=\"±\" align=\"char\">23 ± 6.8</td><td char=\"±\" align=\"char\">16 ± 5</td><td char=\"±\" align=\"char\">27 ± 8.3</td><td char=\"±\" align=\"char\">15 ± 6.5</td><td char=\"±\" align=\"char\">10 ± 3.8</td><td char=\"±\" align=\"char\">18 ± 3.4</td></tr><tr><td align=\"left\"> C Values<sup>‡</sup></td><td char=\"±\" align=\"char\">11 ± 4.1</td><td char=\"±\" align=\"char\">6.8 ± 2.8</td><td char=\"±\" align=\"char\">13 ± 5</td><td char=\"±\" align=\"char\">5.5 ± 3.3</td><td char=\"±\" align=\"char\">4.7 ± 2.2</td><td char=\"±\" align=\"char\">11 ± 5.1</td></tr><tr><td align=\"left\"> O + C Values<sup>‡</sup></td><td char=\"±\" align=\"char\">35 ± 9.7</td><td char=\"±\" align=\"char\">23 ± 8.1</td><td char=\"±\" align=\"char\">40 ± 12</td><td char=\"±\" align=\"char\">21 ± 11</td><td char=\"±\" align=\"char\">15 ± 5.7</td><td char=\"±\" align=\"char\">29 ± 8.2</td></tr><tr><td align=\"left\"> TOT Values<sup>†</sup></td><td char=\"±\" align=\"char\">247 ± 18</td><td char=\"±\" align=\"char\">268 ± 21</td><td char=\"±\" align=\"char\">249 ± 22</td><td char=\"±\" align=\"char\">287 ± 23</td><td char=\"±\" align=\"char\">240 ± 25</td><td char=\"±\" align=\"char\">204 ± 39</td></tr><tr><td align=\"left\"> CON Values<sup>†</sup></td><td char=\"±\" align=\"char\">85 ± 11</td><td char=\"±\" align=\"char\">99 ± 11</td><td char=\"±\" align=\"char\">84 ± 14</td><td char=\"±\" align=\"char\">108 ± 12</td><td char=\"±\" align=\"char\">92 ± 4.1</td><td char=\"±\" align=\"char\">70 ± 22</td></tr><tr><td align=\"left\"> VAR Values<sup>‡</sup></td><td char=\"±\" align=\"char\">14 ± 2</td><td char=\"±\" align=\"char\">12 ± 0.82</td><td char=\"±\" align=\"char\">15 ± 2.3</td><td char=\"±\" align=\"char\">12 ± 1.1</td><td char=\"±\" align=\"char\">12 ± 4.5</td><td char=\"±\" align=\"char\">12 ± 0.63</td></tr><tr><td align=\"left\">Oral SDMT-correct pairings<sup>†</sup></td><td char=\"±\" align=\"char\">25 ± 2.9</td><td char=\"±\" align=\"char\">32 ± 2.6***</td><td char=\"±\" align=\"char\">29 ± 3.3</td><td char=\"±\" align=\"char\">35 ± 2.9**</td><td char=\"±\" align=\"char\">17 ± 4.2</td><td char=\"±\" align=\"char\">25 ± 4.8*</td></tr><tr><td align=\"left\">Oral SDMT-incorrect pairings<sup>‡</sup></td><td char=\"±\" align=\"char\">1.5 ± 0.36</td><td char=\"±\" align=\"char\">1.1 ± 0.31</td><td char=\"±\" align=\"char\">1.5 ± 0.31</td><td char=\"±\" align=\"char\">1.1 ± 0.38</td><td char=\"±\" align=\"char\">1.6 ± 1.1</td><td char=\"±\" align=\"char\">1 ± 0.55</td></tr><tr><td align=\"left\">Oral SDMT-total pairings<sup>†</sup></td><td char=\"±\" align=\"char\">27 ± 2.9</td><td char=\"±\" align=\"char\">34 ± 2.6**</td><td char=\"±\" align=\"char\">30 ± 3.3</td><td char=\"±\" align=\"char\">36 ± 2.7*</td><td char=\"±\" align=\"char\">18 ± 4.2</td><td char=\"±\" align=\"char\">26 ± 4.8</td></tr><tr><td align=\"left\">Digit span-forward<sup>†</sup></td><td char=\"±\" align=\"char\">6.8 ± 0.3</td><td char=\"±\" align=\"char\">7.4 ± 0.56</td><td char=\"±\" align=\"char\">7 ± 0.34</td><td char=\"±\" align=\"char\">7.8 ± 0.62</td><td char=\"±\" align=\"char\">6.2 ± 0.58</td><td char=\"±\" align=\"char\">6.6 ± 1.2</td></tr><tr><td align=\"left\">Digit span-backward<sup>†</sup></td><td char=\"±\" align=\"char\">4.1 ± 0.43</td><td char=\"±\" align=\"char\">4.9 ± 0.58</td><td char=\"±\" align=\"char\">4.2 ± 0.59</td><td char=\"±\" align=\"char\">4.9 ± 0.74</td><td char=\"±\" align=\"char\">4 ± 0.32</td><td char=\"±\" align=\"char\">4.8 ± 0.97</td></tr><tr><td align=\"left\">Digit span-total score<sup>†</sup></td><td char=\"±\" align=\"char\">11 ± 0.62</td><td char=\"±\" align=\"char\">12 ± 1.1</td><td char=\"±\" align=\"char\">11 ± 0.8</td><td char=\"±\" align=\"char\">13 ± 1.3</td><td char=\"±\" align=\"char\">10 ± 0.86</td><td char=\"±\" align=\"char\">11 ± 2.2</td></tr><tr><td align=\"left\">Letter-number sequencing test<sup>†</sup></td><td char=\"±\" align=\"char\">5.4 ± 0.72</td><td char=\"±\" align=\"char\">5.7 ± 0.82</td><td char=\"±\" align=\"char\">5.7 ± 0.9</td><td char=\"±\" align=\"char\">6.5 ± 0.96</td><td char=\"±\" align=\"char\">4.6 ± 1.2</td><td char=\"±\" align=\"char\">3.8 ± 1.4</td></tr><tr><td align=\"left\" colspan=\"7\">Biochemical measurements</td></tr><tr><td align=\"left\"> Ammonia<sup>‡</sup></td><td char=\"±\" align=\"char\">39 ± 6.8</td><td char=\"±\" align=\"char\">55 ± 9.8</td><td char=\"±\" align=\"char\">40 ± 7.8</td><td char=\"±\" align=\"char\">57 ± 13</td><td char=\"±\" align=\"char\">38 ± 15</td><td char=\"±\" align=\"char\">49 ± 13</td></tr><tr><td align=\"left\"> IL6<sup>‡</sup></td><td char=\"±\" align=\"char\">3.7 ± 0.45</td><td char=\"±\" align=\"char\">2.3 ± 0.24**</td><td char=\"±\" align=\"char\">3.8 ± 0.54</td><td char=\"±\" align=\"char\">2.2 ± 0.31**</td><td char=\"±\" align=\"char\">3.7 ± 0.88</td><td char=\"±\" align=\"char\">2.3 ± 0.41</td></tr><tr><td align=\"left\"> IL18<sup>†</sup></td><td char=\"±\" align=\"char\">410 ± 39</td><td char=\"±\" align=\"char\">268 ± 29**</td><td char=\"±\" align=\"char\">450 ± 41</td><td char=\"±\" align=\"char\">233 ± 29 ***</td><td char=\"±\" align=\"char\">307 ± 80</td><td char=\"±\" align=\"char\">359 ± 56</td></tr><tr><td align=\"left\"> CCL20<sup>‡</sup></td><td char=\"±\" align=\"char\">80 ± 14</td><td char=\"±\" align=\"char\">44 ± 6.3**</td><td char=\"±\" align=\"char\">86 ± 15</td><td char=\"±\" align=\"char\">41 ± 6.9**</td><td char=\"±\" align=\"char\">66 ± 33</td><td char=\"±\" align=\"char\">51 ± 15</td></tr><tr><td align=\"left\"> CXCL13<sup>†</sup></td><td char=\"±\" align=\"char\">168 ± 15</td><td char=\"±\" align=\"char\">145 ± 18</td><td char=\"±\" align=\"char\">170 ± 18</td><td char=\"±\" align=\"char\">123 ± 16**</td><td char=\"±\" align=\"char\">164 ± 31</td><td char=\"±\" align=\"char\">204 ± 41</td></tr><tr><td align=\"left\"> IL22<sup>‡</sup></td><td char=\"±\" align=\"char\">70 ± 12</td><td char=\"±\" align=\"char\">47 ± 7.5***</td><td char=\"±\" align=\"char\">73 ± 15</td><td char=\"±\" align=\"char\">48 ± 9.6**</td><td char=\"±\" align=\"char\">63 ± 17</td><td char=\"±\" align=\"char\">46 ± 12</td></tr><tr><td align=\"left\"> IL15<sup>†</sup></td><td char=\"±\" align=\"char\">5.2 ± 0.48</td><td char=\"±\" align=\"char\">3.2 ± 0.27**</td><td char=\"±\" align=\"char\">5.6 ± 0.61</td><td char=\"±\" align=\"char\">3.1 ± 0.36**</td><td char=\"±\" align=\"char\">4.3 ± 0.64</td><td char=\"±\" align=\"char\">3.4 ± 0.26</td></tr><tr><td align=\"left\"> Fractalkine/CX3CL1<sup>†</sup></td><td char=\"±\" align=\"char\">728 ± 82</td><td char=\"±\" align=\"char\">624 ± 75*</td><td char=\"±\" align=\"char\">727 ± 73</td><td char=\"±\" align=\"char\">663 ± 81</td><td char=\"±\" align=\"char\">731 ± 249</td><td char=\"±\" align=\"char\">523 ± 178</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Intra-network clusters showing significant changes and notable trends</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Observed effect</th><th align=\"left\">Affected network</th><th align=\"left\">Cluster name</th><th align=\"left\">N voxels</th><th align=\"left\">p value</th><th align=\"left\">MNI<break/>(X, Y, Z)</th><th align=\"left\">Location</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"7\">General effect of rifaximin</td></tr><tr><td align=\"left\"> Increase of signal after treatment</td><td align=\"left\">Medial sensorimotor network</td><td align=\"left\">MSMN-1</td><td char=\".\" align=\"char\">5</td><td char=\".\" align=\"char\"><bold>0.01</bold></td><td align=\"left\">− 8, 18, 2</td><td align=\"left\">Left Caudate</td></tr><tr><td align=\"left\" rowspan=\"6\"> Decrease of signal after treatment</td><td align=\"left\">Right fronto-parietal network</td><td align=\"left\">RFPN-1</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\">0.096</td><td align=\"left\">− 30, − 62, 30</td><td align=\"left\">Left intraparietal 0 area (IP0)</td></tr><tr><td align=\"left\" rowspan=\"4\">Left fronto-parietal network</td><td align=\"left\">LFPN-1</td><td char=\".\" align=\"char\">11</td><td char=\".\" align=\"char\"><bold>0.02</bold></td><td align=\"left\">− 20, − 40, 58</td><td align=\"left\">Left Brodmann area 2</td></tr><tr><td align=\"left\">LFPN-2</td><td char=\".\" align=\"char\">4</td><td char=\".\" align=\"char\"><bold>0.034</bold></td><td align=\"left\">4,− 52,− 12</td><td align=\"left\">Right cerebellar lobule I-IV</td></tr><tr><td align=\"left\">LFPN-3</td><td char=\".\" align=\"char\">2</td><td char=\".\" align=\"char\">0.051</td><td align=\"left\">− 28,− 52,62</td><td align=\"left\">Left area 7 anterior lateral (7AL)</td></tr><tr><td align=\"left\">LFPN-4</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\">0.094</td><td align=\"left\">26,− 2,30</td><td align=\"left\">Right superior corona radiata</td></tr><tr><td align=\"left\">Lateral sensorimotor network</td><td align=\"left\">LSMN-1</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\"><bold>0.021</bold></td><td align=\"left\">12,− 30,54</td><td align=\"left\">Right corticospinal tract</td></tr><tr><td align=\"left\" colspan=\"7\">Effect of treatment exclusively on responding patients</td></tr><tr><td align=\"left\" rowspan=\"3\"> Decrease of signal after treatment</td><td align=\"left\">Lateral sensorimotor network</td><td align=\"left\">LSMN-2</td><td char=\".\" align=\"char\">2</td><td char=\".\" align=\"char\"><bold>0.01</bold></td><td align=\"left\">2,− 4,46</td><td align=\"left\">Right area 24 prime posterior (p24pr)</td></tr><tr><td align=\"left\" rowspan=\"2\">Left fronto-parietal network</td><td align=\"left\">LFPN-5</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\">0.074</td><td align=\"left\">18,10,16</td><td align=\"left\">Right caudate</td></tr><tr><td align=\"left\">LFPN-6</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\">0.099</td><td align=\"left\">26,− 2,28</td><td align=\"left\">Right superior corona radiata</td></tr><tr><td align=\"left\" colspan=\"7\">Interaction between treatment and response group</td></tr><tr><td align=\"left\" rowspan=\"3\"><p> Relative increase of signal in Responder patients</p><p>and</p><p>Relative decrease of signal in Non-responder patients</p></td><td align=\"left\" rowspan=\"2\">Thalamic network</td><td align=\"left\">THN-1</td><td char=\".\" align=\"char\">10</td><td char=\".\" align=\"char\"><bold>0.021</bold></td><td align=\"left\">− 41,57,4</td><td align=\"left\">Left area 9–46 anterior ventral (a9-46v)</td></tr><tr><td align=\"left\">THN-2</td><td char=\".\" align=\"char\">2</td><td char=\".\" align=\"char\">0.086</td><td align=\"left\">− 3,− 33,12</td><td align=\"left\">Left Corpus callosum splenium</td></tr><tr><td align=\"left\">Lateral visual network</td><td align=\"left\">LVN-1</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\">0.089</td><td align=\"left\">− 26,− 38,52</td><td align=\"left\">Left Brodmann area 2</td></tr><tr><td align=\"left\" colspan=\"7\">Pre-existing differences between response groups</td></tr><tr><td align=\"left\" rowspan=\"2\"> Increased signal in patients who will Respond</td><td align=\"left\" rowspan=\"2\">Basal ganglia network</td><td align=\"left\">BGN-1</td><td char=\".\" align=\"char\">39</td><td char=\".\" align=\"char\"><bold>0.014</bold></td><td align=\"left\">2,− 54,28</td><td align=\"left\">Right area 7 medial (7 M)</td></tr><tr><td align=\"left\">BGN-2</td><td char=\".\" align=\"char\">4</td><td char=\".\" align=\"char\">0.06</td><td align=\"left\">18,− 52,32</td><td align=\"left\">Right posterior corona radiata</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Significant correlations between remarkable intra-network functional clusters and neuropsychological tests or blood biochemical parameters</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Network</th><th align=\"left\">Cluster</th><th align=\"left\">Psychometric tests</th><th align=\"left\">R</th><th align=\"left\">p value</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"12\">Left fronto-parietal network</td><td align=\"left\" rowspan=\"5\">LFPN-1</td><td align=\"left\">PHES</td><td char=\".\" align=\"char\">− 0.595</td><td char=\".\" align=\"char\">0.046</td></tr><tr><td align=\"left\">DST (items completed)</td><td char=\".\" align=\"char\">− 0.583</td><td char=\".\" align=\"char\">0.046</td></tr><tr><td align=\"left\">NCT-A (seconds)</td><td char=\".\" align=\"char\">0.708</td><td char=\".\" align=\"char\">0.015</td></tr><tr><td align=\"left\">Oral SDMT (correct pairings)</td><td char=\".\" align=\"char\">− 0.668</td><td char=\".\" align=\"char\">0.019</td></tr><tr><td align=\"left\">Oral SDMT (total pairings)</td><td char=\".\" align=\"char\">− 0.6</td><td char=\".\" align=\"char\">0.019</td></tr><tr><td align=\"left\" rowspan=\"7\">LFPN-2</td><td align=\"left\">PHES</td><td char=\".\" align=\"char\">− 0.7</td><td char=\".\" align=\"char\">0.015</td></tr><tr><td align=\"left\">DST (items completed)</td><td char=\".\" align=\"char\">− 0.683</td><td char=\".\" align=\"char\">0.019</td></tr><tr><td align=\"left\">LTT (seconds + errors)</td><td char=\".\" align=\"char\">0.597</td><td char=\".\" align=\"char\">0.029</td></tr><tr><td align=\"left\">Stroop-neutral task</td><td char=\".\" align=\"char\">− 0.644</td><td char=\".\" align=\"char\">0.02</td></tr><tr><td align=\"left\">Stroop-incongruent task</td><td char=\".\" align=\"char\">− 0.592</td><td char=\".\" align=\"char\">0.034</td></tr><tr><td align=\"left\">Oral SDMT (correct pairings)</td><td char=\".\" align=\"char\">− 0.769</td><td char=\".\" align=\"char\">0.013</td></tr><tr><td align=\"left\">Oral SDMT (total pairings)</td><td char=\".\" align=\"char\">− 0.704</td><td char=\".\" align=\"char\">0.015</td></tr><tr><td align=\"left\" rowspan=\"2\">Lateral sensorimotor network</td><td align=\"left\" rowspan=\"2\">LSMN-1</td><td align=\"left\">DST (items completed)</td><td char=\".\" align=\"char\">− 0.69</td><td char=\".\" align=\"char\">0.018</td></tr><tr><td align=\"left\">Oral SDMT (correct pairings)</td><td char=\".\" align=\"char\">− 0.669</td><td char=\".\" align=\"char\">0.018</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Network</th><th align=\"left\">Cluster</th><th align=\"left\">Biochemical parameter</th><th align=\"left\">R</th><th align=\"left\">p value</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"5\">Left fronto-parietal network</td><td align=\"left\">LFPN-1</td><td align=\"left\">IL6</td><td char=\".\" align=\"char\">0.66</td><td char=\".\" align=\"char\">0.019</td></tr><tr><td align=\"left\" rowspan=\"4\">LFPN-2</td><td align=\"left\">IL6</td><td char=\".\" align=\"char\">0.815</td><td char=\".\" align=\"char\"> &lt; 0.001</td></tr><tr><td align=\"left\">IL18</td><td char=\".\" align=\"char\">0.565</td><td char=\".\" align=\"char\">0.026</td></tr><tr><td align=\"left\">Mip3/CCL20</td><td char=\".\" align=\"char\">0.59</td><td char=\".\" align=\"char\">0.024</td></tr><tr><td align=\"left\">IL22</td><td char=\".\" align=\"char\">0.656</td><td char=\".\" align=\"char\">0.01</td></tr><tr><td align=\"left\" rowspan=\"11\">Lateral sensorimotor network</td><td align=\"left\" rowspan=\"5\">LSMN-1</td><td align=\"left\">IL6</td><td char=\".\" align=\"char\">0.663</td><td char=\".\" align=\"char\">0.009</td></tr><tr><td align=\"left\">IL18</td><td char=\".\" align=\"char\">0.524</td><td char=\".\" align=\"char\">0.046</td></tr><tr><td align=\"left\">IL22</td><td char=\".\" align=\"char\">0.755</td><td char=\".\" align=\"char\">0.002</td></tr><tr><td align=\"left\">IL15</td><td char=\".\" align=\"char\">0.511</td><td char=\".\" align=\"char\">0.046</td></tr><tr><td align=\"left\">Fractalkine/CX3CL1</td><td char=\".\" align=\"char\">0.519</td><td char=\".\" align=\"char\">0.046</td></tr><tr><td align=\"left\" rowspan=\"6\">LSMN-2</td><td align=\"left\">IL6</td><td char=\".\" align=\"char\">0.68</td><td char=\".\" align=\"char\">0.017</td></tr><tr><td align=\"left\">IL18</td><td char=\".\" align=\"char\">0.824</td><td char=\".\" align=\"char\"> &lt; 0.001</td></tr><tr><td align=\"left\">Mip3/CCL20</td><td char=\".\" align=\"char\">0.711</td><td char=\".\" align=\"char\">0.017</td></tr><tr><td align=\"left\">CXCL13</td><td char=\".\" align=\"char\">0.66</td><td char=\".\" align=\"char\">0.018</td></tr><tr><td align=\"left\">IL22</td><td char=\".\" align=\"char\">0.607</td><td char=\".\" align=\"char\">0.032</td></tr><tr><td align=\"left\">IL15</td><td char=\".\" align=\"char\">0.689</td><td char=\".\" align=\"char\">0.017</td></tr><tr><td align=\"left\" rowspan=\"4\">Medial sensorimotor network</td><td align=\"left\" rowspan=\"4\">MSMN-1</td><td align=\"left\">Ammonia</td><td char=\".\" align=\"char\">0.55</td><td char=\".\" align=\"char\">0.03</td></tr><tr><td align=\"left\">IL6</td><td char=\".\" align=\"char\">− 0.709</td><td char=\".\" align=\"char\">0.003</td></tr><tr><td align=\"left\">IL22</td><td char=\".\" align=\"char\">− 0.715</td><td char=\".\" align=\"char\">0.003</td></tr><tr><td align=\"left\">IL15</td><td char=\".\" align=\"char\">− 0.597</td><td char=\".\" align=\"char\">0.021</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Significant correlations between remarkable inter-network functional connections and neuropsychological tests or blood biochemical parameters</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Connection</th><th align=\"left\">Psychometric tests</th><th align=\"left\">R</th><th align=\"left\">p value</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"13\"><p>Medial visual network</p><p>and</p><p>Thalamic network</p></td><td align=\"left\">PHES</td><td char=\".\" align=\"char\">0.723</td><td char=\".\" align=\"char\">0.01</td></tr><tr><td align=\"left\">DST (items completed)</td><td char=\".\" align=\"char\">0.788</td><td char=\".\" align=\"char\">0.006</td></tr><tr><td align=\"left\">NCT-B (seconds)</td><td char=\".\" align=\"char\">− 0.778</td><td char=\".\" align=\"char\">0.008</td></tr><tr><td align=\"left\">LTT (seconds + errors)</td><td char=\".\" align=\"char\">− 0.57</td><td char=\".\" align=\"char\">0.047</td></tr><tr><td align=\"left\">Stroop-neutral task</td><td char=\".\" align=\"char\">0.666</td><td char=\".\" align=\"char\">0.022</td></tr><tr><td align=\"left\">Stroop-incongruent task</td><td char=\".\" align=\"char\">0.669</td><td char=\".\" align=\"char\">0.022</td></tr><tr><td align=\"left\">Bimanual coordination (min)</td><td char=\".\" align=\"char\">− 0.892</td><td char=\".\" align=\"char\">0.002</td></tr><tr><td align=\"left\">d2 test-TR values</td><td char=\".\" align=\"char\">0.627</td><td char=\".\" align=\"char\">0.047</td></tr><tr><td align=\"left\">d2 test-TA values</td><td char=\".\" align=\"char\">0.638</td><td char=\".\" align=\"char\">0.046</td></tr><tr><td align=\"left\">d2 test-TOT values</td><td char=\".\" align=\"char\">0.681</td><td char=\".\" align=\"char\">0.03</td></tr><tr><td align=\"left\">d2 test-CON values</td><td char=\".\" align=\"char\">0.62</td><td char=\".\" align=\"char\">0.047</td></tr><tr><td align=\"left\">Oral SDMT (correct pairings)</td><td char=\".\" align=\"char\">0.753</td><td char=\".\" align=\"char\">0.009</td></tr><tr><td align=\"left\">Oral SDMT (total pairings)</td><td char=\".\" align=\"char\">0.731</td><td char=\".\" align=\"char\">0.01</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Connection</th><th align=\"left\">Biochemical parameters</th><th align=\"left\">R</th><th align=\"left\">p value</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\"><p>Medial sensorimotor network and</p><p>Dorsal attention network</p></td><td align=\"left\">IL18</td><td char=\".\" align=\"char\">0.581</td><td char=\".\" align=\"char\">0.041</td></tr><tr><td align=\"left\">CXCL13</td><td char=\".\" align=\"char\">0.633</td><td char=\".\" align=\"char\">0.033</td></tr><tr><td align=\"left\" rowspan=\"3\"><p>Right fronto-parietal network and</p><p>Supramarginal gyrus network</p></td><td align=\"left\">IL6</td><td char=\".\" align=\"char\">− 0.633</td><td char=\".\" align=\"char\">0.035</td></tr><tr><td align=\"left\">IL18</td><td char=\".\" align=\"char\">− 0.774</td><td char=\".\" align=\"char\"> &lt; 0.001</td></tr><tr><td align=\"left\">IL15</td><td char=\".\" align=\"char\">− 0.59</td><td char=\".\" align=\"char\">0.043</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p>In brackets: age range</p><p>Comparisons between controls, nMHE, and MHE groups were analysed by one-way ANOVA followed by post-hoc Tukey’s multiple comparison test</p><p>HBV, hepatitis B virus; HCV, hepatitis C virus; MHE, minimal hepatic encephalopathy; MELD, model end stage liver disease</p><p>The Child Pugh Score is derived from a score of 1–3 given for severity of ascites, hepatic encephalopathy, INR, albumin and bilirubin. The higher the score, the greater the liver disease severity</p><p>Significant differences are indicated by *: *p &lt; 0.05</p><p><sup>†</sup>Values are expressed as mean ± SD</p><p><sup>‡</sup>Differences in proportions were analysed with Chi-square test</p><p><sup>§</sup>Differences between groups (nMHE vs. MHE; response vs. no response groups) were analysed with <italic>T</italic>-test</p></table-wrap-foot>", "<table-wrap-foot><p>Values are expressed as mean ± SEM</p><p>PHES, Psychometric Hepatic Encephalopathy Score; DST, Digit Symbol Test; NCT-A, NCT-B: Number Connection Test A and B; SD, Serial Dotting Test; LTT, Line Tracing Test; TR, Total number of characters processed; TA, Total right answers; O, Total omission errors; C, Total commission errors; TOT, Total correctly processed; CON, Concentration performance; VAR, difference between maximum and minimum score. All biochemical parameters are in pg/mL, except ammonia levels, which are in µM. Stroop test: Congruent task: number of words read in 45 s; Neutral task: number of colours read in 45 s; Incongruent task: number of items completed in 45 s. Digit span and Letter-number sequencing: measured as number of right answers</p><p><sup>†</sup>Parametric measurements. <sup>‡</sup> Non-parametric measurements. Differences between pre- and post- treatment were analysed using paired <italic>T-test</italic> for parametric measurements or paired Wilcoxon test for non-parametric measurements</p><p>Differences between responders and non-responders before treatment were analysed using: <italic>T-test</italic> if measurements were parametric or Wilcoxon test if measurements were not parametric</p><p>Resulting levels of significance were corrected using False Discovery Rate (FDR) method, and values of p &lt; 0.05 after FDR correction were considered significant</p><p>Significant pre- and post-treatment differences are indicated by *: *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001</p><p>Significant differences between response groups before treatment are indicated by α: <sup>α</sup>p &lt; 0.05</p></table-wrap-foot>", "<table-wrap-foot><p>The program FSL Randomise was used to analyse all intra-network differences</p><p>FSL Randomise uses Generalized Linear Models (GLM) to analyse differences between groups</p><p>All results were cluster-corrected for multiple comparisons using family-wise error (FWE), in combination with a threshold of p &lt; 0.001 at the uncorrected voxel level</p><p>Clusters were considered significant at p &lt; 0.05 after FWE correction</p><p>MNI: Montreal Neurological Institute; MSMN, medial sensorimotor network; RFPN, right fronto-parietal network; LFPN, left fronto-parietal network; LSMN, lateral sensorimotor network; THN, Thalamic network; LVN, lateral visual network; BGN, basal ganglia network</p><p>Significant p values are in bold</p></table-wrap-foot>", "<table-wrap-foot><p>R and p from significant Spearman correlations are shown</p><p>R, correlation coefficient. Correlations were considered significant at p &lt; 0.05 after FDR correction</p><p>Clusters are named as shown in Table ##TAB##2##3##</p></table-wrap-foot>", "<table-wrap-foot><p>R and p from significant Spearman correlations are shown. R, correlation coefficient. Correlations were considered significant at p &lt; 0.05 after FDR correction</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"12967_2023_4844_Fig1_HTML\" id=\"MO1\"/>", "<graphic xlink:href=\"12967_2023_4844_Fig2_HTML\" id=\"MO2\"/>", "<graphic xlink:href=\"12967_2023_4844_Fig3_HTML\" id=\"MO3\"/>", "<graphic xlink:href=\"12967_2023_4844_Fig4_HTML\" id=\"MO4\"/>" ]
[ "<media xlink:href=\"12967_2023_4844_MOESM1_ESM.docx\"><caption><p><bold>Additional file 1: Table S1.</bold> Psychometric and biochemical characteristics of controls, nMHE patients and MHE patients. <bold>Table S2.</bold> Correlations between changes in THN-1 cluster and psychometric and biochemical parameters in the group of patients responding to rifaximin treatment.</p></caption></media>" ]
[{"label": ["40."], "surname": ["Bajaj", "Heuman", "Wade"], "given-names": ["JS", "DM", "JB"], "article-title": ["Rifaximin improves driving simulator performance in a randomized trial of patients with minimal hepatic encephalopathy"], "source": ["Gastroenterol"], "year": ["2011"], "volume": ["140"], "issue": ["2"], "fpage": ["478"], "lpage": ["487"], "pub-id": ["10.1053/j.gastro.2010.08.061"]}, {"label": ["53."], "surname": ["Baker", "Burks", "Briggs"], "given-names": ["CM", "JD", "RG"], "article-title": ["A connectomic atlas of the human cerebrum"], "source": ["Oper Neurosurg."], "year": ["2018"], "volume": ["15"], "issue": ["1"], "fpage": ["S10"], "lpage": ["S406"], "pub-id": ["10.1093/ons/opy254"]}]
{ "acronym": [ "BGN", "DAN", "DLPFC", "DST", "fMRI", "HE", "ICA", "LFPN", "LSMN", "LVN", "MELODIC", "MHE", "MSMN", "MVN", "nMHE", "Oral SDMT", "PHES", "RFPN", "SPGN", "THN" ], "definition": [ "Basal ganglia network", "Dorsal attention network", "Dorsolateral prefrontal cortex", "Digit Symbol Test", "Functional magnetic resonance imaging", "Hepatic encephalopathy", "Independent component analysis", "Left fronto-parietal network", "Lateral sensorimotor network", "Lateral visual network", "Multivariate Exploratory Linear Optimized Decomposition into Independent Components", "Minimal hepatic encephalopathy", "Medial sensorimotor network", "Medial visual network", "Patients without minimal hepatic encephalopathy", "Symbol Digit Modalities test, oral version", "Psychometric Hepatic Encephalopathy Score", "Right fronto-parietal network", "Supramarginal gyrus network", "Thalamic network" ] }
64
CC BY
no
2024-01-14 23:43:47
J Transl Med. 2024 Jan 12; 22:49
oa_package/45/5e/PMC10787503.tar.gz
PMC10787504
0
[ "<title>Introduction</title>", "<p id=\"Par9\">Currently, cardiovascular diseases (CVDs) continue to be a leading cause of morbidity and mortality on a global scale, presenting an ongoing challenge to healthcare systems and medical research communities [##REF##33309175##1##]. Traditionally, CVD risk has been evaluated by using conventional methods focused on established risk factors, such as hypertension, elevated cholesterol levels, diabetes, or smoking habits [##UREF##0##2##]. While these traditional parameters have provided valuable insights, they have been shown to be insufficient in accounting for the actual incidence of CVD events [##REF##36090536##3##]. Specifically, the number of individuals who ultimately experience cardiovascular events often exceeds those predicted to be at high risk, particularly among populations over the age of 40 [##REF##31035613##4##]. This incongruence, commonly referred to as the “detection gap,” raises questions about the completeness of current risk assessment models and points out the need for additional predictive variables that go beyond these classical factors [##REF##19211497##5##].</p>", "<p id=\"Par10\">Beyond its role as a detection bridge in cardiovascular risk assessment, Early Vascular Aging (EVA) presents a multifaceted challenge, unraveling the intricate complexity of its underlying mechanisms [##REF##26548304##6##]. The accelerated vascular changes observed in EVA, such as arterial stiffening and endothelial dysfunction, pose a complex puzzle for researchers and clinicians alike [##UREF##1##7##]. Understanding the nuanced interplay of these structural and functional alterations is crucial not only for accurate risk assessment but also for devising targeted interventions to impede or reverse the progression of vascular aging [##REF##12749750##8##]. Unraveling the molecular and cellular pathways involved in EVA remains an ongoing pursuit, holding the key to unlocking novel therapeutic avenues in the battle against cardiovascular diseases [##REF##26548304##6##].</p>", "<p id=\"Par11\">EVA has increasingly been recognized as a key factor that could fill the detection gap inherent in traditional cardiovascular risk assessment models [##REF##37592251##9##]. EVA manifests through a collection of both structural and functional alterations in the vascular system. Such changes include arterial stiffness, endothelial dysfunction, and variations in the intima-media thickness of the vessel wall [##REF##28412914##10##]. Moreover, these vascular alterations, while associated with normal ageing, occur at an accelerated rate in EVA, culminating in decreased arterial distensibility and increased arterial stiffness. Consequently, accelerated vascular aging contributes to a number of cardiovascular complications, most notably related to coronary artery disease and stroke [##REF##24729936##11##]. Therefore, the quantification of EVA risk has become a key factor in the strategic prevention and management of cardiovascular diseases [##REF##36738307##12##].</p>", "<p id=\"Par12\">As we delve into the realm of EVA assessment, a paradigm shift towards unsupervised learning techniques unfolds, which can reveal hidden patterns and relationships within vascular risk [##REF##35328275##13##]. Unlike traditional classification methods, which predominantly focus on individuals with documented cardiovascular events, unsupervised techniques cover latent risks that may not manifest explicit symptoms [##UREF##2##14##]. This shift, while promising, is not without its challenges, particularly the absence of a universally accepted gold standard for EVA prediction. Consequently, the validation of unsupervised models becomes paramount, prompting innovative approaches such as comparing emergent risk patterns with external variables like age, body fat percentage, or cholesterol levels. Navigating these uncharted territories in EVA assessment holds the potential to revolutionize risk prediction models and enhance our understanding of vascular aging dynamics [##UREF##3##15##].</p>", "<p id=\"Par13\">Traditionally, vascular risk assessment using EVA has been approached through classification techniques [##REF##35316972##16##]. While these methods have provided valuable insights, their primary focus has been on patients who have either manifested a cardiovascular event or for whom such an event has been documented. However, this methodology might inadvertently omit individuals exhibiting latent risk who have yet to display explicit symptoms or have an event recorded [##REF##36738307##12##]. For this reason, a shift towards unsupervised learning techniques in EVA assessment could uncover underlying risk patterns and identify novel and potentially overlooked relationships between risk factors. However, the absence of a recognized gold standard for EVA prediction presents a significant challenge, emphasizing the need for validating the risk models developed through unsupervised techniques [##REF##37592251##9##]. Given this context, one promising approach for validation involves employing a set of external variables not initially included in the unsupervised model [##REF##26572668##17##]. Specifically, comparing the unsupervised groups against external features, recognized as indicators of premature vascular risk, such as age, body fat percentage or cholesterol, could serve as valuable benchmarks to assess and corroborate the reliability and robustness of these emergent risk models.</p>", "<p id=\"Par14\">In recent research, a construct composed of four variables consisting of pulse pressure (PP), pulse wave velocity (PWv), glycated hemoglobin (HbA1c), and advanced glycation end products (AGEs) measured by skin autofluorescence (SAF) was introduced by our research group as a potential tool for assessing vascular risk [##REF##37592251##9##]. Preliminary analyses of this construct indicated that optimal differentiation into two clusters provided the most coherent grouping of individuals regarding the risk of suffering from EVA. The selection of these variables is based on an understanding of the physiological changes associated with EVA that goes beyond classical risk factors. This comprehensive approach addresses both the structure and function of the vascular system, providing a more complete picture of the mechanisms underlying cardiovascular risk [##REF##22783194##18##]. PP serves as an indicator of the force exerted by the blood on the arterial walls during each heartbeat [##REF##12975261##19##], PWv reflects arterial stiffness [##REF##21612307##20##], HbA1c serves as an indicator of long-term glycaemic control that impacts vascular health [##REF##35130570##21##], and SAF indicate accumulated tissue damage [##REF##20844429##22##].</p>", "<p id=\"Par15\">While this construct offers a novel perspective, it remains essential to further validate its effectiveness. By doing so, we can not only potentially highlight the inherent relationships and patterns within the data but also confirm the robustness and validity of both the construct and the clustering model. Therefore, the objective of this work is to validate the proposed clustering model by comparing its outcomes with external vascular risk indicators, ensuring a comprehensive and practical understanding of EVA. Successful validation of this approach could facilitate its integration into clinical settings, providing healthcare professionals with a robust tool for early and accurate risk assessment.</p>" ]
[ "<title>Materials and methods</title>", "<title>Study design</title>", "<p id=\"Par16\">The EVasCu study was a cross-sectional investigation conducted in the province of Cuenca, Spain, to evaluate the validity of an early vascular aging model as an index of cardiovascular risk in healthy adults [##REF##37592251##9##]. The study adhered to the principles outlined in the Declaration of Helsinki and received prior approval from the Clinical Research Ethics Committee of the Cuenca Health Area (REG: 2022/PI2022). The study design followed the guidelines provided by the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement [##UREF##4##23##].</p>", "<title>Study sample</title>", "<p id=\"Par17\">A total of 390 participants from Cuenca (Spain) were recruited for the EVasCu study between June 2022 and December 2022. Individuals with preexisting conditions that could significantly influence arterial stiffness, including diabetes mellitus, dyslipidemia, and arterial hypertension, were excluded from the study. Participants were provided with detailed information about the purpose and procedures of the study, and written informed consent was obtained. After the data collection, participants received validated reports from a physician, along with appropriate recommendations if needed.</p>", "<title>EVA construct variables</title>", "<p id=\"Par18\">PWv was measured using oscillometric techniques with Mobil-O-Graph® (IEM GmbH). Mobil-O-Graph® measures aortic pulse wave velocity (PWv) calculated as the mean of two repeated measurements, separated by 5 min each. These parameters were measured in a quiet place and after a 5-min rest period using a cuff size according to the participant’s arm/s and/or lower limb circumference.</p>", "<p id=\"Par19\">Pulse pressure (PP) was obtained from the difference between mean systolic blood pressure (SBP) and diastolic blood pressure (DBP). Blood pressure was measured in a quiet place and after a 5-min rest period using the Omron® M5-I monitor (Omron Healthcare UK Ltd.) with a cuff size according to the participant’s arm circumference.</p>", "<p id=\"Par20\">Glycated hemoglobin A1c (HbA1c) was determined by high-performance liquid chromatography using the ADAMS A1c HA-8180 V analyser from A. Menarini Diagnostics®. Samples were collected between 8 a.m. and 9 a.m. and after 12 h of fasting.</p>", "<p id=\"Par21\">AGEs were measured by SAF with the AGE Reader® device. SAF were calculated as the mean of the measurements from both arms. The mean for each arm was calculated as the mean of three repeated measurements.</p>", "<title>External validation variables</title>", "<title>Sociodemographic and lifestyle factors</title>", "<p id=\"Par22\">Age, gender, and smoking status were collected through direct questioning. Smoking status was classified into five groups: smoker, ex-smoker &lt; 1 year, ex-smoker 1–5 years, ex-smoker &gt; 5 years, and nonsmoker.</p>", "<title>Adiposity factors</title>", "<p id=\"Par23\">Weight and height were measured twice using appropriate equipment and averaged for analysis. Body mass index (BMI) was calculated as weight in kilograms divided by the squared height in meters (kg/m2), and participants were classified as underweight, normal weight, overweight, or obese using cut-off points of 18.5, 25.0, and 30.0, respectively [##UREF##5##24##]. Fat percentage was measured by calculating the average of two measurements using Tanita® BC-418 MA 8-electrode electrical bioimpedance.</p>", "<title>Glycemic and inflammatory factors</title>", "<p id=\"Par24\">Glucose and ultrasensitive C-reactive protein (CRP) determinations were measured on a Roche Diagnostics® Cobas 8000 system, and insulin determinations were measured on the Abbott ® Architect platform. Samples were collected between 8 a.m. and 9 a.m. and after 12 h of fasting. Diabetic status was established according to the HbA1c criteria for the diagnosis of diabetes of the American Diabetes Association (ADA) [##UREF##6##25##]: nondiabetic (HbA1c &lt; 5.7%), prediabetic (HbA1c 5.7–6.4%) and diabetic (HbA1c ≥6.5%).</p>", "<title>Lipid profile factors</title>", "<p id=\"Par25\">Total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL) and triglyceride determinations were measured on a Roche Diagnostics® Cobas 8000 system, and insulin determinations were obtained on the Abbott ® Architect platform. Samples were collected between 8 a.m. and 9 a.m. and after 12 h of fasting. Hypercholesterolemia and hypertriglyceridaemia status were established according to the total cholesterol and triglyceride criteria of the American Heart Association (AHA) and the American College of Cardiology (ACC) [##REF##30879339##26##], considering total cholesterol &gt; 200 mg/dL for hypercholesterolemia and triglycerides &gt; 150 mg/dL for hypertriglyceridaemia.</p>", "<title>Vascular factors</title>", "<p id=\"Par26\">The augmentation index (AIx75) was measured using Mobil-O-Graph®, which was calculated as the mean of two repeated measurements, separated by 5 min each. Cardio-ankle vascular index (CAVI) and ankle-brachial index (ABI) were measured with the VaSera system VS-1500 N (Fukuda Denshi UK Ltd.). Finally, intima-media thickness (IMT) was measured by ultrasound with the Sonosite SII device (Sonosite Inc., Bothell, Washington, USA). IMT was calculated as the mean measurement of the right and left carotid arteries.</p>", "<title>Blood pressure factors</title>", "<p id=\"Par27\">Blood pressure was measured in a quiet place and after a 5-min rest period using the Omron® M5-I monitor (Omron Healthcare UK Ltd. with a cuff size according to the participant’s arm circumference. SBP and DBP were calculated as the mean of two repeated measurements, separated by 5 min each. Hypertension status was established according to the SBP and DBP criteria of the AHA and ACC [##REF##30879339##26##]: Optima blood pressure (SBP &lt; 120 mmHg and/or DBP &lt; 80 mmHg), normal blood pressure (SBP 120–129 mmHg and/or DBP 80–84 mmHg), normal-high blood pressure (SBP 130–139 mmHg and/or DBP 85–89 mmHg), hypertension grade I (SBP 140–159 mmHg and/or DBP 90–99 mmHg), hypertension grade II (SBP ≥160 mmHg and/or DBP ≥100 mmHg) and hypertension grade III (SBP ≥180 mmHg and/or DBP ≥110 mmHg).</p>", "<p id=\"Par28\">It should be clarified that all subjects included in the EVasCu study were healthy subjects, so diabetic, hypercholesterolemia, hypertriglyceridaemia and hypertension status were established on the basis of the guidelines mentioned above and would not be subjects diagnosed with these diseases at the time of data collection, so they were not treated for these diseases and were informed with a report to be diagnosed by a physician.</p>", "<title>Statistical analysis</title>", "<title>Data description and preprocessing</title>", "<p id=\"Par29\">Before any analyses, a preprocessing stage was conducted on the dataset. First, when features presented missing values, they were imputed using their respective medians for each feature. Second, to ensure a consistent scale across variables, all features were standardized using z score normalization.</p>", "<title>Cluster formation and data representation</title>", "<p id=\"Par30\">Initially, the optimal number of risk groups, denoted as K, was determined based on the inherent characteristics of the four-variable construct. Different values of K (ranging from 2 to 5) were tested using the Calinski‒Harabasz, Davies‒Bouldin, and Silhouette indices to evaluate the quality and coherence of the formed clusters [##UREF##7##27##, ##UREF##8##28##]. These indices were also crucial to determine the cohesion and separation within and between the clusters once they were computed, thus measuring the quality of the grouping [##UREF##9##29##].</p>", "<p id=\"Par31\">Later, patients were assigned to these groups using the K-means algorithm [##UREF##10##30##]. Mathematically, this algorithm minimizes the sum of squared Euclidean distances between each point and its assigned centroid. In this process, each patient was allocated to the nearest group or centroid based on the shortest Euclidean distance. This assignment process was rigorously iterated until the centroid positions experienced negligible changes or until a maximum of 100 iterations was reached. To further elucidate the structure of the data and to visualize the delineation between clusters, principal component analysis (PCA) was utilized, especially emphasizing the distinction between clusters derived from the original four-variable construct.</p>", "<title>External validation analysis</title>", "<p id=\"Par32\">To assess the distribution of external variables within each cluster, Shapiro-Wilks and Levene’s tests were employed, which determine the normality and homoscedasticity of the data, respectively. Based on the outcomes of these preliminary tests, the appropriate statistical analysis to discern differences between clusters was chosen. Thus, when a quantitative variable exhibited a normal distribution and homogenous variances across both clusters, Student’s t test was applied. Conversely, when the assumptions of normality or homoscedasticity were not met, the nonparametric Mann‒Whitney U test was chosen. For categorical variables, the chi-square test was used. These tests ensure robust identification of significant differences between clusters based on the data characteristics.</p>", "<p id=\"Par33\">Clustering modelling was conducted using Python version 3.10 and the scikit-learn package [##UREF##11##31##], whereas statistical analyses were performed with SPSS version 28.</p>" ]
[ "<title>Results</title>", "<title>Characteristics of study participants</title>", "<p id=\"Par34\">The EVasCu study sample included a total of 390 participants, of whom 246 (63.1%) were women. The mean age of the participants was 42.0 ± 13.1 years. Table ##TAB##0##1## shows the baseline characteristics of the enrolled population.</p>", "<p id=\"Par35\">\n\n</p>", "<title>Cluster analysis</title>", "<p id=\"Par36\">This study is constructed upon a clustering model previously established in preceding research [##REF##26548304##6##]. In that study, the robustness and coherence of the clustering groups were rigorously demonstrated, showcasing the model’s reliability [##REF##26548304##6##]. Building on this solid foundation, the following analyses seek to further validate the clustering, with the objective of affirming its applicability and consistency across practical applications. Figure ##FIG##0##1## shows a 2-D visual representation of clusters using the first two principal components with normalized data, which explains more than 75% of the total data variability of the construct variables. Notably, the most robust model configuration was identified when utilizing two distinct groups.</p>", "<p id=\"Par37\">\n\n</p>", "<title>Identifying health and risk groups in the cluster model</title>", "<p id=\"Par40\">Table ##TAB##1##2## presents the definitions for the two clusters identified within the EVasCu study, referred to as Cluster 1 and Cluster 2 (see Fig. ##FIG##0##1##), based on the selected construct variables. The median values and interquartile ranges (IQRs) for each variable are presented, and <italic>p</italic> values are provided to highlight any statistically significant differences between the two clusters. The data in Table ##TAB##1##2##, complemented by the visual representation in Fig. ##FIG##1##2##, reveal marked disparities between the two clusters across all the construct variables. Indeed, Cluster 2 exhibited higher values of pulse pressure, HbA1c, PWv, and SAF than Cluster 1, with these differences achieving statistical significance (<italic>p</italic> &lt; 0.001). Consequently, Cluster 1 can be confidently associated with the HVA, while Cluster 2 can be associated with the EVA. In subsequent analyses, this grouping aims to be further validated, employing external variables not used in the initial construct of the model, thus ensuring the robustness and applicability of this assignment.</p>", "<p id=\"Par41\">\n\n</p>", "<title>Quantitative and categorical risk indicators</title>", "<p id=\"Par42\">Tables ##TAB##2##3## and ##TAB##3##4## provide a comprehensive analysis of quantitative and categorical risk indicators, categorized into distinct factors, enabling an in-depth exploration of the multifaceted risk factors present within the EVasCu study.</p>", "<p id=\"Par43\">\n\n</p>", "<p id=\"Par44\">\n\n</p>", "<p id=\"Par38\">\n\n</p>", "<p id=\"Par45\">In terms of sociodemographic and lifestyle factors, age emerged as a substantial differentiator, with individuals in the EVA cluster showing a marked increase in age compared to their HVA counterparts (<italic>p</italic> &lt; 0.001). In contrast, the gender distribution remained statistically homogeneous between the two clusters. However, a notable trend emerged in smoking status, where although similar prevalences were observed in current and ex-smoker (0–1 year) categories, there were more nonsmokers in the HVA cluster than in the EVA cluster, resulting in a significant distinction (<italic>p</italic> &lt; 0.001) (Fig. ##FIG##2##3##).</p>", "<p id=\"Par46\">\n\n</p>", "<p id=\"Par47\">Within the adiposity factors, our analysis revealed that individuals in the EVA cluster had significantly higher BMI values than those in the HVA cluster (<italic>p</italic> &lt; 0.001). Similarly, waist circumference showed a substantial increase in the EVA cluster, emphasizing the role of adiposity in vascular aging (<italic>p</italic> &lt; 0.001). Furthermore, a discernible elevation in fat percentage was observed in the EVA cluster, suggesting the importance of adipose tissue in the early vascular aging process (<italic>p</italic> &lt; 0.001). In terms of weight status, the EVA cluster exhibited a higher prevalence of individuals classified as overweight and obese in comparison to the HVA cluster (<italic>p</italic> &lt; 0.001) (Fig. ##FIG##3##4##).</p>", "<p id=\"Par48\">\n\n</p>", "<p id=\"Par49\">Regarding glycemic and inflammatory factors, our examination showed a significant increase in glucose levels in the EVA cluster compared to the HVA cluster (<italic>p</italic> &lt; 0.001). While insulin levels and CRP showed slight increases in the EVA cluster, these differences were statistically significant (<italic>p</italic> = 0.034 and <italic>p</italic> = 0.031, respectively). Notably, although the majority of individuals in both clusters were nondiabetic, the prevalence of prediabetes and diabetes was slightly higher in the EVA cluster (<italic>p</italic> = 0.012) (Fig. ##FIG##4##5##).</p>", "<p id=\"Par50\">\n\n</p>", "<p id=\"Par51\">In terms of lipid profile factors, our findings highlighted substantial elevations in the levels of total cholesterol, LDL, and triglycerides in the EVA cluster compared to the HVA cluster (<italic>p</italic> &lt; 0.001). In particular, no statistically significant variations in HDL levels were observed between the two clusters. The EVA cluster also had a higher proportion of individuals with hypercholesterolemia than the HVA cluster (<italic>p</italic> &lt; 0.001). Additionally, a modest inclination in hypertriglyceridaemia was observed in the EVA cluster, although it did not reach statistical significance (<italic>p</italic> = 0.051) (Fig. ##FIG##5##6##).</p>", "<p id=\"Par52\">\n\n</p>", "<p id=\"Par53\">Among the vascular factors, AIx-75 showed a significant increase in the EVA cluster compared to the HVA cluster, suggesting altered arterial stiffness and wave reflection properties in early vascular aging (<italic>p</italic> &lt; 0.001). However, no statistically significant differences in ABI were observed between the two clusters. CAVI showed a significant elevation in the EVA cluster, signifying greater vascular stiffness (<italic>p</italic> &lt; 0.001). Additionally, our analysis revealed that IMT was slightly greater in the EVA cluster, with statistically significant differences (<italic>p</italic> = 0.015) (Fig. ##FIG##6##7##).</p>", "<p id=\"Par54\">\n\n</p>", "<p id=\"Par55\">Finally, in terms of blood pressure factors, both SBP and DBP were substantially higher in the EVA cluster than in the HVA cluster (<italic>p</italic> &lt; 0.001). Importantly, our analysis revealed a higher proportion of hypertension, especially in the categories of hypertension grade I and grade II, in the EVA cluster (<italic>p</italic> &lt; 0.001) (Fig. ##FIG##7##8##).</p>", "<p id=\"Par56\">\n\n</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par57\">In summary, our findings provide valuable insights into the potential utility of the clustering model for the assessment of EVA and its associated cardiovascular risk. The results indicate that the clustering model effectively stratifies individuals into two distinct groups, HVA and EVA, based on a set of construct variables. These findings align with the growing recognition of EVA as a key factor in cardiovascular risk assessment. EVA manifests through structural and functional alterations in the vascular system, including arterial stiffness, endothelial dysfunction, and variations in intima-media thickness. Our results confirm that individuals with EVA exhibit significant differences in various risk factors, including age, adiposity, glycemic control, lipid profiles, vascular health, and blood pressure, compared to those in the HVA group.</p>", "<p id=\"Par58\">Age is a well-established risk factor for cardiovascular disease, but its specific role in EVA has been a subject of interest in recent research [##REF##31035613##4##]. In this study, individuals in the EVA cluster were significantly older than those in the HVA group. This finding aligns with the concept that EVA is a process characterized by accelerated vascular aging [##REF##28966804##32##]. As individuals age, a number of structural and functional changes occur in the vascular system, including increased arterial stiffness and endothelial dysfunction [##UREF##1##7##]. EVA may represent an extreme manifestation of these age-related changes. Early detection and intervention in older populations is crucial because these individuals may already be in an advanced stage of vascular aging, which may result in an increased risk of cardiovascular events [##REF##31203728##33##]. This finding underscores the need for age-specific interventions and risk assessments in clinical practice [##REF##18827905##34##].</p>", "<p id=\"Par59\">The significantly higher levels of adiposity factors, including BMI, waist circumference, and fat percentage, in the EVA group suggest a strong association between adipose tissue and early vascular aging. Adipose tissue is known to produce various bioactive substances, such as cytokines and adipokines, which can contribute to inflammation and endothelial dysfunction [##REF##20671929##35##]. Moreover, adiposity is often associated with other risk factors, such as insulin resistance, dyslipidemia, and hypertension, all of which are known to accelerate vascular aging [##REF##33882682##36##]. Therefore, addressing weight management and reducing adiposity may be crucial in EVA prevention. Lifestyle modifications, including dietary changes and regular physical activity, may play a significant role in mitigating the adverse effects of adiposity on vascular health [##UREF##12##37##].</p>", "<p id=\"Par60\">The elevated glucose levels and the observed higher prevalence of prediabetes and diabetes in the EVA group suggest a strong link between glycemic control and EVA. Chronic hyperglycemia is known to induce oxidative stress and inflammation, which can contribute to endothelial dysfunction and vascular damage [##REF##23448484##38##]. Moreover, the inclusion of HbA1c in the construct is primarily based on the analysis of the EVA construct previously analysed [##REF##37592251##9##]. HbA1c in apparently healthy individuals is crucial because of its ability to provide an integrative measure of long-term glycaemic control [##REF##27398023##39##]. Since chronic hyperglycaemia is a known risk factor for the development and progression of cardiovascular disease [##REF##28760792##40##], the inclusion of HbA1c in the EVA construct may more accurately identify those individuals who, despite having apparently normal fasting glucose values, may experience detrimental fluctuations in their glucose levels over time. Consequently, integrating HbA1c in apparently healthy subjects may improve the predictive ability of cardiovascular risk by more effectively capturing chronic glycaemic load, thus contributing to more personalised and effective preventive strategies [##REF##24195452##41##].</p>", "<p id=\"Par61\">Similarly, the elevated lipid profiles (total cholesterol, LDL, and triglycerides) in the EVA group highlight the significance of lipid management in EVA prevention. Dyslipidemia can lead to atherosclerosis and increased vascular stiffness, contributing to early vascular aging [##REF##33356026##42##]. The findings related to vascular factors (arterial stiffness and IMT) reinforce the importance of assessing vascular health for the early detection and management of EVA. The higher prevalence of hypertension in the EVA cluster underlines the role of blood pressure management in EVA risk reduction. Elevated blood pressure can lead to structural changes in blood vessels and increased vascular resistance, further accelerating vascular aging [##REF##25896391##43##].</p>", "<p id=\"Par62\">The external validation of the clustering model is crucial for several reasons. First, it confirms that the model’s clusters align with established risk factors. This strengthens the model’s clinical utility and suggests that it can identify individuals at risk of cardiovascular events based on readily available parameters. Second, the external validation underscores the importance of these traditional risk factors in EVA. This suggests that EVA is not an isolated phenomenon but rather a manifestation of cardiovascular risk factors that can be identified and managed using established guidelines. This, in turn, highlights the potential for early intervention in individuals showing signs of EVA.</p>", "<p id=\"Par63\">However, important limitations should be acknowledged for this study. First, the size of the study population, deemed too small, may impact the statistical power and generalizability of the findings. Additionally, the age composition, primarily comprising young to middle-age subjects with a low rate of cardiovascular events in the general population, introduces a potential limitation in extrapolating the results to broader age groups with different cardiovascular risk profiles. The cross-sectional design of the research restricts our ability to test the proposed hypothesis thoroughly. Longitudinal studies are imperative to elucidate causal relationships and comprehensively assess the impact of identified risk factors on the development of early vascular aging (EVA) over time. Additionally, the study’s findings are based on a specific population, and their generalizability to other demographic groups should be investigated. Furthermore, the clustering model’s performance may vary in different clinical and research settings, and its robustness needs further validation in diverse populations.</p>", "<p id=\"Par64\">In conclusion, the results of this study support the validity and utility of the clustering model for assessing EVA and its association with cardiovascular risk. The differentiation between the HVA and EVA groups based on various risk factors provides a comprehensive understanding of EVA’s distinct characteristics. These findings have implications for clinical practice, as they suggest that interventions targeting age, adiposity, glycemic control, lipid profile, vascular health, and blood pressure may be particularly relevant for individuals at risk of EVA. Further research and longitudinal studies are needed to confirm these associations and to assess the long-term impact of EVA on cardiovascular outcomes. Successful validation of this clustering model could confirm its utility for model integration into clinical settings, offering healthcare professionals a robust tool for early and accurate cardiovascular risk assessment and management.</p>" ]
[]
[ "<title>Background</title>", "<p id=\"Par2\">Cardiovascular diseases (CVDs) remain a major global health concern, necessitating advanced risk assessment beyond traditional factors. Early vascular aging (EVA), characterized by accelerated vascular changes, has gained importance in cardiovascular risk assessment.</p>", "<title>Methods</title>", "<p id=\"Par4\">The EVasCu study in Spain examined 390 healthy participants using noninvasive measurements. A construct of four variables (Pulse Pressure, Pulse Wave Velocity, Glycated Hemoglobin, Advanced Glycation End Products) was used for clustering. K-means clustering with principal component analysis revealed two clusters, healthy vascular aging (HVA) and early vascular aging (EVA). External validation variables included sociodemographic, adiposity, glycemic, inflammatory, lipid profile, vascular, and blood pressure factors.</p>", "<title>Results</title>", "<p id=\"Par6\">EVA cluster participants were older and exhibited higher adiposity, poorer glycemic control, dyslipidemia, altered vascular properties, and higher blood pressure. Significant differences were observed for age, smoking status, body mass index, waist circumference, fat percentage, glucose, insulin, C-reactive protein, diabetes prevalence, lipid profiles, arterial stiffness, and blood pressure levels. These findings demonstrate the association between traditional cardiovascular risk factors and EVA.</p>", "<title>Conclusions</title>", "<p id=\"Par8\">This study validates a clustering model for EVA and highlights its association with established risk factors. EVA assessment can be integrated into clinical practice, allowing early intervention and personalized cardiovascular risk management.</p>", "<title>Keywords</title>" ]
[]
[ "<title>Acknowledgements</title>", "<p>Not applicable.</p>", "<title>Author contributions</title>", "<p>Conceptualization, IC-R and AS-L; methodology, ICR and AM-R; software, AM‐R; validation, IM‐G and IO‐L; formal analysis, IC‐R and AM‐R; investigation, IC‐R; resources, AL‐S; data curation, IC‐R and AL‐S; writing—original draft preparation, IC‐R and AM‐R; writing—review and editing, AL‐S; visualization, IM‐G and IO‐L; supervision, AS‐L. All authors have read and agreed to the published version of the manuscript.</p>", "<title>Funding</title>", "<p>This study was funded by 42nd edition of the nursing award of the University Pontificia Comillas and Escuela de Enfermeria y Fisioterapia San Juan de Dios, and co-funded by the European Union (ERDF/ESF) and by the Carvascare Research Group from the Universidad de Castilla‐La Mancha (2023‐GRIN‐ 34459). IMG is supported by a grant from the Ministry of Science, Innovation and Universities. (FPU19/06866).</p>", "<title>Data Availability</title>", "<p>All data generated or analysed during this study are included in this published article.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par66\">The research protocol of this study was approved by the Clinical Research Ethics Committee of the Cuenca Health Area (REG: 2022/PI2022). Written informed consent to participate was obtained from all subjects included in the study.</p>", "<title>Consent for publication</title>", "<p id=\"Par67\">Written informed consent for publication was obtained from all subjects included in the study.</p>", "<title>Competing interests</title>", "<p id=\"Par65\">The authors declare no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>2D PCA view with K-means cluster</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Differences between the cluster categories (HVA and EVA) for sociodemographic and lifestyle factors</p><p>For <bold>A</bold>, <bold>B</bold>, <bold>C</bold> and <bold>D</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test. HVA, healthy vascular aging; EVA, early vascular aging; HbA1c: glycated hemoglobin A1c; PWv, pulse wave velocity; SAF: advanced glycation products measured by skin autofluorescence</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Differences between the cluster categories (HVA and EVA) for sociodemographic and lifestyle factors</p><p>For <bold>A</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test, and for <bold>B</bold> and <bold>C</bold>, <italic>p</italic> values were estimated using the chi-squared test. HVA, healthy vascular aging; EVA, early vascular aging</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Differences between the cluster categories (HVA and EVA) for adiposity factors</p><p>For <bold>A</bold>, <bold>B</bold> and <bold>C</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test, and for <bold>D</bold>, <italic>p</italic> values were estimated using the chi-squared test. HVA, healthy vascular aging; EVA, early vascular aging; BMI, body mass index</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Differences between the cluster categories (HVA and EVA) for glycemic and inflammatory factors</p><p>For <bold>A</bold>, <bold>C</bold> and <bold>D</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test, and for <bold>B</bold>, <italic>p</italic> values were estimated using the chi-squared test. HVA, healthy vascular aging; EVA, early vascular aging; CRP, c-reactive protein</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Differences between the cluster categories (HVA and EVA) for lipid profile factors</p><p>For <bold>A</bold>, <bold>B</bold>, <bold>C</bold> and <bold>E</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test, and for <bold>D</bold> and <bold>F</bold>, <italic>p</italic> values were estimated using the chi-squared test. HVA, healthy vascular aging; EVA, early vascular aging; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>Differences between the cluster categories (HVA and EVA) for vascular factors</p><p>For <bold>A</bold>, <bold>B</bold>, <bold>C</bold> and <bold>D</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test. HVA, healthy vascular aging; EVA, early vascular aging; AIx@75, augmentation index; ABI, ankle-brachial index; CAVI, cardio-ankle vascular index; IMT: intima media thickness</p></caption></fig>", "<fig id=\"Fig8\"><label>Fig. 8</label><caption><p>Differences between the cluster categories (HVA and EVA) for blood pressure factors</p><p>For <bold>A</bold> and <bold>B</bold>, <italic>p</italic> values were estimated using the Mann‒Whitney U test, and for <bold>C</bold>, <italic>p</italic> values were estimated using the chi-squared test. HVA, healthy vascular aging; EVA, early vascular aging; SBP, systolic blood pressure; DBP, diastolic blood pressure</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Characteristics of the participants in the EVasCu study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Total</th><th align=\"left\">HVA</th><th align=\"left\">EVA</th><th align=\"left\"><italic>p</italic>-value</th></tr></thead><tbody><tr><td align=\"left\">Sample (n)</td><td align=\"left\">390</td><td align=\"left\">232</td><td align=\"left\">158</td><td align=\"left\"/></tr><tr><td align=\"left\">Age, years</td><td align=\"left\">42.1 (13.2)</td><td align=\"left\">34.9 (10.9)</td><td align=\"left\">52.5 (8.2)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Gender, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Women</td><td align=\"left\">63.1</td><td align=\"left\">64.2</td><td align=\"left\">61.4</td><td char=\".\" align=\"char\" rowspan=\"2\">0.549</td></tr><tr><td align=\"left\"> Men</td><td align=\"left\">36.9</td><td align=\"left\">35.8</td><td align=\"left\">38.6</td></tr><tr><td align=\"left\">Smoking status, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Nonsmoker</td><td align=\"left\">63.6</td><td align=\"left\">72.8</td><td align=\"left\">50.0</td><td char=\".\" align=\"char\" rowspan=\"5\">&lt; 0.001</td></tr><tr><td align=\"left\"> Ex-smoker (&gt; 5 years)</td><td align=\"left\">18.7</td><td align=\"left\">11.2</td><td align=\"left\">29.7</td></tr><tr><td align=\"left\"> Ex-smoker (1–5 years)</td><td align=\"left\">3.3</td><td align=\"left\">0.9</td><td align=\"left\">7.0</td></tr><tr><td align=\"left\"> Ex-smoker (0–1 year)</td><td align=\"left\">1.8</td><td align=\"left\">1.7</td><td align=\"left\">1.9</td></tr><tr><td align=\"left\"> Current smoker</td><td align=\"left\">12.6</td><td align=\"left\">13.4</td><td align=\"left\">11.4</td></tr><tr><td align=\"left\">Weight, kg</td><td align=\"left\">70.1 (14.3)</td><td align=\"left\">67.9 (13.6)</td><td align=\"left\">73.7 (15.3)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Height, m</td><td align=\"left\">1.7 (0.1)</td><td align=\"left\">1.7 (0.1)</td><td align=\"left\">1.7 (0.1)</td><td char=\".\" align=\"char\">0.197</td></tr><tr><td align=\"left\">BMI, kg/m<sup>2</sup></td><td align=\"left\">24.8 (4.2)</td><td align=\"left\">23.9 (3.7)</td><td align=\"left\">26.3 (4.5)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Waist circumference, cm</td><td align=\"left\">82.7 (12.8)</td><td align=\"left\">79.1 (10.9)</td><td align=\"left\">87.9 (13.7)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Fat percentage. %</td><td align=\"left\">27.2 (9.4)</td><td align=\"left\">25.5 (8.9)</td><td align=\"left\">29.8 (9.5)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Weight status, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Underweight</td><td align=\"left\">2.8</td><td align=\"left\">3.9</td><td align=\"left\">1.2</td><td char=\".\" align=\"char\" rowspan=\"4\">&lt; 0.001</td></tr><tr><td align=\"left\"> Normal weight</td><td align=\"left\">52.4</td><td align=\"left\">60.8</td><td align=\"left\">41.1</td></tr><tr><td align=\"left\"> Overweight</td><td align=\"left\">32.1</td><td align=\"left\">27.6</td><td align=\"left\">37.3</td></tr><tr><td align=\"left\"> Obesity</td><td align=\"left\">12.6</td><td align=\"left\">7.8</td><td align=\"left\">20.3</td></tr><tr><td align=\"left\">PWv, m/s</td><td align=\"left\">6.3 (1.4)</td><td align=\"left\">5.6 (0.9)</td><td align=\"left\">7.5 (1.1)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">CAVI, m/s</td><td align=\"left\">7.1 (1.2)</td><td align=\"left\">6.7 (1.0)</td><td align=\"left\">7.6 (1.2)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">ABI</td><td align=\"left\">1.1 (0.1)</td><td align=\"left\">1.1 (0.1)</td><td align=\"left\">1.1 (0.1)</td><td char=\".\" align=\"char\">0.195</td></tr><tr><td align=\"left\">AIx-75, %</td><td align=\"left\">16.8 (11.9)</td><td align=\"left\">14.8 (11.9)</td><td align=\"left\">19.5 (11.6)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">IMT, mm</td><td align=\"left\">0.2 (0.1)</td><td align=\"left\">0.2 (0.1)</td><td align=\"left\">0.2 (0.1)</td><td char=\".\" align=\"char\">0.009</td></tr><tr><td align=\"left\">SBP, mmHg</td><td align=\"left\">116.6 (15.2)</td><td align=\"left\">112.6 (12.4)</td><td align=\"left\">122.7 (16.9)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">DBP, mmHg</td><td align=\"left\">70.3 (10.6)</td><td align=\"left\">68.2 (9.6)</td><td align=\"left\">73.5 (11.2)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">PP,mmHg</td><td align=\"left\">46.3 (9.9)</td><td align=\"left\">44.4 (9.3)</td><td align=\"left\">48.8 (10.8)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Hypertension status, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Optima</td><td align=\"left\">61.1</td><td align=\"left\">71.4</td><td align=\"left\">45.9</td><td char=\".\" align=\"char\" rowspan=\"6\">&lt; 0.001</td></tr><tr><td align=\"left\"> Normal</td><td align=\"left\">19.6</td><td align=\"left\">17.7</td><td align=\"left\">22.2</td></tr><tr><td align=\"left\"> Normal-High</td><td align=\"left\">9.8</td><td align=\"left\">6.9</td><td align=\"left\">13.9</td></tr><tr><td align=\"left\"> Hypertension Grade I</td><td align=\"left\">7.5</td><td align=\"left\">3.0</td><td align=\"left\">13.9</td></tr><tr><td align=\"left\"> Hypertension Grade II</td><td align=\"left\">1.8</td><td align=\"left\">0.9</td><td align=\"left\">3.2</td></tr><tr><td align=\"left\"> Hypertension Grade III</td><td align=\"left\">0.3</td><td align=\"left\">0.0</td><td align=\"left\">0.6</td></tr><tr><td align=\"left\">HbA1c, %</td><td align=\"left\">5.2 (0.3)</td><td align=\"left\">5.0 (0.3)</td><td align=\"left\">5.4 (0.3)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Glucose, mg/dL</td><td align=\"left\">89.4 (9.9)</td><td align=\"left\">86.3 (8.4)</td><td align=\"left\">94.0 (10.3)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Insulin, mg/dL</td><td align=\"left\">8.5 (6.1)</td><td align=\"left\">7.9 (5.7)</td><td align=\"left\">9.5 (6.6)</td><td char=\".\" align=\"char\">0.04</td></tr><tr><td align=\"left\">Diabetes status, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Nondiabetic</td><td align=\"left\">98.4</td><td align=\"left\">100</td><td align=\"left\">96.2</td><td char=\".\" align=\"char\" rowspan=\"3\">0.012</td></tr><tr><td align=\"left\">Prediabetic</td><td align=\"left\">1.3</td><td align=\"left\">0.0</td><td align=\"left\">3.2</td></tr><tr><td align=\"left\">Diabetic</td><td align=\"left\">0.3</td><td align=\"left\">0.0</td><td align=\"left\">0.6</td></tr><tr><td align=\"left\">SAF, au</td><td align=\"left\">1.9 (0.4)</td><td align=\"left\">1.7 (0.3)</td><td align=\"left\">2.2 (0.4)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">CRP, mg/L</td><td align=\"left\">1.8 (4.1)</td><td align=\"left\">1.9 (4.7)</td><td align=\"left\">1.6 (2.9)</td><td char=\".\" align=\"char\">0.600</td></tr><tr><td align=\"left\">Total cholesterol, mg/dL</td><td align=\"left\">187.6 (36.2)</td><td align=\"left\">179.3 (35.0)</td><td align=\"left\">199.8 (34.6)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">HDL, mg/dL</td><td align=\"left\">61.5 (13.7)</td><td align=\"left\">62.0 (13.8)</td><td align=\"left\">60.8 (13.8)</td><td char=\".\" align=\"char\">0.392</td></tr><tr><td align=\"left\">LDL, mg/dL</td><td align=\"left\">118.9 (32.9)</td><td align=\"left\">110.9 (31.0)</td><td align=\"left\">130.6 (32.3)</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">Triglycerides, mg/dL</td><td align=\"left\">87.2 (48.8)</td><td align=\"left\">80.7 (43.7)</td><td align=\"left\">96.9 (54.1)</td><td char=\".\" align=\"char\">0.002</td></tr><tr><td align=\"left\">Hypercholesterolemia status, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Non-hypercholesterolemia</td><td align=\"left\">64.9</td><td align=\"left\">73.3</td><td align=\"left\">52.5</td><td char=\".\" align=\"char\" rowspan=\"2\">&lt; 0.001</td></tr><tr><td align=\"left\"> Hypercholesterolemia</td><td align=\"left\">35.1</td><td align=\"left\">26.7</td><td align=\"left\">47.5</td></tr><tr><td align=\"left\">Hypertriglyceremia status, %</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Non-hypertriglyceridaemia</td><td align=\"left\">90.3</td><td align=\"left\">92.7</td><td align=\"left\">86.7</td><td char=\".\" align=\"char\" rowspan=\"2\">0.051</td></tr><tr><td align=\"left\"> Hypertriglyceridaemia</td><td align=\"left\">9.7</td><td align=\"left\">7.3</td><td align=\"left\">13.3</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Differences between the cluster categories (HVA and EVA) in the variables included in the EVA construct model</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\" colspan=\"2\">HVA</th><th align=\"left\" colspan=\"2\">EVA</th><th align=\"left\" rowspan=\"2\"><italic>p</italic> value</th></tr><tr><th align=\"left\">Construct variable</th><th align=\"left\">Median</th><th align=\"left\">IQR</th><th align=\"left\">Median</th><th align=\"left\">IQR</th></tr></thead><tbody><tr><td align=\"left\">Pulse pressure, mmHg</td><td align=\"left\">43,0</td><td char=\".\" align=\"char\">38.0–50.0</td><td char=\".\" align=\"char\">48.0</td><td char=\".\" align=\"char\">42.5–55.5</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">HbA1c, %</td><td align=\"left\">5.1</td><td char=\".\" align=\"char\">4.9–5.2</td><td char=\".\" align=\"char\">5.4</td><td char=\".\" align=\"char\">5.2–5.6</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">PWv, m/s</td><td align=\"left\">5.5</td><td char=\".\" align=\"char\">4.8–6.3</td><td char=\".\" align=\"char\">7.4</td><td char=\".\" align=\"char\">6.7-8.0</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr><tr><td align=\"left\">SAF, au</td><td align=\"left\">1.7</td><td char=\".\" align=\"char\">1.5–1.8</td><td char=\".\" align=\"char\">2.2</td><td char=\".\" align=\"char\">2.0-2.4</td><td char=\".\" align=\"char\">&lt; 0.001</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Differences between the cluster categories (HVA and EVA) in the quantitative risk factors</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Variables</th><th align=\"left\" colspan=\"2\">HVA</th><th align=\"left\" colspan=\"2\">EVA</th><th align=\"left\" rowspan=\"2\"><italic>p</italic> value</th></tr><tr><th align=\"left\">Median</th><th align=\"left\">IQR</th><th align=\"left\">Median</th><th align=\"left\">IQR</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"6\">\n<italic>Sociodemographic factors</italic>\n</td></tr><tr><td align=\"left\">Age, years</td><td align=\"left\">35.0</td><td align=\"left\">24.3–44.0</td><td align=\"left\">53.0</td><td align=\"left\">48.0–58.0</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\" colspan=\"6\">Adiposity factors</td></tr><tr><td align=\"left\">BMI, kg/m2</td><td align=\"left\">23.6</td><td align=\"left\">21.3–23.8</td><td align=\"left\">25.9</td><td align=\"left\">23.3–29.3</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">Waist circumference, cm</td><td align=\"left\">78.4</td><td align=\"left\">70.3–86.2</td><td align=\"left\">87.2</td><td align=\"left\">79.3–96.9</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">Fat percentage. %</td><td align=\"left\">24.9</td><td align=\"left\">19.2–31.8</td><td align=\"left\">29.9</td><td align=\"left\">23.9–37.5</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\" colspan=\"6\">\n<italic>Glycemic and inflammatory factors</italic>\n</td></tr><tr><td align=\"left\">Glucose, mg/dL</td><td align=\"left\">87.0</td><td align=\"left\">81.0-90.8</td><td align=\"left\">93.0</td><td align=\"left\">86.0–99.0</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">Insulin, mg/dL</td><td align=\"left\">6.6</td><td align=\"left\">4.5–9.4</td><td align=\"left\">7.1</td><td align=\"left\">4.8–11.6</td><td align=\"left\">0.034</td></tr><tr><td align=\"left\">CRP, mg/L</td><td align=\"left\">0.8</td><td align=\"left\">0.4–1.5</td><td align=\"left\">1.0</td><td align=\"left\">0.5–1.5</td><td align=\"left\">0.031</td></tr><tr><td align=\"left\" colspan=\"6\">\n<italic>Lipid profile factors</italic>\n</td></tr><tr><td align=\"left\">Total cholesterol, mg/dL</td><td align=\"left\">176.0</td><td align=\"left\">152.3-202.7</td><td align=\"left\">197.5</td><td align=\"left\">176.5-223.3</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">HDL, mg/dL</td><td align=\"left\">61.5</td><td align=\"left\">52.0–70.0</td><td align=\"left\">61.0</td><td align=\"left\">50.5–69.1</td><td align=\"left\">0.498</td></tr><tr><td align=\"left\">LDL, mg/dL</td><td align=\"left\">108.0</td><td align=\"left\">87.0-132.0</td><td align=\"left\">127.0</td><td align=\"left\">109.0-152.0</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">Triglycerides, mg/dL</td><td align=\"left\">68.5</td><td align=\"left\">56.0–91.0</td><td align=\"left\">81.5</td><td align=\"left\">63.8–114.0</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\" colspan=\"6\">\n<italic>Vascular factors</italic>\n</td></tr><tr><td align=\"left\">AIx-75, %</td><td align=\"left\">16.5</td><td align=\"left\">5.0–24.0</td><td align=\"left\">19.0</td><td align=\"left\">10.0–29.0</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">ABI</td><td align=\"left\">1.1</td><td align=\"left\">1.1–1.2</td><td align=\"left\">1.1</td><td align=\"left\">1.1–1.2</td><td align=\"left\">0.115</td></tr><tr><td align=\"left\">CAVI, m/s</td><td align=\"left\">6.8</td><td align=\"left\">6.1–7.3</td><td align=\"left\">7.4</td><td align=\"left\">7.0-8.4</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">IMT, mm</td><td align=\"left\">0.2</td><td align=\"left\">0.1–0.3</td><td align=\"left\">0.2</td><td align=\"left\">0.2–0.3</td><td align=\"left\">0.015</td></tr><tr><td align=\"left\" colspan=\"6\">\n<italic>Blood pressure factors</italic>\n</td></tr><tr><td align=\"left\">SBP, mmHg</td><td align=\"left\">111.0</td><td align=\"left\">104.0-120-0</td><td align=\"left\">122.0</td><td align=\"left\">110.0-134.5</td><td align=\"left\">&lt; 0.001</td></tr><tr><td align=\"left\">DBP, mmHg</td><td align=\"left\">68.0</td><td align=\"left\">62.0–74.0</td><td align=\"left\">73.0</td><td align=\"left\">65.0–82.0</td><td align=\"left\">&lt; 0.001</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Differences between the cluster categories (HVA and EVA) in the categorical risk factors</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\">HVA</th><th align=\"left\">EVA</th><th align=\"left\" rowspan=\"2\"><italic>p</italic> value</th></tr><tr><th align=\"left\">n (%)</th><th align=\"left\">n (%)</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"4\">\n<italic>Sociodemographic factors</italic>\n</td></tr><tr><td align=\"left\">Gender</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"3\">0.549</td></tr><tr><td align=\"left\"> Women</td><td align=\"left\">149 (64.2)</td><td align=\"left\">97 (61.4)</td></tr><tr><td align=\"left\"> Men</td><td align=\"left\">83 (35.8)</td><td align=\"left\">61 (38.6)</td></tr><tr><td align=\"left\">Smoking status</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"6\">&lt; 0.001</td></tr><tr><td align=\"left\"> Nonsmoker</td><td align=\"left\">169 (72.8)</td><td align=\"left\">79 (50.0)</td></tr><tr><td align=\"left\"> Ex-smoker (&gt; 5 years)</td><td align=\"left\">26 (11.2)</td><td align=\"left\">47 (29.7)</td></tr><tr><td align=\"left\"> Ex-smoker (1–5 years)</td><td align=\"left\">2 (0.9)</td><td align=\"left\">11 (7.0)</td></tr><tr><td align=\"left\"> Ex-smoker (0–1 year)</td><td align=\"left\">4 (1.7)</td><td align=\"left\">3 (1.9)</td></tr><tr><td align=\"left\"> Current smoker</td><td align=\"left\">31 (13.4)</td><td align=\"left\">18 (11.4)</td></tr><tr><td align=\"left\" colspan=\"4\">\n<italic>Adiposity factors</italic>\n</td></tr><tr><td align=\"left\">Weight status</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"5\">&lt; 0.001</td></tr><tr><td align=\"left\"> Underweight</td><td align=\"left\">9 (3.9)</td><td align=\"left\">2 (1.2)</td></tr><tr><td align=\"left\"> Norma weight</td><td align=\"left\">141 (60.8)</td><td align=\"left\">65 (41.1)</td></tr><tr><td align=\"left\"> Overweight</td><td align=\"left\">64 (27.6)</td><td align=\"left\">59 (37.3)</td></tr><tr><td align=\"left\"> Obesity</td><td align=\"left\">18 (7.8)</td><td align=\"left\">32 (20.3)</td></tr><tr><td align=\"left\" colspan=\"4\">\n<italic>Glycemic factors</italic>\n</td></tr><tr><td align=\"left\">Diabetes status</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"4\">0.012</td></tr><tr><td align=\"left\"> Nondiabetic</td><td align=\"left\">232 (100)</td><td align=\"left\">152 (96.2)</td></tr><tr><td align=\"left\"> Prediabetic</td><td align=\"left\">0 (0.0)</td><td align=\"left\">5 (3.2)</td></tr><tr><td align=\"left\"> Diabetic</td><td align=\"left\">0 (0.0)</td><td align=\"left\">1 (0.6)</td></tr><tr><td align=\"left\" colspan=\"4\">\n<italic>Lipid profile factors</italic>\n</td></tr><tr><td align=\"left\">Hypercholesterolemia status</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"3\">&lt; 0.001</td></tr><tr><td align=\"left\"> Non-Hypercholesterolemia</td><td align=\"left\">170 (73.3)</td><td align=\"left\">83 (52.5)</td></tr><tr><td align=\"left\"> Hypercholesterolemia</td><td align=\"left\">62 (26.7)</td><td align=\"left\">75 (47.5)</td></tr><tr><td align=\"left\">Hypertriglyceremia status</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"3\">0.051</td></tr><tr><td align=\"left\"> Non-Hypertriglyceremia</td><td align=\"left\">215 (92.7)</td><td align=\"left\">137 (86.7)</td></tr><tr><td align=\"left\"> Hypertriglyceremia</td><td align=\"left\">17 (7.3)</td><td align=\"left\">21 (13.3)</td></tr><tr><td align=\"left\" colspan=\"4\">\n<italic>Bloopd pressure factors</italic>\n</td></tr><tr><td align=\"left\">Hypertension status</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\" rowspan=\"7\">&lt; 0.001</td></tr><tr><td align=\"left\"> Optima</td><td align=\"left\">166 (71.4)</td><td align=\"left\">73 (45.9)</td></tr><tr><td align=\"left\"> Normal</td><td align=\"left\">41 (17.7)</td><td align=\"left\">35 (22.2)</td></tr><tr><td align=\"left\"> Normal-High</td><td align=\"left\">16 (6.9)</td><td align=\"left\">22 (13.9)</td></tr><tr><td align=\"left\"> Hypertension Grade I</td><td align=\"left\">7 (3.0)</td><td align=\"left\">22 (13.9)</td></tr><tr><td align=\"left\"> Hypertension Grade II</td><td align=\"left\">2 (0.9)</td><td align=\"left\">5 (3.2)</td></tr><tr><td align=\"left\"> Hypertension Grade III</td><td align=\"left\">0 (0.0)</td><td align=\"left\">1 (0.6)</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>Data are shown as the mean (SD) (continuous variables) and percentage (categorical variables). HVA, healthy vascular aging; EVA, early vascular aging; BMI, body mass index; PWv, pulse wave velocity; CAVI, cardio-ankle vascular index; ABI, ankle-brachial index; AIx@75, augmentation index; IMT: intima media thickness; SBP, systolic blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; HbA1c: glycated hemoglobin A1c; SAF: advanced glycation products measured by skin autofluorescence; CRP, c-reactive protein; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol</p></table-wrap-foot>", "<table-wrap-foot><p>HVA, healthy vascular aging; EVA, early vascular aging; IQR: interquartile range; HbA1c: glycated hemoglobin A1c; PWv, pulse wave velocity; SAF: advanced glycation products measured by skin autofluorescence</p></table-wrap-foot>", "<table-wrap-foot><p>HVA, healthy vascular aging; EVA, early vascular aging; IQR: interquartile range; BMI, body mass index; CRP, c-reactive protein; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; AIx@75, augmentation index; ABI, ankle-brachial index; CAVI, cardio-ankle vascular index; IMT: intima media thickness; SBP, systolic blood pressure; DBP, diastolic blood pressure</p></table-wrap-foot>", "<table-wrap-foot><p>HVA, healthy vascular aging; EVA, early vascular aging</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[{"label": ["2."], "mixed-citation": ["deGoma EM, Knowles JW, Angeli F, Budoff MJ, Rader DJ. The evolution and refinement of traditional risk factors for Cardiovascular Disease. Cardiol Rev. 2012 May-Jun;20(3):118\u201329."]}, {"label": ["7."], "mixed-citation": ["Laurent S. Defining vascular aging and cardiovascular risk. J Hypertens. 2012;30 Suppl:S3-8."]}, {"label": ["14."], "mixed-citation": ["\u0160muc T, Gamberger D, Krsta\u010di\u0107 G. Combining unsupervised and supervised machine learning in analysis of the CHD patient database. Lect Notes Comput Sci. 2001; 109\u201312."]}, {"label": ["15."], "mixed-citation": ["Bednarski B, Williams M, Pieszko K, Miller R, Huang C, Kwiecinski J et al. Unsupervised machine learning improves risk stratification of patients with visual normal SPECT myocardial perfusion imaging assessments. Eur Heart J. 2022."]}, {"label": ["23."], "surname": ["Von Elm", "Altman", "Egger", "Pocock", "G\u00f8tzsche", "Vandenbroucke"], "given-names": ["E", "DG", "M", "SJ", "PC", "JP"], "article-title": ["The strengthening the reporting of Observational studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies"], "source": ["The Lancet"], "year": ["2007"], "volume": ["370"], "issue": ["9596"], "fpage": ["1453"], "lpage": ["7"], "pub-id": ["10.1016/S0140-6736(07)61602-X"]}, {"label": ["24."], "mixed-citation": ["NHLBI Obesity Education Initiative Expert Panel on the Identification., Evaluation, and Treatment of Obesity in Adults (US). Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults: The Evidence Report. Bethesda (MD): National Heart, Lung, and Blood Institute; 1998 Sep. Available from: "], "ext-link": ["https://www.ncbi.nlm.nih.gov/books/NBK2003/"]}, {"label": ["25."], "collab": ["American Diabetes Association Professional Practice Committee"], "article-title": ["2. Classification and diagnosis of Diabetes: standards of Medical Care in Diabetes-2022"], "source": ["Diabetes Care"], "year": ["2022"], "volume": ["45"], "issue": ["Suppl 1"], "fpage": ["17"], "lpage": ["S38"], "pub-id": ["10.2337/dc22-S002"]}, {"label": ["27."], "surname": ["Calinski", "Harabasz"], "given-names": ["T", "J"], "article-title": ["A dendrite method for cluster analysis"], "source": ["Commun Stat Theory Methods"], "year": ["1974"], "volume": ["3"], "issue": ["1"], "fpage": ["1"], "lpage": ["27"], "pub-id": ["10.1080/03610927408827101"]}, {"label": ["28."], "surname": ["Davies", "Bouldin"], "given-names": ["DL", "DW"], "article-title": ["A cluster separation measure"], "source": ["TPAMI"], "year": ["1979"], "volume": ["1"], "issue": ["2"], "fpage": ["224"], "lpage": ["7"], "pub-id": ["10.1109/TPAMI.1979.4766909"]}, {"label": ["29."], "mixed-citation": ["Vendramin L, Campello RJ, Hruschka ER. Relative clustering validity criteria: a comparative overview. Statistical analysis and data mining: the ASA data science journal. 2010;3(4):209\u201335."]}, {"label": ["30."], "mixed-citation": ["Arthur D, Vassilvitskii S. K-means++: the advantages of careful seeding. In: ACM\u2010SIAM. 2007. p.\u00a01027\u201335."]}, {"label": ["31."], "surname": ["Pedregosa", "Varoquaux", "Gramfort", "Michel", "Thirion", "Grisel"], "given-names": ["F", "G", "A", "V", "B", "O"], "article-title": ["Scikit-learn: machine learning in Python"], "source": ["J Mach Learn Res"], "year": ["2011"], "volume": ["12"], "fpage": ["2825"], "lpage": ["30"]}, {"label": ["37."], "surname": ["Fruh", "Obesity"], "given-names": ["SM"], "article-title": ["Risk factors, Complications, and strategies for sustainable long-term weight management"], "source": ["J Am Assoc Nurse Pract"], "year": ["2017"], "volume": ["29"], "issue": ["S1"], "fpage": ["3"], "lpage": ["S14"], "pub-id": ["10.1002/2327-6924.12510"]}]
{ "acronym": [ "ABI", "ACC", "ADA", "AGEs", "AHA", "Aix-75", "BMI", "CAVI", "CRP", "CVD", "DBP", "EVA", "HDL", "HbA1c", "HVA", "IMT", "IQR", "LDL", "PCA", "PCR", "PP", "PWv", "SAF", "SBP", "STROBE" ], "definition": [ "Ankle-brachial index", "American College of Cardiology", "American Diabetes Association", "Advanced glycation end products", "American Heart Association", "Augmentation index", "Body mass index", "Cardio ankle vascular index", "Ultrasensitive C-reactive protein", "Cardiovascular disease", "Diastolic blood pressure", "Early vascular aging", "High-density lipoprotein cholesterol", "Glycated hemoglobin A1c", "Healthy vascular aging", "Intima media thickness", "Interquartile range", "Low-density lipoprotein cholesterol", "Principal component analysis", "Protein c-reactive", "Pulse pressure", "Pulse wave velocity", "Skin autofluorescence", "Systolic blood pressure", "Strengthening the Reporting of Observational Studies in Epidemiology" ] }
43
CC BY
no
2024-01-14 23:43:47
Cardiovasc Diabetol. 2024 Jan 13; 23:33
oa_package/47/55/PMC10787504.tar.gz
PMC10787505
0
[ "<title>Introduction</title>", "<title>Background and rationale</title>", "<p id=\"Par24\">Major depressive disorder (MDD) is a common mental disorder among adolescents worldwide [##UREF##0##1##], By the end of puberty, up to 20% of adolescents may have experienced it [##UREF##1##2##], leading to a significant burden in both low-income and high-income countries [##REF##34559991##3##, ##REF##32758528##4##]. The COVID-19 pandemic has further increased the prevalence of anxiety and depression, with youth being one of the most affected groups [##UREF##2##5##]. Depression in adolescents is closely associated with suicide and other adverse outcomes, such as recurrent MDD in adulthood, impaired social function, and physical illness [##REF##32758528##4##, ##REF##36219399##6##, ##UREF##3##7##].</p>", "<p id=\"Par25\">Anhedonia, the inability to experience pleasure, is a key marker of depression vulnerability [##REF##20202041##8##] and significantly contributes to the severity of depression. It predicts reduced treatment efficacy and an elevated risk of relapse and suicidality [##REF##22449641##9##–##REF##25802984##11##]. Despite access to empirically supported treatments like cognitive-behavioral therapy (CBT), selective serotonin reuptake inhibitors (SSRIs), or their combination, many adolescents with depression exhibit treatment resistance [##REF##22449646##12##]. Anhedonia, therefore, plays a detrimental role in the onset, management, and prognosis of MDD in adolescents. Yet, the therapeutic outcomes for addressing anhedonia remain far from optimal.</p>", "<p id=\"Par26\">Previous researches have underscored a connection between anhedonia and irregularities within the reward circuitry. Individuals with MDD [##REF##26487590##13##, ##REF##31712555##14##], who frequently experience anhedonia [##REF##26379600##15##] show weaker neurobiological responses to anticipating rewards when compared to healthy controls. This is most pronounced in the nucleus accumbens (NAc), which plays a crucial role in reward processing [##REF##28870407##16##]. A comprehensive longitudinal study involving adolescents has found that a diminished response in the ventral striatum during reward anticipation can presage the onset of anhedonia two years later in adolescents who were initially healthy. This predictive relationship is distinct from the one involving low mood without anhedonia [##REF##35582783##17##, ##REF##26085042##18##]. This indicates that the ventral striatum, particularly the NAc, could be a biomarker and potential target for anhedonia. However, conventional noninvasive techniques for treating the NAc can be difficult due to its deep-seated position in the brain. Repetitive transcranial magnetic stimulation (rTMS) may offer a solution by indirectly influencing the NAc through functional connectivity (FC) with the brain’s superficial cortex [##REF##32973580##19##]. Studies employing rTMS to target the most positively correlated FC between the dorsolateral prefrontal cortex (DLPFC) and the NAc have shown significant amelioration in anhedonia in adults subjects [##REF##34157193##20##]. However, effective intervention targets for anhedonia in adolescent patients, a crucial period for managing MDD, remain elusive. Therefore, testing this hypothesis specifically within this demographic is paramount and forms the basis of our study.</p>", "<p id=\"Par27\">RTMS is a safe and noninvasive neuromodulation technology that has received FDA approval [##REF##34609737##21##]. By intensifying synaptic connections and modifying FC and activity patterns in distant regions of the same brain region, high-frequency rTMS has demonstrated the potential to treat mental disorders, particularly MDD [##REF##34711062##22##, ##REF##32252538##23##], through a novel approach called individual target-transcranial magnetic stimulation (IT-TMS) [##REF##35582784##24##–##REF##36779330##26##]. It is believed to be the mechanism by which rTMS addresses anhedonia, a hallmark symptom of MDD, involves enhancing the FC between the left DLPFC and the NAc using IT-TMS [##REF##32973580##19##].</p>", "<p id=\"Par28\">We are planning to conduct a randomized controlled trial (RCT) to evaluate the efficacy of rTMS in managing anhedonia, which is a core symptom of MDD, in adolescents. The specific target areas for this study will be the DLPFC-NAc. We will use an intelligent neural navigation system to generate personalized targets based on resting-state fcMRI analysis, which will involve identifying the left DLPFC region that has the highest functional connectivity with the NAc in each participant. Our research aims to determine the preliminary efficacy, safety, and tolerability of the rTMS protocol using fcMRI-guided targeting. We hypothesized that adolescents who suffer from MDD and anhedonia will have altered neural functional patterns. We anticipate that these patterns will normalize after exposure to stimuli. Additionally, we aimed to explore changes in FC and neuropsychological functioning that are associated with anhedonia and MDD. By conducting this study, we hope to demonstrate that our hypothesis has the potential to improve the treatment of anhedonia.</p>", "<title>The explanation for the choice of comparators</title>", "<p id=\"Par29\">All patients will receive pharmacological treatments and be randomly assigned to either the active rTMS group or the sham rTMS group with a 1:1 distribution. They will then undergo consecutive intervention treatment for 15 days.</p>", "<title>Objective</title>", "<p id=\"Par30\">This study aims to evaluate the efficacy and safety of targeted rTMS in managing anhedonia in adolescents with MDD by precisely targeting DLPFC-NAc FC.</p>", "<title>Trial design</title>", "<p id=\"Par31\">This study is a double-blind, placebo-controlled, parallel-group, randomized superiority trial sponsored by the Department of Psychiatry at Xijing Hospital. The Ethics Committee of Xijing Hospital in Shaanxi Province, China, has granted approval for this study, which is also registered with ClinicalTrials.gov (reference number: NCT05544071). The study’s design is depicted in Fig. ##FIG##0##1##. Furthermore, we will follow the guidelines set by SPIRIT for reporting throughout the trial [##REF##23303884##27##] (Additional file ##SUPPL##1##2##).</p>" ]
[ "<title>Methods: participants, interventions, and outcomes</title>", "<title>Study setting</title>", "<p id=\"Par32\">This study will be conducted at Xijing Hospital (Xian, China). It is an affiliated hospital of the Air Force Medical University and sees an average 12,000 outpatients per day. From February 2023 to December 2023, eligible adolescent MDD patients will be screened for participation in both outpatient and inpatient settings. A total of 88 patients will be recruited for trial.</p>", "<title>Eligibility criteria</title>", "<p id=\"Par33\">Researchers will conduct eligibility screening based on inclusion and exclusion criteria. All participants must provide voluntary consent and sign an informed consent form after qualification is confirmed (Additional file ##SUPPL##0##1##).</p>", "<title>Inclusion criteria</title>", "<p id=\"Par34\">Participants are eligible for this study if they meet all of the following criteria.<list list-type=\"order\"><list-item><p id=\"Par35\">Aged between 13 (inclusive) and 18 (exclusive) years.</p></list-item><list-item><p id=\"Par36\">Diagnosed with MDD and currently experiencing a major depressive episode, as per the Diagnostic and Statistical Manual of Mental Disorders, 5th edition.</p></list-item><list-item><p id=\"Par37\">Must secure a total score of 17 or higher on the Hamilton Rating Scale for Depression (HAMD-17) during both the screening and baseline visits (day − 7 and day 0).</p></list-item><list-item><p id=\"Par38\">As per academic requirements, participants should achieve a total score of 20 or more on the Snaith-Hamilton Pleasure Scale (SHAPS) during both the screening and baseline visits, which occur on day − 7 and day 0 respectively [##REF##33653118##28##].</p></list-item><list-item><p id=\"Par39\">Good overall health status, as verified by medical records.</p></list-item><list-item><p id=\"Par40\">After gaining a comprehensive understanding of the rTMS treatment, participants must express their eagerness to actively participate in the therapy and be capable of providing informed consent.</p></list-item></list></p>", "<title>Exclusion criteria</title>", "<p id=\"Par41\">The study will exclude participants who meet any of the following criteria.<list list-type=\"order\"><list-item><p id=\"Par42\">Current diagnosis of a substance use disorder, excluding nicotine and caffeine dependency.</p></list-item><list-item><p id=\"Par43\">Current diagnosis of any mental disorders other than dysthymic disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, agoraphobia, or specific phobia. If these disorders are clinically unstable or have been the primary focus of treatment for the past 6 months or more, they should be excluded.</p></list-item><list-item><p id=\"Par44\">History of schizophrenia or schizoaffective disorders, or presence of psychotic symptoms during current or past episodes of depression.</p></list-item><list-item><p id=\"Par45\">Presence of any additional mental disorders, personality disorders, or intellectual disabilities that are clinically more significant than MDD at screening.</p></list-item><list-item><p id=\"Par46\">Clinically abnormality detected during screening that could potentially affect safety, interfere with study participation, or complicate the interpretation of study results.</p></list-item><list-item><p id=\"Par47\">Any current or past physical condition that, in the investigator’s judgment, could pose a risk to the participant or interfere with the interpretation of study results.</p></list-item><list-item><p id=\"Par48\">Participation in other clinical trials involving an investigational drug or device within the past month or during the study period.</p></list-item><list-item><p id=\"Par49\">Presence of electronic devices or metal implants in the skull.</p></list-item><list-item><p id=\"Par50\">History of epilepsy.</p></list-item><list-item><p id=\"Par51\">Previous history of cardiovascular disease or cardiac event.</p></list-item><list-item><p id=\"Par52\">Prior diagnosis of obsessive–compulsive disorder.</p></list-item><list-item><p id=\"Par53\">History of autism spectrum disorder.</p></list-item><list-item><p id=\"Par54\">Previously exposed to rTMS.</p></list-item><list-item><p id=\"Par55\">Use of any antidepressants within 2 weeks prior to the screening phase.</p></list-item><list-item><p id=\"Par56\">Any other circumstances deemed by the investigators as inappropriate for the study.</p></list-item></list></p>", "<title>Interventions: description</title>", "<p id=\"Par57\">The primary interventions in this study encompass both rTMS and pharmacological treatments.</p>", "<title>rTMS</title>", "<p id=\"Par58\">The study will employ a Black Dolphin TMS Robot (Spirit Dolphin, SLD-YXRJ-V1.0, Solide company, Xi'an, China) equipped with a figure-of-eight-shaped coil to apply rTMS. A personalized 3D facial tracer will be created based on participants’ MRI data, enabling the robotic arm to position the rTMS probe accurately onto the target stimulation area during treatment. Before initiating treatment, the rest motor threshold (rMT) will be determined. The rMT is the minimum intensity needed to generate 5 motor-evoked potential responses out of 10 attempts.</p>", "<p id=\"Par59\">In the active rTMS group, we will administer 50 sessions of stimulation. Each session will include 6-s trains operating at 10 Hz, with a 30-s interval between trains. The stimulation will specifically target left DLPFC, which has the strongest functional correlation with NAc. Each session will deliver 3000 pulses over 15 consecutive days, resulting in a total of 45,000 pulses. The stimulation will be administered at an intensity of 120% of the rMT.</p>", "<p id=\"Par60\">The procedure will be identical in the sham group, except that the coil will be positioned at a 90° angle to the scalp surface, thereby mimicking the procedure without delivering active stimulation.</p>", "<title>Medication</title>", "<p id=\"Par61\">During the rTMS treatment, participants will receive sertraline hydrochloride tablets, as the China National Medical Products Administration has approved sertraline for treating MDD in adolescents. Participants will begin taking sertraline hydrochloride tablets post-screening. The initial dosage will be 25 mg/day for the first 3 days, 50 mg/day for the subsequent 4 days, further increased to 100 mg /day after 1 week, assuming no adverse events that limit dosage. The dose can be flexibly adjusted between 100 and 150 mg/day until an optimal clinical response. Subsequently, participants will be required to maintain a stable antidepressant medication regimen. Concomitant medication for insomnia, such as benzodiazepines or nonbenzodiazepine hypnotics, will be permitted in this study. Any other medications taken will be reported to the principal investigator throughout the study period. Furthermore, the case report form (CRF) will meticulously document all administered medications.</p>", "<title>MRI</title>", "<p id=\"Par62\">The study will utilize a 3.0-T UNITED 770 scanner for MRI data acquisition before and after treatment. Subjects will lie quietly on the bed during the procedure, using cotton pads to minimize head motion and earplugs to block out extraneous noise. The parameters for 3D-T1-weighted structural imaging will be set as follows:<list list-type=\"bullet\"><list-item><p id=\"Par63\">Number of slices = 192</p></list-item><list-item><p id=\"Par64\">Repetition time = 7.24 ms</p></list-item><list-item><p id=\"Par65\">Echo time = 3.10 ms</p></list-item><list-item><p id=\"Par66\">Slice thickness = 1.0 mm</p></list-item><list-item><p id=\"Par67\">Matrix size = 512 × 512</p></list-item><list-item><p id=\"Par68\">Field of view = 256 × 256 mm<sup>2</sup></p></list-item><list-item><p id=\"Par69\">Flip angle = 10°</p></list-item></list></p>", "<p id=\"Par70\">The acquisition parameters will be set as follows:<list list-type=\"bullet\"><list-item><p id=\"Par71\">Number of slices = 35</p></list-item><list-item><p id=\"Par72\">Repetition time = 2000 ms</p></list-item><list-item><p id=\"Par73\">Echo time = 30 ms</p></list-item><list-item><p id=\"Par74\">Slice thickness = 4 mm</p></list-item><list-item><p id=\"Par75\">Matrix size = 64 × 64</p></list-item><list-item><p id=\"Par76\">Field of view = 224 × 224 mm<sup>2</sup></p></list-item><list-item><p id=\"Par77\">Flip angle = 90°</p></list-item></list></p>", "<p id=\"Par78\">These settings will ensure high-resolution imaging necessary for precise analysis of structural brain changes.</p>", "<title>Interventions: modifications</title>", "<p id=\"Par79\">There is no intervention modification plan except for adjusting drug dosage due to changes in symptoms.</p>", "<title>Interventions: adherence</title>", "<p id=\"Par80\">Before intervention implementation, the operator will provide participants with a detailed introduction to the treatment to dispel patient concerns, including its nature, expected effects, and possible adverse reactions. During treatment, they will also be reminded of follow-up treatment time. The purpose of these measures is to improve adherence to intervention protocols.</p>", "<title>Interventions: concomitant care</title>", "<p id=\"Par81\">Concomitant medication for insomnia, such as benzodiazepines or nonbenzodiazepine hypnotics, will be permitted in this study. Any other medications taken will be reported to the principal investigator and meticulously documented in the case report form (CRF) throughout the study period.</p>", "<title>Outcome</title>", "<title>Primary outcome</title>", "<p id=\"Par82\">The primary outcome measure for this study will be the percentage difference in the SHAPS score between the initial (baseline) assessment and the 8-week follow-up assessment. The SHAPS is a widely adopted 14-item self-assessment questionnaire utilized to gauge the severity of anhedonia symptoms in individuals diagnosed with mood disorders. The scale ranges from 14 to 56, with higher scores indicating more severe anhedonia symptoms.</p>", "<title>Secondary outcome</title>", "<p id=\"Par83\">Secondary outcomes of this research will include the following:<list list-type=\"order\"><list-item><p id=\"Par84\">Seventeen-item Hamilton Depression Rating Scale (HAMD-17): Percent decrease in the HAMD-17 and the Montgomery Asberg Depression Rating Scale (MADRS) to assess remission and recovery from depression. The HAMD is the most commonly used clinician-administered scale for evaluating depression [##REF##14399272##29##]. This study will use the 17-item, where a higher score denotes more severe symptoms.</p></list-item><list-item><p id=\"Par85\">Montgomery Asberg Depression Rating Scale (MADRS): This examiner-rated scale can be used to evaluate the depressive symptoms of MDD in conjunction with HAMD-17 [##REF##444788##30##]. The scale ranges from 0 to 60, with a higher score indicating more significant depressive symptomology.</p></list-item><list-item><p id=\"Par86\">The Chinese Version of Temporal Experience of Pleasure Scale (CV-TEPS): The TEPS includes 20 items, 11 measuring anticipatory anhedonia and 9 evaluating consummatory anhedonia [##UREF##4##31##, ##REF##34320935##32##]. The Chinese version (CV-TEPS) has been confirmed as a reliable and valid instrument for assessing anhedonia in Chinese students [##REF##34320935##32##]. The scale ranges from 20 to 120, with a lower score representing more significant anhedonia symptomology.</p></list-item><list-item><p id=\"Par87\">The Chinese Version of Beck Scale for Suicide Ideation (BSI-CV). The BSI-CV is a 19-item questionnaire used to reliably and validly measure the severity of suicidal ideation [##REF##469082##33##, ##REF##17453698##34##]. The scale includes two subscales: current suicidal ideation and past suicidal ideation (Suicidal ideation at one’s worst point). The scale ranges from 0 to 38, with higher scores indicating more severe suicidal ideation in the last week or at the most serious point.</p></list-item><list-item><p id=\"Par88\">The Insomnia Severity Index (ISI): The ISI questionnaire has good reliability and validity for estimating insomnia severity. It includes 7 items: the severity of insomnia symptoms, satisfaction with sleep patterns, the effects of insomnia on daytime function, the effects of insomnia on subjects’ quality of life, and the degree of worry or distress caused by insomnia [##REF##21493134##35##, ##REF##11438246##36##]. The scale ranges from 0 to 28, with a higher score illustrating more significant insomnia symptomology.</p></list-item><list-item><p id=\"Par89\">The Clinical Global Impression (CGI): The CGI scale is designed to assess clinical efficacy and includes three parts: severity of illness, global improvement, and effect index [##UREF##5##37##]. A higher score suggests a more severe disease and poorer efficacy.</p></list-item><list-item><p id=\"Par90\">FC: Functional connectivity refers to the extent to which two brain regions are simultaneously activated and communicating with each other [##REF##30831580##38##]. It is recorded using fMRI techniques, such as the blood oxygen level-dependent (BOLD) signal.</p></list-item><list-item><p id=\"Par91\">THINC-it: THINC-it is a cognitive screening tool comprised of objective and subjective measures of cognitive function with high levels of reliability and stability [##REF##30088298##39##].</p></list-item></list></p>", "<title>Participant timeline</title>", "<p id=\"Par92\">This study will be divided into a 7-day screening period, a 15-day stimulation period, and a 6-week follow-up period. After qualification confirmation, the subjects will sign an informed consent form, undergo MRI scans, and begin sertraline titration. Subsequently, they will be randomly assigned to one of two groups for a 15-day stimulation period with a 1:1 ratio. All patients will undergo a second MRI scan after the stimulation period. Finally, there will be a 6-week follow-up period. We will conduct clinical scale evaluation and cognitive testing at baseline (D0), 7 and 15 days of rTMS treatment (D7&amp;D15), and 2 and 6 weeks after the end of the stimulation period (D28&amp;D56). We will track the number of participants excluded, refused consent, or withdrawn from the study and their reasons. The timeline is shown in Fig. ##FIG##1##2##.</p>", "<title>Sample size</title>", "<p id=\"Par93\">The objective of this study is to investigate the efficacy and underlying mechanism of rTMS, explicitly targeting the DLPFC and NAc in adolescents suffering from MDD and anhedonia. Based on previous studies [##UREF##6##40##], we anticipate the SHAPS score to be 29.59 ± 7.677 in the active treatment group compared to 34.82 ± 7.283 in the sham treatment group. A power analysis (with a power of 0.8, <italic>α</italic> of 0.05, and a two-sided test) estimates that a minimum of 34 patients per group will be required to detect a significant difference. Taking into account a projected dropout rate of 20% and blocked randomization, the plan is to recruit a total of 88 patients. As for the fMRI studies, earlier studies suggest a sample size between 15 and 30 patients suffices to validate the hypothesis [##REF##26884746##41##]. Therefore, this study will proceed with the computed maximum sample size of 88.</p>", "<title>Recruitment</title>", "<p id=\"Par94\">This study will recruit participants who fulfill our criteria from both the outpatient and inpatient departments of Xijing Hospital, utilizing recruitment advertisements for outreach.</p>" ]
[ "<title>Dissemination policy: trial results</title>", "<p id=\"Par121\">The results of the study will be prepared for submission to international, peer-reviewed journals. This process involves assembling the data into a comprehensive manuscript that outlines the study’s methodology, findings, and implications.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par110\">This study is a randomized, double-blind, sham stimulation-controlled, parallel-group trial. Its goal is to investigate the effectiveness of rTMS in improving anhedonia in adolescent patients diagnosed with MDD. Previous researches have indicated that rTMS could be a promising therapeutic intervention for anhedonia [##REF##34453491##42##]. However, the efficacy of fcMRI-guided rTMS in mitigating anhedonia in adolescent patients is yet to be explored. The brain’s neural network is a complex system that has been partially explored. Advances in fMRI have provided compelling evidence suggesting that individuals exhibit substantial activation in striatal regions (specifically, the NAc, caudate, and putamen) during reward processing. In contrast, individuals suffering from anhedonia often show diminished activity in these regions, regardless of whether they have MDD or not [##REF##33965516##43##–##UREF##7##46##]. Randy et al. found that reduced reward-related activation in the NAc was linked to anhedonia in adolescents with first-episode depression [##REF##33965516##43##]. Additionally, a recent study reported that activating the left ventral striatum, particularly the NAc, improves anhedonia-related symptoms [##UREF##8##47##]. This suggests that Nac could be a potential target for anhedonia [##UREF##8##47##–##UREF##9##49##]. As discussed earlier, the effectiveness of antidepressant medication and evidence-based psychotherapy remains unsatisfactory. However, rTMS shows promise as a precise intervention for the NAc. Due to the positively correlated functional connection between the left DLPFC and NAc [##REF##32973580##19##], it is crucial to conduct additional clinical studies to clarify the effectiveness and potential neural mechanisms of fcMRI-guided rTMS through DLPFC-NAc FC for anhedonia in adolescent MDD patients. This clinical study will generate the necessary data to support these hypotheses.</p>", "<p id=\"Par111\">We hypothesize a potential association between the efficacy of the intervention and the modulation of FC between the DLPFC and NAc. If this hypothesis is correct, the active treatment group should show higher modulation in the FC maps of reward-related regions than the sham treatment group. These results could provide insights into the potential mechanism of rTMS for this specific symptom among adolescents suffering from MDD.</p>" ]
[]
[ "<title>Background</title>", "<p id=\"Par1\">Anhedonia, which is defined as the inability to feel pleasure, is considered a core symptom of major depressive disorder (MDD). It can lead to several adverse outcomes in adolescents, including heightened disease severity, resistance to antidepressants, recurrence of MDD, and even suicide. Specifically, patients who suffer from anhedonia may exhibit a limited response to selective serotonin reuptake inhibitors (SSRIs) and cognitive behavioral therapy (CBT). Previous researches have revealed a link between anhedonia and abnormalities within the reward circuitry, making the nucleus accumbens (NAc) a potential target for treatment. However, since the NAc is deep within the brain, repetitive transcranial magnetic stimulation (rTMS) has the potential to modulate this specific region. Recent advances have enabled treatment technology to precisely target the left dorsolateral prefrontal cortex (DLPFC) and modify the functional connectivity (FC) between DLPFC and NAc in adolescent patients with anhedonia. Therefore, we plan to conduct a study to explore the safety and effectiveness of using resting-state functional connectivity magnetic resonance imaging (fcMRI)-guided rTMS to alleviate anhedonia in adolescents diagnosed with MDD.</p>", "<title>Methods</title>", "<p id=\"Par2\">The aim of this article is to provide a study protocol for a parallel-group randomized, double-blind, placebo-controlled experiment. The study will involve 88 participants who will be randomly assigned to receive either active rTMS or sham rTMS. The primary object is to measure the percentage change in the severity of anhedonia, using the Snaith-Hamilton Pleasure Scale (SHAPS). The assessment will be conducted from the baseline to 8-week post-treatment period. The secondary outcome includes encompassing fMRI measurements, scores on the 17-item Hamilton Rating Scale for Depression (HAMD-17), the Montgomery Asberg Depression Rating Scale (MADRS), the Chinese Version of Temporal Experience of Pleasure Scale (CV-TEPS), and the Chinese Version of Beck Scale for Suicide Ideation (BSI-CV). The Clinical Global Impression (CGI) scores will also be taken into account, and adverse events will be monitored. These evaluations will be conducted at baseline, as well as at 1, 2, 4, and 8 weeks.</p>", "<title>Discussion</title>", "<p id=\"Par3\">If the hypothesis of the current study is confirmed, (fcMRI)-guided rTMS could be a powerful tool to alleviate the core symptoms of MDD and provide essential data to explore the mechanism of anhedonia.</p>", "<title>Trial registration</title>", "<p id=\"Par4\">ClinicalTrials.gov NCT05544071. Registered on 16 September 2022.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s13063-023-07814-y.</p>", "<title>Keywords</title>" ]
[ "<title>Methods: assignment of interventions</title>", "<title>Allocation: sequence generation</title>", "<p id=\"Par95\">The study will adopt a blocked randomization approach. The random allocation sequence will be generated using statistical analysis software (version 9.4, SAS Institute Inc., Cary, NC, USA). The purpose of generating sequence numbers is to divide participants evenly and randomly into two groups.</p>", "<title>Allocation: concealment mechanism</title>", "<p id=\"Par96\">Computer-assisted generation of opaque envelopes will be marked with sequence numbers externally and containing a concealed randomization list internally. Upon confirmation of each patient’s eligibility, researchers will open the envelope and inform operators responsible for implementing treatment of the grouping results.</p>", "<title>Allocation: implementation</title>", "<p id=\"Par97\">The unblinded research members will create the allocation sequence and assign participants to either the active or sham treatment groups. RTMS is performed by trained operators subsequently.</p>", "<title>Blinding</title>", "<p id=\"Par98\">The treatment assignments will be hidden from participants, clinical evaluators, and statistical analysts. While aware of participant allocation, the unblinded research members and experienced rTMS operators will be deliberately excluded from the evaluations and data analysis processes.</p>", "<title>Blinding: emergency unblinding</title>", "<p id=\"Par99\">They are barring exceptional circumstances such as serious adverse events or other emergent situations necessitating the breaking of the blinding protocol.</p>", "<title>Methods: data collection, management, and analysis</title>", "<title>Data collection plan</title>", "<p id=\"Par100\">A paper case report form (CRF) will record all experimental processes and results. After each participant completes the research process, two researchers will independently input the original paper records into an Excel sheet to verify the completeness and accuracy of the paper records and data entry with each other. MRI scan will be implemented at the Yunying Medical Imaging Diagnosis Center (Xian, China).</p>", "<title>Data collection plan: retention</title>", "<p id=\"Par101\">When signing the informed consent form, researchers will provide participants with a detailed introduction to the trial situation, including the purpose, process, requirements, and disease-related information, including etiology, treatment methods, and outcomes. During the study process, they will also be reminded to complete subsequent actions. The purpose of these measures is to improve subject compliance. Subjects can request withdrawal for any reason, while researchers will collect as much outcome data on this group of patients as possible.</p>", "<title>Data management</title>", "<p id=\"Par102\">Clinical data will be accurately, comprehensively, and promptly recorded on CRF as soon as they are acquired. Each participant will be assigned a unique identifier, replacing the need for personal names in the data collection process. After each participant completes the research, two researchers will independently input the original paper records into an Excel sheet to verify the completeness and accuracy of the paper records and data entry with each other. This database will allow the application for the corresponding author to access the final trial dataset. Data will be directly imported into IBM SPSS (version 26.0) to facilitate the statistical analysis.</p>", "<title>Statistics: outcomes</title>", "<p id=\"Par103\">Statistical analyses will be conducted on clinical data using IBM SPSS version 26 (Armonk, NY). Current research results are expected to be consistent with the two-factor (active and sham rTMS) and multilevel (baseline and all follow-up time points) analysis of variance of repeated measurement data. We will evaluate the normality of continuous data through the Shapiro–Wilk test. To examine the assumption of sphericity, we will employ Mauchly’s test of sphericity. A two-sided test will be deemed statistically significant if the <italic>P</italic>-value is less than or equal to 0.05. Any missing data in a specific group will be substituted with the corresponding group’s mean value. Mean ± standard deviation or frequency (%) will be used to present sociodemographic information and clinical outcomes in both the active and sham treatment groups. The neuroimaging indexes will be analyzed by MATLAB R2013b and SPM12 software to explore the changes in FC from baseline to posttreatment. We used correlations between imaging data and questionnaire scores to identify predictors potentially affecting rTMS outcomes.</p>", "<title>Statistics: additional analyses</title>", "<p id=\"Par104\">There are no additional analysis plans.</p>", "<title>Statistics: analysis population and missing data</title>", "<p id=\"Par105\">Any missing data in a specific group will be substituted with the corresponding group’s mean value.</p>", "<title>Methods: monitoring</title>", "<title>Data monitoring: formal committee</title>", "<p id=\"Par106\">A data monitoring committee (DMC) is crucial in maintaining researchers’ and clinicians’ blindness and ensuring the research study’s integrity. To address potential adverse reactions, an emergency epilepsy rescue team will be established under the purview of the DMC. Before enrollment, all researchers involved in the study will undergo training to understand the clinical manifestations and management processes related to seizures. In the event of any adverse reactions, these will be promptly reported to the DMC and relevant regulatory agencies. This procedure ensures the study participants’ safety and the study results’ validity.</p>", "<title>Data monitoring: interim analysis</title>", "<p id=\"Par107\">There are no interim analysis plans.</p>", "<title>Harms</title>", "<p id=\"Par108\">Possible adverse reactions associated with rTMS may include headache, fatigue, tingling, muscle twitching or soreness, syncope (fainting), mania, epilepsy, etc. All adverse reactions during the study will be meticulously recorded in the CRF. Each case will be evaluated for severity and possible causal relationships with the treatment. In the event of any adverse reactions, the information will be promptly reported to the DMC and relevant regulatory agencies. This ensures a high standard of safety and transparency throughout the study and allows for appropriate action if required.</p>", "<title>Auditing</title>", "<p id=\"Par109\">The sponsoring agency will appoint independent supervisors to oversee the research supervision work of the clinical trials. The role of these inspectors is to ensure that researchers adhere strictly to the experimental protocol, relevant standard operating procedures, guiding principles, and regulatory requirements throughout the research process. The supervisors conduct monthly site visits to check the research progress. These visits will involve verification of original records, CRF, and other pertinent information. These checks aim to ensure the researcher’s compliance with the research protocol, procedures, and regulations and guarantee that the obtained research data is objective, truthful, and lawful. At the end of the study, the inspectors will be responsible for verifying and archiving all documents about the research conducted at the center.</p>", "<title>Trial status</title>", "<p id=\"Par112\">The Ethics Committee of Xijing Hospital approved the study protocol in September 2022 (Protocol ID: KY20222165-F-1). The current version is version 4.0, dated 11 May 2023. This trial has been registered with the ClinicalTrials.gov database under the reference number NCT05544071. The trial commenced on 1 February 2023. Participant recruitment for the trial is ongoing. We have enrolled and randomly assigned 51 participants as of the latest update. We anticipate completing the recruitment phase by December 2023.</p>", "<title>Ethics and dissemination</title>", "<title>Research ethics approval</title>", "<p id=\"Par113\">The study protocol has received approval from the Ethics Committee of Xijing Hospital in Shaanxi Province, China. and was registered at ClinicalTrials. Gov (URL: the Safety and Effectiveness of Precise rTMS Based on Neuroimaging in the Treatment of Adolescent Depression With Anhedoniadepression With Anhedonia—Full Text View—ClinicalTrials.gov reference number: NCT05544071). This study will be conducted in accordance with the ethical principles stated in the Declaration of Helsinki.</p>", "<title>Protocol amendments</title>", "<p id=\"Par114\">Any significant modifications to the protocol will be promptly reported to the Ethics Committee of Xijing Hospital and updated on ClinicalTrials.gov.</p>", "<title>Consent or assent</title>", "<p id=\"Par115\">Eligibility screening for adolescent MDD patients will be conducted in outpatient and inpatient at Xijing Hospital. The researchers will provide participants with a detailed introduction to the trial situation, including the purpose, process, and requirements after qualification confirmation. All participants will voluntarily participate in the study and sign an informed consent.</p>", "<title>Consent or assent: ancillary studies</title>", "<p id=\"Par116\">There are no relevant plans.</p>", "<title>Confidentiality</title>", "<p id=\"Par117\">After enrollment, each participant will be assigned a unique identifier, replacing the need for personal names in the data collection process. This unique identifier will be meaningful only to the research team, ensuring participant confidentiality. On completion of the study, the principal investigator will be responsible for securely storing and protecting these unique identifiers, in accordance with research guidelines. Any publications resulting from this study will not include any personally identifiable information, maintaining participant privacy at all times.</p>", "<title>Declaration of interests</title>", "<p id=\"Par118\">The submitter declares that they have no competing interests.</p>", "<title>Data access</title>", "<p id=\"Par119\">This database will allow the application for Huaning Wang to access the final trial dataset.</p>", "<title>Ancillary and post-trial care</title>", "<p id=\"Par120\">If any harm related to this study occur, participants can receive free treatment provided by Xijing Hospital, which will compensate in accordance with relevant laws and regulations.</p>", "<title>Dissemination policy: authorship</title>", "<p id=\"Par122\">See the author’s contribution section below.</p>", "<title>Dissemination policy: reproducible research</title>", "<p id=\"Par123\">The public can access the complete protocol through ClinicalTrials.gov website (NCT05544071), but it does not include the personal information of participants. This database will allow the reasonable application for corresponding author to access the final trial dataset.</p>", "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>We express our gratitude to Professor Chong Lei for her invaluable guidance regarding the manuscript. Additionally, we extend our thankfulness to all participants in the clinical trial and their families.</p>", "<title>Authors’ contributions</title>", "<p>All authors participated in the protocol design. The study was conceived and designed by RL, NT, YuyuZ, NL, TH, YaochiZ, MC, and HW. RL drafted the protocol, whereas NT, YS, CL, MC, and HW reviewed and revised the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This study received financial support from the National Natural Science Foundation of China (grant number 81974215). The funder has no role in collecting, analyzing, and interpreting data or writing manuscripts.</p>", "<title>Availability of data and materials</title>", "<p>All study data will be deposited at the Department of Psychiatry of Xijing Hospital. The datasets that have been analyzed during the current study will be available for future secondary analysis with the approval from the corresponding author.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par124\">The study protocol was approved by the Ethics Committee of Xijing Hospital in Shaanxi Province, China, and was registered at ClinicalTrials.gov (URL: the Safety and Effectiveness of Precise rTMS Based on Neuroimaging in the Treatment of Adolescent Depression With Anhedoniadepression With Anhedonia—Full Text View—ClinicalTrials.gov reference number: NCT05544071). Participants will be required to provide informed consent forms.</p>", "<title>Consent for publication</title>", "<p id=\"Par125\">This manuscript and the provided sample of the informed consent form do not contain the personal data of the patients. Except for current information, participant information materials and informed consent forms will be available from the corresponding author upon request.</p>", "<title>Competing interests</title>", "<p id=\"Par126\">The authors declare that they have no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Flow chart to interpret the format of the trial</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Case report form (CRF)</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>" ]
[ "<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"13063_2023_7814_Fig1_HTML\" id=\"MO1\"/>", "<graphic xlink:href=\"13063_2023_7814_Fig2_HTML\" id=\"MO2\"/>" ]
[ "<media xlink:href=\"13063_2023_7814_MOESM1_ESM.zip\"><caption><p><bold>Additional file 1.</bold></p></caption></media>", "<media xlink:href=\"13063_2023_7814_MOESM2_ESM.docx\"><caption><p><bold>Additional file 2.</bold></p></caption></media>" ]
[{"label": ["1."], "mixed-citation": ["WHO. Adolescent mental health 2020 [Available from: "], "ext-link": ["https://www.who.int/news-room/fact-sheets/detail/adolescent-mental-health"]}, {"label": ["2."], "surname": ["Thapar", "Collishaw", "Pine", "Thapar"], "given-names": ["A", "S", "DS", "AK"], "article-title": ["Depression in adolescence"], "source": ["The Lancet"], "year": ["2012"], "volume": ["379"], "issue": ["9820"], "fpage": ["1056"], "lpage": ["1067"], "pub-id": ["10.1016/S0140-6736(11)60871-4"]}, {"label": ["5."], "mixed-citation": ["organization wh. COVID-19 pandemic triggers 25% increase in prevalence of anxiety and depression worldwide 2022 [Available from: "], "ext-link": ["https://www.who.int/news/item/02-03-2022-covid-19-pandemic-triggers-25-increase-in-prevalence-of-anxiety-and-depression-worldwide"]}, {"label": ["7."], "surname": ["Leone", "Kuja-Halkola", "Leval", "D\u2019Onofrio", "Larsson", "Lichtenstein"], "given-names": ["M", "R", "A", "BM", "H", "P"], "article-title": ["Association of youth depression with subsequent somatic diseases and premature death"], "source": ["JAMA Psychiat"], "year": ["2021"], "volume": ["78"], "issue": ["3"], "fpage": ["302"], "lpage": ["310"], "pub-id": ["10.1001/jamapsychiatry.2020.3786"]}, {"label": ["31."], "surname": ["Gard", "Gard", "Kring", "John"], "given-names": ["DE", "MG", "AM", "OP"], "article-title": ["Anticipatory and consummatory components of the experience of pleasure: a scale development study"], "source": ["J Res Pers"], "year": ["2006"], "volume": ["40"], "issue": ["6"], "fpage": ["1086"], "lpage": ["1102"], "pub-id": ["10.1016/j.jrp.2005.11.001"]}, {"label": ["37."], "surname": ["Guy"], "given-names": ["W"], "source": ["ECDEU assessment manual for psychopharmacology: US Department of Health, Education, and Welfare, Public Health Service"], "year": ["1976"]}, {"label": ["40."], "surname": ["Xuemin"], "given-names": ["Z"], "source": ["Efficacy and safety of robot-assisted repetitive transcranial magnetic stimulation combined with sertraline in adolescent depressive disorder [master]: Department of Psychiatry, The First Hospital of Shanxi Medical University, Taiyuan, China"], "year": ["2022"]}, {"label": ["46."], "surname": ["Whitton", "Pizzagalli", "Pizzagalli"], "given-names": ["AE", "DA", "DA"], "article-title": ["Anhedonia in depression and bipolar disorder"], "source": ["Anhedonia: Preclinical, Translational, and Clinical Integration"], "year": ["2022"], "publisher-loc": ["Cham"], "publisher-name": ["Springer International Publishing"], "fpage": ["111"], "lpage": ["127"]}, {"label": ["47."], "surname": ["Eckstrand", "Forbes", "Bertocci", "Chase", "Greenberg", "Lockovich"], "given-names": ["KL", "EE", "MA", "HW", "T", "J"], "article-title": ["Anhedonia reduction and the association between left ventral striatal reward response and 6-month improvement in life satisfaction among young adults"], "source": ["JAMA Psychiat"], "year": ["2019"], "volume": ["76"], "issue": ["9"], "fpage": ["958"], "lpage": ["965"], "pub-id": ["10.1001/jamapsychiatry.2019.0864"]}, {"label": ["49."], "surname": ["Tang", "Harrewijn", "Benson", "Haller", "Guyer", "Perez-Edgar"], "given-names": ["A", "A", "B", "SP", "AE", "KE"], "article-title": ["Striatal activity to reward anticipation as a moderator of the association between early behavioral inhibition and changes in anxiety and depressive symptoms from adolescence to adulthood"], "source": ["JAMA Psychiat"], "year": ["2022"], "volume": ["79"], "issue": ["12"], "fpage": ["1199"], "lpage": ["1208"], "pub-id": ["10.1001/jamapsychiatry.2022.3483"]}]
{ "acronym": [ "MDD", "SSRI", "CBT", "NAc", "rTMS", "DLPFC", "FC", "MRI", "SHAPS", "HAMD-17", "MADRS", "CV-TEPS", "BSI-CV", "ISI", "CGI", "IT-TMS", "RCT", "CRF", "RMT", "DMC" ], "definition": [ "Major depressive disorder", "Selective serotonin reuptake inhibitor", "Cognitive behavioral therapy", "Nucleus accumbens", "Repetitive transcranial magnetic stimulation", "Dorsolateral prefrontal cortex", "Functional connectivity", "Magnetic resonance imaging", "Snaith-Hamilton pleasure scale", "Hamilton Rating Scale for Depression", "Montgomery Asberg Depression Rating Scale", "Chinese Version of the Temporal Experience of Pleasure Scale", "Chinese Version of Beck Scale for Suicide Ideation", "Insomnia Severity Index", "Clinical Global Impression", "Individual target-transcranial magnetic stimulation", "Randomized controlled trial", "Case report form", "Rest motor threshold", "Data Monitoring Committee" ] }
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2024-01-14 23:43:47
Trials. 2024 Jan 13; 25:44
oa_package/9a/0d/PMC10787505.tar.gz
PMC10787506
38217030
[ "<title>Background</title>", "<p id=\"Par47\">Breast cancer, one of the most common types of malignant tumors affecting women, has high incidence and mortality rates worldwide [##REF##31421262##1##]. Breast cancer is a highly heterogeneous disease, which is molecularly classified into the luminal, HER2-overexpressing (HER2 +), and triple-negative subtypes. Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer, which is defined as estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and human epidermal growth factor receptor type 2 (HER2)-negative disease [##REF##21067385##2##]. Because of the lack of ER, PR, and HER2, TNBC is insensitive to established endocrine and anti-HER2 therapies, which carry a particularly unfavorable prognosis. Thus, there remains a great need to further explore the mechanisms of TNBC progression and identify potential therapeutic targets.</p>", "<p id=\"Par48\">Aerobic glycolysis, also known as the Warburg effect, is among the most important mechanisms in tumor growth and progression [##REF##34014531##3##]. During this process, cancer cells are characterized by increased glucose uptake and lactate production [##REF##30626875##4##]. There are three important rate-limiting enzymes including hexokinase (HK), pyruvate kinase (PK), and phosphofructokinase (PFK) to control aerobic glycolysis. Human PFK exists as three isoforms: liver (PFKL), muscle (PFKM), and platelet (PFKP), which catalyzes the phosphorylation of fructose-6-phosphate to fructose-1, 6-bisphosphate during the glycolytic pathway [##UREF##0##5##]. PFKP is overexpressed in various types of cancers and its elevated expression is associated with poor prognosis. For example, PFKP alleviates glucose starvation-induced metabolic stress by regulating long-chain fatty acid oxidation via the AMPK-ACC2 axis in lung cancer [##REF##35641476##6##]. In addition, PFKP levels are higher in hepatocellular carcinoma (HCC) as compared to normal hepatic tissues, which promotes HCC proliferation and contributes to the maintenance of HCC stemness [##REF##34418458##7##]. A recent study in colorectal cancer also validates that PFKP is significantly overexpressed in cancer tissues and overexpression of PFKP contributes to the growth and invasion of cancer cells by regulating cell cycle progression [##REF##37151384##8##]. However, the functions and mechanisms underlying PFKP expression in TNBC are still unclear, in particular the mechanisms regulating PFKP protein deubiquitination. Thus, further elucidating the underlying mechanisms of PFKP protein deubiquitination will identify new therapeutic strategies to deplete PFKP expression and develop personalized targeted therapies for TNBC.</p>", "<p id=\"Par49\">Deubiquitinases (DUBs) are proteases of the ubiquitin–proteasome system that cleave ubiquitin from substrates as well as disassemble polyubiquitin chains [##REF##19626045##9##]. Numerous pieces of evidence suggest that dysregulation of DUBs has been implicated in various human diseases, including cancer. For example, ATXN3 promotes prostate cancer progression by interacting with, deubiquitylating, and stabilizing YAP protein [##REF##37349820##10##]. USP8 positively regulates hepatocellular carcinoma tumorigenesis and confers ferroptosis resistance through β-catenin stabilization [##REF##37311739##11##]. In addition, USP13 regulates glycolytic reprogramming and progression in osteosarcoma by stabilizing METTL3/m6A/ATG5 axis [##REF##37151889##12##]. Notably, growing evidence has indicated that several inhibitors of DUBs displayed potent anticancer activities. For example, the USP5 inhibitor EOAI prevents non-small cell lung cancer progression by inducing DNA damage [##REF##36611139##13##]. And USP8 inhibitor DUB-IN-1 induces DNA damage, cell cycle arrest, apoptosis, and autophagy in esophageal squamous cell carcinoma [##REF##35022897##14##]. Together, these studies indicate that DUBs are potential therapeutic targets in the treatment of cancer progression.</p>", "<p id=\"Par50\">In the current study, we aim to investigate the expression and functions of PFKP in TNBC, furthermore, elucidate the underlying mechanisms of PFKP deubiquitination to identify new therapeutic strategies that deplete PFKP expression, and provide personalized targeted therapies for TNBC.</p>" ]
[ "<title>Methods</title>", "<title>Database analysis</title>", "<p id=\"Par51\">Protein expression data of PFKP in TNBC and normal samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) cohort were analyzed based on the UALCAN (<ext-link ext-link-type=\"uri\" xlink:href=\"http://ualcan.path.uab.edu/\">http://ualcan.path.uab.edu/</ext-link>) website [##REF##35562349##15##]. The association between PFKP protein expression and the prognosis of TNBC patients was analyzed using the Kaplan–Meier plotter (KM plotter) database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://kmplot.com/analysis/)\">http://kmplot.com/analysis/)</ext-link> [##REF##34408238##16##]. The correlation between PFKP, deubiquitinase, and E3 ubiquitinase protein expression was analyzed by the LinkedOmics platform [##REF##29136207##17##].</p>", "<title>Tissue microarray and IHC staining</title>", "<p id=\"Par52\">A total of 172 cases of human TNBC and 130 cases of para-carcinoma tissues were obtained from Shanghai Superbiotek Pharmaceutical Technology Co., Ltd. The IHC staining and evaluation were performed as previously described [##REF##32296027##18##], using 1:100 dilution of an anti-PFKP antibody (Santa Cruz, sc-514824) and 1:200 dilution of an anti-USP5 antibody (Proteintech, 10,473-1-AP).</p>", "<title>Cell culture</title>", "<p id=\"Par53\">MDA-MB-231, SUM-159, and 293T Cell lines information and cell culture conditions were described previously [##REF##32296027##18##]. The MCF-10A cell line was obtained from the American Type Culture Collection and cultured with DMEM/F12 medium supplemented with 5% horse serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, and 100 ng/mL cholera toxin in a humidified incubator at 37 °C with 5% CO<sub>2</sub>.</p>", "<title>Western blotting</title>", "<p id=\"Par54\">Western blotting assay was performed as described previously [##REF##35058607##19##], using 1:1000 dilution of an anti-PFKP antibody (Santa Cruz, sc-514824), 1:1000 dilution of an anti-USP5 antibody (Proteintech, 10,473-1-AP), and 1:1000 dilution of an anti-β actin antibody (Santa Cruz, sc-47778).</p>", "<title>RNA extraction and quantitative RT-PCR</title>", "<p id=\"Par55\">RNA extraction and quantitative RT-PCR were performed as previously described [##REF##32296027##18##]. β-actin was used as a normalization control. The primer sequences are listed in Additional file ##SUPPL##0##1##: Table S1.</p>", "<title>Lentiviral overexpression and knockdown systems</title>", "<p id=\"Par56\">Lentiviral systems for overexpression of PFKP and knockdown of PFKP and USP5 were performed as described previously [##REF##32296027##18##]. The primer sequences of PFKP and USP5 are listed in Additional file ##SUPPL##0##1##: Table S1.</p>", "<title>Cell proliferation assay</title>", "<p id=\"Par57\">Cell proliferation was detected with CCK-8, colony formation, and EDU incorporation assays, which were performed as described previously [##REF##36119494##20##].</p>", "<title>Tumor xenograft experiments</title>", "<p id=\"Par58\">All experiments with mice were approved by the Ethics Committee at the Jiangxi Provincial People's Hospital. Six-week-old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. MDA-MB-231 cells (2 × 10<sup>6</sup> cells in 100μL PBS) were injected into the fourth mammary fat pads of mice. The tumors were measured every 3 days after sizeable tumor formation. Then, the mice were euthanized, and tumors were dissected, weighed, and photographed after 20 days.</p>", "<title>Ubiquitination assay</title>", "<p id=\"Par59\">Cells were treated with the proteasome inhibitor MG132 for 6 h before cell lysis. The cell lysates were prepared in RIPA buffer and incubated with an anti-PFKP antibody or IgG at 4 °C overnight. The immunoprecipitates were then used in a Western blotting assay with 1:1000 dilution of anti-ubiquitin antibody (Santa Cruz, sc-8017).</p>", "<title>Co-immunoprecipitation assay</title>", "<p id=\"Par60\">Co-immunoprecipitation assay was performed as described previously [##REF##32296027##18##], using 2 µg of anti-PFKP antibody (Santa Cruz, sc-514824) and 3 µg of anti-USP5 antibody (Proteintech, 10,473-1-AP) per 500 µg of total protein.</p>", "<title>Immunofluorescence microscopy</title>", "<p id=\"Par61\">Immunofluorescence microscopy was performed as described previously [##REF##29352223##21##], using 1:200 dilution of an anti-PFKP antibody (Santa Cruz, sc-514824) and 1:200 dilution of an anti-USP5 antibody (Proteintech, 10,473–1-AP).</p>", "<title>In vitro<italic> binding assay</italic></title>", "<p id=\"Par62\">Purified His-USP5 (MCE, HY-P74479) was incubated with purified GST-PFKP (Novus, H00005214-P01) or GST-Tag protein (Novus, NBC1-18,537) at 4 °C overnight. Then, the Glutathione Sepharose beads were added and incubated for 4 h. The bound proteins were then eluted in boiling 2 × SDS sample buffer and used in western blotting analysis.</p>", "<title>Glucose uptake, pyruvate, lactate, and ATP measurements</title>", "<p id=\"Par63\">Glucose uptake was quantified using a commercially available Glucose Uptake Assay Kit (Abcam, ab136955). MDA-MB-231 and SUM-159 cells were seeded in six-well plates at a density of 3 × 10<sup>5</sup> cells per well and incubated at 37 °C for 24 h. Prior to the assay, cells were subjected to serum deprivation to enhance glucose uptake. Subsequently, the cells were washed thrice with PBS buffer and starved in Krebs–Ringer-Phosphate-HEPES buffer for 40 min. Following this, the cells were stimulated with 100 nM insulin for 30 min. Finally, glucose uptake was assessed according to the manufacturer's instructions, with all results being normalized to cell number.</p>", "<p id=\"Par64\">The Pyruvate Assay Kit (Abcam, ab65342) and Lactate Assay Kit (Abcam, ab65331) were utilized to perform the pyruvate and lactate assay, respectively. MDA-MB-231 and SUM-159 cells were seeded in six-well plates at a density of 3 × 10<sup>5</sup> cells per well and incubated at 37 °C for 24 h. Subsequently, the cells were homogenized using the lysis buffer provided in each respective assay kit. The processed samples were then centrifuged, and the resulting supernatants were collected in fresh tubes. Finally, the measurement of pyruvate and lactate production was conducted according to the manufacturer’s instructions, with all results being normalized to the number of cells.</p>", "<p id=\"Par65\">The ATP assay was conducted utilizing an ATP Assay Kit (Abcam, ab83355). Briefly, MDA-MB-231 and SUM-159 cells (1 × 10<sup>6</sup> cells) were homogenized in ATP assay buffer and subjected to deproteinization using a Deproteinization Sample Preparation Kit (Abcam, ab284939). Ultimately, the ATP levels were quantified in accordance with the guidelines provided by the manufacturer. All outcomes were adjusted based on the number of cells.</p>", "<title>Extracellular acidification rate (ECAR) assays</title>", "<p id=\"Par66\">The extracellular acidification rate (ECAR) was quantified utilizing the Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies). Experimental procedures were conducted in accordance with the guidelines provided by the manufacturer. Specifically, MDA-MB-231 and SUM-159 cells were seeded in Seahorse XF24 cell plates at a density of 2 × 10<sup>4</sup> cells per well and incubated at 37 °C for 24 h. Prior to the assay, the cell plates were placed in a non-CO2 incubator for 30 min. Subsequently, the ECAR of the cells was assessed following the manufacturer's instructions. In the experimental procedure, glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM) were introduced into the wells in a sequential manner following each cycle. The resulting data were expressed as extracellular acidification rate (ECAR) normalized to protein concentration.</p>", "<title>Statistical analysis</title>", "<p id=\"Par67\">Data were analyzed using GraphPad Prism 9 (GraphPad Software Inc) and SPSS-22 (SPSS Inc). The data from all of the experiments were presented as the means ± SEM. Spearman’s rank correlation test was used to analyze the correlation of gene expression in tissue samples. Student’s <italic>t</italic>-test for two groups was applied. The survival analysis was conducted using the Kaplan–Meier and log-rank tests. A <italic>P</italic>-value &lt; 0.05 was considered significant. The <italic>r</italic>-value was used to evaluate the correlation analysis.</p>" ]
[ "<title>Results</title>", "<title>PFKP protein expression is frequently upregulated in TNBC and is associated with poor prognosis in TNBC patients</title>", "<p id=\"Par68\">To explore whether PFKP protein has a potential role in the progression of TNBC, we queried the Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets using the UALCAN website. The results of the analysis showed that PFKP protein expression was significantly higher in TNBC samples compared with normal breast samples (Fig. ##FIG##0##1##a). Notably, survival analysis based on the KM plotter database showed a negative correlation of PFKP protein expression with overall survival in TNBC patients (Fig. ##FIG##0##1##b). We further detected the expression of PFKP in TNBC carcinoma and para-carcinoma tissues by immunohistochemistry, and the results showed that the protein expression level of PFKP was higher in carcinoma tissues than that in para-carcinoma tissues in patients with TNBC (Fig. ##FIG##0##1##c, d). Then, the TNBC carcinoma tissue samples were stratified into two groups, according to high or low expression of the PFKP protein (Fig. ##FIG##0##1##e). As shown in Fig. ##FIG##0##1##f, the protein expression level of PFKP was positively associated with the TNM stage in TNBC patients. Moreover, immunohistochemical staining showed that the patients with high PFKP expression exhibited poorer overall survival compared with patients with low PFKP expression (Fig. ##FIG##0##1##g). In addition, we further interrogated the PFKP protein expression in TNBC cell lines (MDA-MB-231 and SUM-159) and normal breast cell lines (MCF-10A) and identified the level of PFKP protein was higher in TNBC cell lines compared with normal breast cell line (Fig. ##FIG##0##1##h). Together these results demonstrated that PFKP protein expression was frequently upregulated in TNBC and was associated with poor prognosis in TNBC patients.</p>", "<title><italic>Inhibition of PFKP reduces TNBC progression </italic>in vitro<italic> and </italic>in vivo</title>", "<p id=\"Par69\">To further confirm the role of PFKP in TNBC progression, we used lentiviral shRNA to knockdown PFKP in MDA-MB-231 and SUM-159 cells and found that the shRNAs specific for PFKP efficiently downregulated PFKP expression (Fig. ##FIG##1##2##a, b and Additional file ##SUPPL##0##1##: Figure S1A, S1B). Then, we examined the effect of PFKP on TNBC progression by CCK-8 assay, colony-formation assay, and EDU incorporation assay in MDA-MB-231 and SUM-159 cells. The results showed that inhibition of PFKP significantly reduced TNBC progression in vitro (Fig. ##FIG##1##2##c–g and Additional file ##SUPPL##0##1##: Figure S1C–S1G). To further determine the role of PFKP on TNBC progression in vivo, we established xenograft tumor models in nude mice. To do so, MDA-MB-231 cells stably expressing control shRNA or PFKP shRNA were implanted into the mammary fat pad of female BALB/c nude mice, and tumor growth was examined. The results showed that PFKP inhibition significantly suppressed tumor growth, tumor volume, and tumor weight (Fig. ##FIG##1##2##h–j). Moreover, an immunohistochemistry staining assay was used to investigate the expression of PFKP protein in each sample of mice tumor tissue. As shown in Fig. ##FIG##1##2##k, PFKP protein expression was significantly decreased in PFKP-knockdown tumors. Collectively, these results suggested that PFKP played a key role in TNBC progression and the inhibition of PFKP was an effective strategy to curb TNBC progression.</p>", "<title>USP5 is a bona fide deubiquitinase that targets PFKP protein for deubiquitination and stabilization in TNBC</title>", "<p id=\"Par70\">To explore the underlying mechanisms involved in the regulation of PFKP protein expression in TNBC, we performed an ubiquitin assay to detect the ubiquitination level of PFKP protein in TNBC cell line MDA-MB-231 and normal breast cell line MCF-10A. As shown in Fig. ##FIG##2##3##a, the ubiquitination level of PFKP protein in MDA-MB-231 cells was significantly lower than that in MCF-10A cells. In line, the PFKP protein stability (half-life) was significantly increased in MDA-MB-231 cells in response to the protein synthesis inhibitor cycloheximide (CHX) treatment, compared to that in MCF-10A cells (Fig. ##FIG##2##3##b, c). Together, these observations demonstrated that high expression of PFKP protein in TNBC might be regulated by the posttranslational modification: ubiquitination-mediated degradation.</p>", "<p id=\"Par71\">Given that the E3 ubiquitin ligases and deubiquitinases play central roles in protein degradation and turnover through protein ubiquitination and deubiquitination [##REF##25444757##22##], we performed the Co-immunoprecipitation (Co-IP) and mass spectrometry-based protein identification, to identify E3 ubiquitin ligases and deubiquitinases that interacted with PFKP. The results showed that three deubiquitinases (USP5, USP14, and OTUB1) and three E3 ubiquitin ligases (HUWE1, TRIM21, and TRIM25) could potentially interact with PFKP in MDA-MB-231 cells (Fig. ##FIG##2##3##d). Notably, the correlation between USP5 protein and PFKP protein expression levels was much higher as compared with the five other E3 ubiquitin ligases and deubiquitinases in TNBC (Fig. ##FIG##2##3##e and Additional file ##SUPPL##0##1##: Figure S2A-2E), indicating that USP5 might regulate PFKP protein deubiquitination. To substantiate this finding, we performed the immunoblotting and ubiquitination assay in MDA-MB-231 and SUM-159 cells with or without USP5 knockdown. The results showed that the knockdown of USP5 by specific shRNAs decreased PFKP protein expression while increasing the ubiquitination level of PFKP protein (Fig. ##FIG##2##3##f, g and Additional file ##SUPPL##0##1##: Figure S2F, S2G). In addition, USP5 depletion promoted PFKP protein degradation in response to treatment with CHX in MDA-MB-231 and SUM-159 cells (Fig. ##FIG##2##3##h–k and Additional file ##SUPPL##0##1##: Figure S2H-S2K). Furthermore, we performed a Co-IP assay and immunofluorescence assay to investigate the interaction of USP5 and PFKP proteins in MDA-MB-231 and SUM-159 cells. The results of the Co-IP assay showed that USP5 and PFKP proteins were bound to each other (Fig. ##FIG##2##3##l and Additional file ##SUPPL##0##1##: Figure S2L), and the immunofluorescence assay indicated that USP5 co-localized with PFKP proteins in the cytoplasm (Fig. ##FIG##2##3##m and Additional file ##SUPPL##0##1##: Figure S2M). Notably, an in vitro binding assay proved that the purified proteins of His-USP5 and GST-PFKP could directly bind to each other under cell-free conditions (Fig. ##FIG##2##3##n). Collectively, our data demonstrated that USP5 was a bona fide deubiquitinase that directly targeted PFKP protein for deubiquitination and stabilization in TNBC.</p>", "<title>USP5 regulates aerobic glycolysis in TNBC</title>", "<p id=\"Par72\">Aerobic glycolysis is a common feature of glucose metabolism in cancer cells, and PFKP is an important rate-limiting enzyme that regulates aerobic glycolysis. Given that our study demonstrated that USP5 deubiquitinated and stabilized PFKP protein in TNBC, we next explore whether USP5 regulates aerobic glycolysis in TNBC. As shown in Fig. ##FIG##3##4##a–d and Additional file ##SUPPL##0##1##: Figure S3A—S3D, USP5 depletion reduced the glucose uptake, lactate level, pyruvate level, and ATP level in MDA-MB-231 and SUM-159 cells. Moreover, the potential regulatory role of USP5 in TNBC glucose metabolism was further explored by ECAR assay, and results showed that the knockdown of USP5 significantly reduced ECAR and glycolytic capacity (Fig. ##FIG##3##4##e, f and Additional file ##SUPPL##0##1##: Figure S3E, S3F). Taken together, our results demonstrated that USP5 regulated aerobic glycolysis in TNBC cells.</p>", "<title><italic>USP5 promotes TNBC progression by regulating PFKP protein expression </italic>in vitro<italic> and </italic>in vivo</title>", "<p id=\"Par73\">Given that USP5 deubiquitinates and stabilizes PFKP protein in TNBC, we predict whether USP5 promotes TNBC progression by regulating PFKP protein expression. To do so, PFKP was overexpressed based on USP5 knockdown in TNBC cells. The immunoblotting assay showed that the decrease of PFKP caused by the USP5 knockdown could be rescued by exogenous overexpression of PFKP (Fig. ##FIG##4##5##a and Additional file ##SUPPL##0##1##: Figure S4A). Next, CCK-8 assay, colony-formation assay, and EDU incorporation assay showed that USP5 depletion significantly inhibited the proliferation of MDA-MB-231 and SUM-159 cells in vitro, while this inhibitory effect was reversed by exogenous overexpression of PFKP (Fig. ##FIG##4##5##b–f and Additional file ##SUPPL##0##1##: Figure S4B-S4F). In line, the xenograft nude mice model was applied to determine whether USP5 promotes TNBC progression by regulating PFKP protein expression in vivo. And we found that the knockdown of USP5 significantly decreased the growth, volume, weight, and PFKP protein expression of tumors in mice; however, this effect was attenuated in mice carrying PFKP-overexpressing tumors (Fig. ##FIG##4##5##g–j), confirming that the knockdown of USP5 reduces TNBC progression through the regulation of PFKP in vivo. Collectively, these results revealed that USP5 promoted TNBC progression by regulating PFKP protein expression in vitro and in vivo.</p>", "<title>USP5 is positively correlated with PFKP expression in TNBC and is associated with poor prognosis in TNBC patients</title>", "<p id=\"Par74\">To confirm the role of the USP5-PFKP signaling pathway in TNBC progression, we performed immunohistochemistry to detect the expression of USP5 and PFKP in the 172 cases of human TNBC carcinoma tissues and analyzed the correlations between USP5 and PFKP in TNBC tissues. The results indicated a strong positive correlation between the expression of USP5 and PFKP (Fig. ##FIG##5##6##a, b). Importantly, we demonstrated concomitantly high expression of USP5 and PFKP in TNBC patients with advanced TNM stage (Fig. ##FIG##5##6##c), highlighting that dysregulated USP5-PFKP signaling was functionally linked to cancer progression in TNBC. In addition, the survival analysis indicated that TNBC patients with concomitantly high expression of USP5 and PFKP in their tumors had shorter overall survival than those with low USP5 and PFKP expression (Fig. ##FIG##5##6##d). Altogether, these results revealed that aberrant functionality of the USP5-PFKP axis might contribute to TNBC progression and could be used to predict poor clinical outcomes in TNBC patients.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par75\">Although an increase in PFKP expression is a characteristic feature of malignant tissues, little is known about how PFKP expression is regulated during the development and progression of cancers. Only a handful of studies have explored the molecular mechanisms changing the expression of PFKP in cancer. For example, at the transcriptional level, BRCA1/ZBRK1 complex and Snail restrain PFKP expression [##REF##32470015##23##, ##REF##28176759##24##], while KLF4 increases the expression of PFKP by directly binding to the PFKP promoter [##REF##21586797##25##]. In addition, at the posttranslational level, E3 ubiquitin ligase HRD1 and TRIM21 interact and target PFKP for ubiquitination and degradation, which ultimately reduces PFKP expression [##REF##33588886##26##, ##REF##29038421##27##]. However, the mechanisms regulating PFKP protein deubiquitination, a process opposing ubiquitination, remain unexplored. Here, we identified that PFKP was a bona fide target of deubiquitinase USP5 and USP5 directly deubiquitinated and stabilized PFKP in TNBC, which provided the new therapeutic strategies to deplete PFKP expression and to develop personalized targeted therapies of TNBC. Notably, besides USP5, based on Co-immunoprecipitation (Co-IP)/mass spectrometry-based protein identification and correlation analysis (Fig. ##FIG##2##3##d and Additional file ##SUPPL##0##1##: Figure S2A), our study also showed that another deubiquitinase USP14 was a potential deubiquitinating enzyme of PFKP, but whether it regulates PFKP protein deubiquitination and stabilization needs to be further validated in future studies.</p>", "<p id=\"Par76\">PFKP is a significant enzyme that serves as a rate-limiting factor in the regulation of aerobic glycolysis. Our study provides evidence that USP5 plays a role in deubiquitinating and stabilizing the PFKP protein in TNBC, prompting us to investigate the impact of USP5 on the regulation of aerobic glycolysis in TNBC. Consistent with our expectations, we observed that depletion of USP5 effectively suppressed aerobic glycolysis in TNBC cells. It is worth noting that previous research has shown that USP5 also targets the Hypoxia-inducible factor (HIF) protein [##REF##35102545##28##], which is known to regulate aerobic glycolysis [##REF##21508971##29##]. Thus, it is hypothesized that USP5 plays a role in the regulation of aerobic glycolysis through its deubiquitinating and stabilizing effects on the PFKP protein, as well as its regulation of the HIF protein. Notably, a previous study has demonstrated that PFKP is a downstream target of the HIF protein [##REF##34367973##30##]. Therefore, it can be inferred that USP5 influences breast cancer progression by targeting either the HIF protein [##REF##35102545##28##] or the PFKP protein, which in turn may directly or indirectly impact the function of PFKP. This finding also provides an explanation for the significant rescue of TNBC progression inhibition observed upon exogenous overexpression of PFKP following USP5 knockdown in the present study. Furthermore, it is worth conducting further investigation to determine if other downstream targets of USP5 also play a role in regulating the function of PFKP.</p>", "<p id=\"Par77\">USP5, also referred to as isopeptidase T (ISOT), is a deubiquitinase of the ubiquitin–proteasome system, which removes unanchored polyubiquitin chains on protein substrates and plays an essential role in several diseases [##REF##31756387##31##]. Notably, a growing body of evidence suggests that USP5 is widely expressed in various cancers and contributes significantly to cancer progression. For example, USP5 protein is highly expressed in non-small cell lung cancer tissues and closely correlates with poor prognosis of these patients; and USP5 facilitates non-small cell lung cancer progression by directly interacting with, deubiquitinating, and stabilizing PD-L1 protein [##REF##34741014##32##]. In hepatocellular carcinoma, USP5 interacts with and stabilizes SLUG to promote the EMT and malignant progression of cancer [##REF##30809294##33##]. Consistent with these previous studies, our study showed that USP5 was positively correlated to PFKP in TNBC tissues, and concomitantly high expression of USP5 and PFKP were correlated with poor prognosis of TNBC patients. Moreover, the knockdown of USP5 significantly reduced TNBC progression through the regulation of PFKP, suggesting that USP5 may serve as a potential therapeutic target for patients with cancer.</p>", "<p id=\"Par78\">Cancer cells reprogram their glucose metabolism that produce energy predominantly by glycolysis rather than by mitochondrial oxidative phosphorylation even under aerobic conditions, which is termed as Warburg effect or aerobic glycolysis [##REF##21376230##34##]. In addition to providing cellular energy, the metabolic intermediates generated during aerobic glycolysis can be used for macromolecular biosynthesis, which facilitates the rapid growth of cancer cells [##REF##19460998##35##]. Studies have shown that metabolic changes in cancer cells from cytoplasm-based glycolysis to mitochondria-based glucose oxidation can effectively decrease tumor growth [##REF##17222789##36##], indicating that targeting aerobic glycolysis in cancer cells is a promising therapeutic strategy. Taken together with our results showed that knockdown of USP5 significantly inhibited the aerobic glycolysis process, further development of USP5 inhibitors to reverse the aerobic glycolysis of cancer cells and reduce cancer progression may contribute to future clinical treatment of cancer.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"Par79\">In this study, we showed that PFKP protein was highly expressed in TNBC tissues and cells, which was associated with TNBC progression and poor prognosis. In addition, using a series of experiments, we demonstrated that PFKP depletion significantly inhibited the TNBC progression in vitro and in vivo. Importantly, we identified that PFKP was a bona fide target of deubiquitinase USP5, and the USP5-mediated deubiquitination and stabilization of PFKP were essential for cancer cell aerobic glycolysis and TNBC progression. Thus, our study established the USP5-PFKP axis as an important regulatory mechanism of TNBC progression and provided a rationale for future therapeutic interventions in the treatment of TNBC (Fig. ##FIG##5##6##e).</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Triple-negative breast cancer (TNBC) remains the most challenging subtype of breast cancer and lacks definite treatment targets. Aerobic glycolysis is a hallmark of metabolic reprogramming that contributes to cancer progression. PFKP is a rate-limiting enzyme involved in aerobic glycolysis, which is overexpressed in various types of cancers. However, the underlying mechanisms and roles of the posttranslational modification of PFKP in TNBC remain unknown.</p>", "<title>Methods</title>", "<p id=\"Par2\">To explore whether PFKP protein has a potential role in the progression of TNBC, protein levels of PFKP in TNBC and normal breast tissues were examined by CPTAC database analysis, immunohistochemistry staining (IHC), and western blotting assay. Further CCK-8 assay, colony formation assay, EDU incorporation assay, and tumor xenograft experiments were used to detect the effect of PFKP on TNBC progression. To clarify the role of the USP5-PFKP pathway in TNBC progression, ubiquitin assay, co-immunoprecipitation (Co-IP), mass spectrometry-based protein identification, western blotting assay, immunofluorescence microscopy, in vitro binding assay, and glycolysis assay were conducted.</p>", "<title>Results</title>", "<p id=\"Par3\">Herein, we showed that PFKP protein was highly expressed in TNBC, which was associated with TNBC progression and poor prognosis of patients. In addition, we demonstrated that PFKP depletion significantly inhibited the TNBC progression in vitro and in vivo. Importantly, we identified that PFKP was a bona fide target of deubiquitinase USP5, and the USP5-mediated deubiquitination and stabilization of PFKP were essential for cancer cell aerobic glycolysis and TNBC progression. Moreover, we found a strong positive correlation between the expression of USP5 and PFKP in TNBC samples. Notably, the high expression of USP5 and PFKP was significantly correlated with poor clinical outcomes.</p>", "<title>Conclusions</title>", "<p id=\"Par4\">Our study established the USP5-PFKP axis as an important regulatory mechanism of TNBC progression and provided a rationale for future therapeutic interventions in the treatment of TNBC.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s13058-024-01767-z.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>Not applicable.</p>", "<title>Author contributions</title>", "<p>ZZ was involved in the conceptualization; ZZ, C-XY and J-JL acquired the funding; Z-MP, X-JH and TW contributed to the investigation; Z-MP, X-JH and TW contributed to the methodology; F-FT and ZZ assisted in the project administration; F-FT and ZZ contributed to the supervision; J-JL and C-XY assisted in the validation; ZZ was involved in writing—original draft; F-FT contributed to writing—review and editing. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This work is supported by grants from the National Natural Science Foundation of China (No. 82203342), the Jiangxi Provincial Natural Science Foundation (Nos. 20232ACB216013, 20224BAB216073 and 20212BAB216059), the Science and Technology Research Project of Jiangxi Provincial Department of Education (No. GJJ218906), and the Foundation of Health Care Rejuvenation by Science and Education (No. KJXW2022002).</p>", "<title>Availability of data and materials</title>", "<p>The data and materials of this study are available from the corresponding author upon reasonable request.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par80\">All experiments were approved by the Ethics Committee at the Jiangxi Provincial People's Hospital (Approval Number: KT2023-016).</p>", "<title>Consent for publication</title>", "<p id=\"Par81\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par82\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>PFKP protein expression is frequently upregulated in TNBC and is associated with poor prognosis in TNBC patients. <bold>a</bold> The protein expression of PFKP is higher in TNBC than in normal breast tissues in CPTAC data. ***<italic>P</italic> &lt; 0.001. <bold>b</bold> The survival curves of TNBC patients with high or low expression of PFKP protein based on the Kaplan–Meier plotter platform. <bold>c</bold> Representative images of IHC staining of PFKP in TNBC carcinoma and para-carcinoma tissues. Scale bars, 100 μm. <bold>d</bold> PFKP protein expression in TNBC carcinoma tissues (<italic>n</italic> = 172) was significantly higher than that in para-carcinoma tissues (<italic>n</italic> = 130). <italic>r</italic> = 0.268, <italic>P</italic> &lt; 0.001. <bold>e</bold> Representative images of IHC staining of PFKP in TNBC carcinoma tissues. Scale bars, 100 μm. <bold>f</bold> The expression of PFKP correlates positively with the TNM stage in human TNBC carcinoma samples. <italic>r</italic> = 0.206, <italic>P</italic> = 0.026. <bold>g</bold> High expression of PFKP is correlated with poor overall survival of TNBC patients. Log-rank <italic>P</italic> = 0.0037. <bold>h</bold> The protein expression of PFKP in TNBC cell lines (MDA-MB-231 and SUM-159) and normal breast cell line (MCF-10A)</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Inhibition of PFKP reduces TNBC progression in <italic>vitro</italic> and in vivo. <bold>a</bold>, <bold>b</bold> qRT-PCR (<bold>a</bold>) and western blot (<bold>b</bold>) analysis of PFKP expression with PFKP knockdown in MDA-MB-231 cells. ***<italic>P</italic> &lt; 0.001. <bold>c</bold> CCK-8 assay determination of MDA-MB-231 cell proliferation in response to PFKP knockdown. ***<italic>P</italic> &lt; 0.001. <bold>d</bold>, <bold>e</bold> Clone formation assay determination of MDA-MB-231 cell proliferation in response to PFKP knockdown. ***<italic>P</italic> &lt; 0.001, **<italic>P</italic> &lt; 0.01. <bold>f</bold>, <bold>g</bold> EDU incorporation assay determination of MDA-MB-231 cell proliferation in response to PFKP knockdown. **<italic>P</italic> &lt; 0.01. <bold>h</bold> Representative images of the xenograft tumors in each group. <bold>i</bold>, <bold>j</bold> PFKP knockdown reduces tumor volume (<bold>i</bold>) and tumor weight (<bold>j</bold>) of nude mice. ***<italic>P</italic> &lt; 0.001. <bold>k</bold> The PFKP protein expression in each sample of mice tumor tissue was analyzed by IHC staining. Scale bars, 100 μm</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>USP5 is a bona fide deubiquitinase that targets PFKP protein for deubiquitination and stabilization in TNBC. <bold>a</bold> Co-IP analysis of PFKP protein ubiquitination in MCF-10A and MDA-MB-231 cells. <bold>b</bold> MCF-10A and MDA-MB-231 cells were treated with CHX for the indicated time points and analyzed by Western blot. <bold>c</bold> Quantification of PFKP protein levels normalized to β-actin in time after the addition of CHX. <bold>d</bold> The diagram showing three deubiquitinases (USP5, USP14, and OTUB1) and three E3 ubiquitin ligases (HUWE1, TRIM21, and TRIM25) could interact with PFKP identified by a combination of Co-IP and MS. <bold>e</bold> The correlation between USP5 protein expression and PFKP protein expression in TNBC was analyzed by LinkedOmics platform. <bold>f</bold> The USP5 and PFKP protein expression in control and USP5 knockdown MDA-MB-231 cells were analyzed by Western blot. <bold>g</bold> Co-IP analysis of PFKP protein ubiquitination in control and USP5 knockdown MDA-MB-231 cells. <bold>h–k</bold> Control and USP5 knockdown MDA-MB-231 cells were treated with CHX for the indicated time points and analyzed by Western blot <bold>h</bold>, <bold>j</bold>, quantification of PFKP protein levels normalized to β-actin in time after the addition of CHX (<bold>i</bold>, <bold>k</bold>). <bold>l</bold> The binding of USP5 to PFKP was detected by Co-IP assay in MDA-MB-231 cells. <bold>m</bold> Immunofluorescence co-localization of USP5 and PFKP in MDA-MB-231 cells. <bold>n</bold> In vitro binding assays of purified GST-PFKP and His-USP5 proteins</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>USP5 regulates aerobic glycolysis in TNBC. <bold>a</bold>–<bold>d</bold> Glucose update (<bold>a</bold>), lactate level (<bold>b</bold>), pyruvate level (<bold>c</bold>), and ATP level (<bold>d</bold>) were significantly decreased after USP5 knockdown in MDA-MB-231 cells. <bold>e</bold>, <bold>f</bold> ECAR values (<bold>e</bold>) and calculated glycolytic capacity (<bold>f</bold>) of control and USP5 knockdown MDA-MB-231 cells</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>USP5 promotes TNBC progression by regulating PFKP protein expression in vitro and in vivo. <bold>a</bold> The USP5 and PFKP protein expression in control and USP5 knockdown MDA-MB-231 cells in the presence or absence of overexpressed PFKP were analyzed by Western blot. <bold>b–f</bold> MDA-MB-231 cell proliferation following USP5 knockdown in the presence or absence of overexpressed PFKP was evaluated using CCK8 assay (<bold>b</bold>), Clone formation assay (<bold>c</bold>, <bold>d</bold>), and EDU incorporation assay (<bold>e</bold>, <bold>f</bold>). ***<italic>P</italic> &lt; 0.001, **<italic>P</italic> &lt; 0.01. <bold>g</bold> Representative images of the xenograft tumors in each group. <bold>h</bold>, <bold>i</bold> Tumor volume (<bold>h</bold>) and tumor weight (<bold>i</bold>) of nude mice in each group. **<italic>P</italic> &lt; 0.01, *<italic>P</italic> &lt; 0.05. <bold>j</bold> The PFKP protein expression in each sample of mice tumor tissue was analyzed by IHC staining. Scale bars, 100 μm</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>USP5 is positively correlated with PFKP expression in TNBC and is associated with poor prognosis of TNBC patients. <bold>a</bold> Representative images of IHC staining of USP5 and PFKP in serial sections of the same TNBC tumor. Scale bars, 100 μm. <bold>b</bold> A positive correlation between the expression of USP5 and PFKP in the 172 human TNBC carcinoma samples. <italic>r</italic> = 0.223, <italic>P</italic> = 0.003. <bold>c</bold> Concomitantly high expression of USP5 and PFKP correlates positively with the TNM stage in human TNBC carcinoma samples. <italic>r</italic> = 0.369, <italic>P</italic> &lt; 0.001. <bold>d</bold> Kaplan–Meier curves showed that the overall survival of patients with concomitantly high USP5 and PFKP expression in TNBC tumors was shorter than that of those with concomitantly low USP5 and PFKP expression. Log-rank <italic>P</italic> = 0.0004. <bold>e</bold> A schematic diagram of USP5 promotes TNBC progression and aerobic glycolysis through deubiquitinating and stabilizing PFKP</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"13058_2024_1767_Fig1_HTML\" id=\"MO1\"/>", "<graphic xlink:href=\"13058_2024_1767_Fig2_HTML\" id=\"MO2\"/>", "<graphic xlink:href=\"13058_2024_1767_Fig3_HTML\" id=\"MO3\"/>", "<graphic xlink:href=\"13058_2024_1767_Fig4_HTML\" id=\"MO4\"/>", "<graphic xlink:href=\"13058_2024_1767_Fig5_HTML\" id=\"MO5\"/>", "<graphic xlink:href=\"13058_2024_1767_Fig6_HTML\" id=\"MO6\"/>" ]
[ "<media xlink:href=\"13058_2024_1767_MOESM1_ESM.pdf\"><caption><p><bold>Additional file 1</bold>. Supplementary Information of PFKP deubiquitination and stabilization by USP5 activate aerobic glycolysis to promote triple-negative breast cancer progression, Figures S1–S4 and Table S1.</p></caption></media>" ]
[{"label": ["5."], "surname": ["Vora", "Halper", "Knowles"], "given-names": ["S", "JP", "DM"], "article-title": ["Alterations in the activity and isozymic profile of human phosphofructokinase during malignant transformation in vivo and in vitro: transformation- and progression-linked discriminants of malignancy"], "source": ["Can Res"], "year": ["1985"], "volume": ["45"], "issue": ["7"], "fpage": ["2993"], "lpage": ["3001"]}]
{ "acronym": [ "PFKP", "USP5", "TNBC", "CPTAC", "IHC", "CCK-8", "EDU", "HER2", "ER", "PR", "HK", "PK", "PFK", "AMPK", "ACC2", "HCC", "DUBs", "ATXN3", "YAP", "USP8", "USP13", "METTL3", "m6A", "ATG5", "ATP", "CHX", "shRNAs", "ECAR", "TNM", "USP14", "OTUB1", "HUWE1", "TRIM21", "TRIM25", "BRCA1", "ZBRK1", "KLF4", "HRD1", "HIF", "PD-L1", "SLUG", "EMT" ], "definition": [ "ATP-dependent 6-phosphofructokinase, platelet type", "Ubiquitin carboxyl-terminal hydrolase 5", "Triple-negative breast cancer", "Clinical proteomic tumor analysis consortium", "Immunohistochemistry", "Cell counting Kit-8", "5-Ethynyl-2’-deoxyuridine", "Human epidermal growth factor receptor type 2", "Estrogen receptor", "Progesterone receptor", "Hexokinase", "Pyruvate kinase", "Phosphofructokinase", "AMP-activated protein kinase", "Acetyl-CoA carboxylase 2", "Hepatocellular carcinoma", "Deubiquitinases", "Ataxin-3", "Yes-associated protein", "Ubiquitin carboxyl-terminal hydrolase 8", "Ubiquitin carboxyl-terminal hydrolase 13", "N6-adenosine-methyltransferase catalytic subunit", "N6-methyladenosine", "Autophagy protein 5", "Adenosine 5'-triphosphate", "Cycloheximide", "Small hairpin RNAs", "Extracellular acidification rate", "Tumor node metastasis classification", "Ubiquitin carboxyl-terminal hydrolase 14", "OTU domain-containing ubiquitin aldehyde-binding protein 1", "HECT, UBA and WWE domain-containing protein 1", "Tripartite motif-containing protein 21", "Tripartite motif-containing protein 25", "Breast cancer type 1 susceptibility protein", "Zinc finger and BRCA1-interacting protein with a KRAB domain 1", "Kruppel-like factor 4", "HMG-CoA reductase degradation protein 1", "Hypoxia-inducible factor", "Programmed cell death 1 ligand 1", "Neural crest transcription factor Slug", "Epithelial-mesenchymal transition" ] }
36
CC BY
no
2024-01-14 23:43:47
Breast Cancer Res. 2024 Jan 12; 26:10
oa_package/47/78/PMC10787506.tar.gz
PMC10787507
38217019
[ "<title>Introduction</title>", "<p id=\"Par2\">The ultraviolet (UV) component of sunlight can be divided into ultraviolet C (UVC; wavelength: 100–290 nm), ultraviolet B (UVB; wavelength: 290–320 nm), and ultraviolet A (UVA; wavelength: 320–400 nm), according to their different wavelengths [##REF##32230973##1##]. UVC is mostly blocked by the ozone layer and rarely reaches the human skin, while both UVB and UVA can penetrate the ozone layer, accounting for about 95% and 5% of UV radiation (UVR) that reaches the skin, respectively [##UREF##0##2##] (Fig. ##FIG##0##1##). UVR can contribute to dermal photodamage via DNA damage, inflammation, oxidative stress, and apoptosis, leading to external signs of skin damage. For example, exogenous aging, which accounts for 80% of skin aging, manifests with the development of wrinkles, skin relaxation, hyperpigmentation [##REF##35645796##3##].</p>", "<p id=\"Par3\">In recent years, many studies have indicated that reactive oxygen species (ROS), which are molecules with short lives that compromised of unpaired electrons [##REF##27666490##4##], play crucial roles in regulating UV-induced photodamage of the skin. Hence, to better address the harmful effects of UV-induced photodamage, understanding how it is regulated by ROS is crucial. In this review, we elaborate and focus on the biological characteristics of ROS and its role in regulating UV-induced photodamage of the skin.</p>" ]
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[ "<title>Conclusion</title>", "<p id=\"Par27\">UV has been discovered as the main reason for the induction of photodamage, and there are many mechanisms involved in UV-induced photodamage. ROS, as key signaling molecules of life, play an increasingly central role in UV-induced photodamage, in which, elevated ROS levels have been documented. However, to the best of our knowledge, the detailed signaling pathways, especially the apoptosis mechanisms in which ROS are involved have not been reviewed previously. Hence, in this review, we discussed ROS, UV, and UV-induced photodamage. Moreover, the associated signal transduction of ROS in UV-induced photodamage has been summarized. ROS play a crucial role in inflammation, activating the MAPK signaling pathway, along with NF-κB and AP-1, which leads to the release of inflammatory factors, such as IL-1β, IL-6 and TNF-α; upregulation of MMPs, and degradation of collagen fibers. Consequently, the photoaged skin exhibits clinical symptoms, such as inflammatory erythema, skin relaxation, and wrinkles. Furthermore, ROS are involved in DNA damage, including mtDNA damage, and induces mitochondrial apoptosis. They can also disrupt the balance between members of the Bax protein family, accelerating cell death, which is also closely related to the pathogenesis of skin cancer. The information highlighted in this review will be helpful to future research and development programs of relevant antioxidants and can contribute to the discovery of targets that can reduce or prevent photodamage. Moreover, since many anticancer drugs destroy DNA in a similar manner to UVR [##REF##17213957##64##], further research into the mechanisms underlying UV damage could also contribute to the understanding of the mechanisms of action of these highly relevant drugs.</p>" ]
[ "<p id=\"Par1\">Reactive oxygen species (ROS), such as superoxides (O<sub>2</sub> •−) and hydroxyl groups (OH·), are short-lived molecules containing unpaired electrons. Intracellular ROS are believed to be mainly produced by the mitochondria and NADPH oxidase (NOX) and can be associated with various physiological processes, such as proliferation, cell signaling, and oxygen homeostasis. In recent years, many studies have indicated that ROS play crucial roles in regulating ultraviolet (UV)-induced photodamage of the skin, including exogenous aging, which accounts for 80% of aging. However, to the best of our knowledge, the detailed signaling pathways, especially those related to the mechanisms underlying apoptosis in which ROS are involved have not been reviewed previously. In this review, we elaborate on the biological characteristics of ROS and its role in regulating UV-induced photodamage of the skin.</p>", "<title>Keywords</title>" ]
[ "<title>Reactive oxygen species</title>", "<p id=\"Par4\">ROS, which are the molecules containing highly unstable oxygen radicals, such as superoxide (O<sub>2</sub> •−) and hydroxyl group (OH•), usually can be rapidly transformed to more stable non-radicals like hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and hypochlorous acid, which can diffuse easily [##REF##27666490##4##] [##REF##24987008##5##]. Hence, ROS can oxidize sulfhydryl groups with cysteine residues, including proteins such as kinases, phosphatases and transcription factors [##REF##19558209##6##]. They can also participate in physiological processes, such as cell signaling, proliferation, and tumor suppression, supporting the immune system for pathogen resistance and oxygen homeostasis [##REF##32352946##7##]. The effects of ROS depend on the dose and persistence of the ROS particles, as well as the type of cells involved. A low level of ROS can lead to mutation, a medium level of ROS can cause senescence, and a high level of ROS usually lead to cell death, such as apoptosis or necrosis [##REF##25653189##8##] (Fig. ##FIG##1##2##).</p>", "<title>ROS production</title>", "<p id=\"Par5\">Intracellular ROS can be produced by various systems, including mitochondrial electron transport chain (ETC), NADPH oxidase (NOX), cytochrome P450, lipoxygenase, xanthine oxidase, nitric oxide synthase, and cyclooxygenase [##REF##20370557##9##].</p>", "<p id=\"Par6\">Large part of endogenous ROS are produced from mitochondria, which contain 10 sites that are known to be capable of producing O<sub>2</sub> •− , and 1% of the consumed O<sub>2</sub> is used to produce O<sub>2</sub> •− [##REF##25389297##10##]. Mitochondria produce ATP via the oxidation process of glucose, amino acids, and lipids. The Krebs cycle involves removing an electron from these metabolites and transferring it to the ETC, thereby reducing O<sub>2</sub> to O<sub>2</sub> •− [##REF##25342630##11##]. The energy released by the transport of electrons is utilized to expel protons (H +) from the mitochondrial matrix across the mitochondrial inner membrane (at multisubunit protein complexes include complex I, complex III, and complex IV) into the intermembrane space [##REF##35613354##12##]. And O<sub>2</sub> •− can be released both into the intermembrane space and mitochondrial matrix by complex III, involving in many biological processes [##REF##23102266##13##]. Meanwhile, most of the O<sub>2</sub> •− produced by mitochondria are changed to H<sub>2</sub>O<sub>2</sub> by manganese-superoxide dismutase in the mitochondrial matrix [##REF##19061483##14##]. With the facility of specific members of the aquaporin family, H<sub>2</sub>O<sub>2</sub> can be highly diffused through the mitochondrial membrane [##REF##24060746##15##]. Members of the NOX family are considered to have the primary function of producing ROS and serve as the primary source of ROS [##REF##26861575##16##]. The NOX family contains seven isomers, which share similar molecular structure and function, are all transmembrane proteins. It includes NOX1–5 and dual oxidases 1 and 2, with one NADPH-binding site, one flavin adenine dinucleotide (FAD)-binding site, six transmembrane domains, and four heme-binding histidines [##REF##17237347##17##]. NOX transmits electrons through biofilms to produce O<sub>2</sub> •− , which is also transformed to H<sub>2</sub>O<sub>2</sub> quickly [##REF##17237347##17##] (Fig. ##FIG##2##3##).</p>", "<title>ROS removal</title>", "<p id=\"Par7\">The homeostasis of ROS is important for cell survival and cell signaling of prevention of cell damage. The involvements of non-enzymatic or enzymatic antioxidants facilitate elimination of different types of ROS to achieve ROS detoxification, which contribute to the homeostasis of ROS. The most important antioxidant enzyme is glutathione peroxidase(GPX), which can regulate intracellular H<sub>2</sub>O<sub>2</sub> level, maintain reduced and oxidized glutathione (GSH/GSSG) balance, and promote antioxidant enzyme activity by allowing sulfhydryl reaction with glutathione (GSH) to remove singlet oxygen, hydrogen peroxide, and organic peroxides, thus regulating ROS homeostasis. Enzymatic antioxidants include superoxide dismutase (SOD), catalase, GSH, peroxiredoxin(PRX), and thioredoxin [##REF##28587975##18##]. SOD enzymes can dismutate O<sub>2</sub> •− into H<sub>2</sub>O<sub>2</sub>, which then be reduced and converted to form H<sub>2</sub>O and OH•, an extremely reactive free radical that can damage DNA, proteins, and lipids. Non-enzymatic antioxidants include GSH; flavonoids; vitamins A, C, and E; and ubiquinone [##REF##29145191##19##]. Antioxidants play are key in degrading O<sub>2</sub> •− and H<sub>2</sub>O<sub>2</sub>, thus reducing the damage of oxidation (Fig. ##FIG##2##3##).</p>", "<title>Role of ROS in UV-induced photodamage of the skin</title>", "<p id=\"Par8\">Normal skin tissue mainly includes keratinocytes in epidermis, melanocytes near the basement membrane, fibroblasts in dermis and some extracellular matrix. The morphology of the aged skin is aging and atrophy, with a decrease in the thickness of epidermis and dermis, a decrease in the composition of collagen and elastin in extracellular matrix, and a decrease in the total number of fibroblasts [##REF##22707767##20##]. Photoaged skin usually presents with proliferation of skin cells, and increased thickness of stratum corneum, epidermis and dermis. Photoaged epidermis is characterized by acanthosis and excessive keratinization, and the clinical manifestation is rough and dry skin. Fibroblasts and inflammatory cells were increased in the photoaged dermis. The distribution of melanocytes is not uniform, resulting in pigmentation and pigmentation spots. The amount of interstitial collagen in photoaging is decreased and damaged, leading to the disorder of skin molecular structure [##REF##25561721##21##].</p>", "<p id=\"Par9\">Long-term exposure to UV can cause DNA damage, oxidative stress, inflammation, and cell apoptosis [##REF##29124687##22##]. UV can interact with chromogenic groups and photosensitizers in cells to produce ROS, such as superoxide anion radical and hydroxyl radical [##REF##25611805##23##]. ROS may act as second messengers to regulate phosphorylation of multiple proteins in signal transduction pathways [##REF##32323267##24##]. While the exact mechanism of photodamage is still being studied, it has been reported that increased ROS production, collagen degradation, and mitochondrial DNA damage are its key features. ROS can regulate DNA damage and cell signaling pathways, leading to an imbalance of skin antioxidants, thus accelerating skin photodamage [##REF##24714202##25##]. ROS signaling regulates transcription factors, such as AP-1 and nuclear factor-kappa B (NF-κB), inducing the expression of matrix metalloproteinases (MMPs) that can induce collagen oxidation and reduce the expression of types I and III collagen [##REF##35316219##26##]. In addition, ROS can regulate collagen metabolism, resulting in skin relaxation, deepening of wrinkles, and decreased skin elasticity [##REF##35316219##26##]. Moreover, ROS are directly or indirectly involved in UV-induced mitochondrial apoptosis (Fig. ##FIG##3##4##).</p>", "<p id=\"Par10\">In addition, ROS produced by ultraviolet radiation can also affect intracellular DNA through MAPK-induced signaling. Studies have found that ROS generated by ultraviolet radiation can not only regulate MAPK signaling pathway, but also simultaneously regulate signaling pathways such as JAK/STAT and extracellular signal-regulated kinase (ERK) [##REF##26783703##27##, ##REF##32721499##28##]. Further studies have found in UV-mediated inflammation, ROS activates MAPK pathway and downstream factors NF-κB and AP-1, thus can regulate the release of inflammatory cytokines such as IL-1β, IL-6 and TNF-α [##REF##25465718##29##]. P38 and c-Jun N-terminal kinase (JNK) are two important downstream factors in MAPK signaling pathway [##REF##28495448##30##]. After the activation of p38 and JNK in MAPK pathway, they can also enter the nucleus and upregulate the expression of related inflammatory cytokines, induce the expression of MMPs and downregulate TGF-β expression, thus playing an important role in the UV-mediated photodamage [##REF##25607638##31##, ##REF##32172136##32##] (Fig. ##FIG##4##5##).</p>", "<title>DNA damage by ROS</title>", "<p id=\"Par11\">Both UVA and UVB can cause photodamage by inducing ROS production and oxidative damage [##REF##29575554##33##], and ROS can directly or indirectly result in DNA damage.</p>", "<p id=\"Par12\">The DNA damage caused by UV mainly affects the electronic structure of DNA by promoting chemical reactions between bases, especially the dimerization of thymine (cyclobutane derivative) [##UREF##0##2##]. The absorption of UV irradiation directly by DNA can result in adducts between adjacent pyrimidine nucleotides, forming cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4)pyrimidone photoproducts [(6–4)PPs] [##REF##25370518##34##]. 6-4PPs are larger and more massive in size, but more efficiently repaired than CPDs [##REF##27935061##35##]. However, with larger numbers and slower repair rate, CPDs have more obvious mutagenicity than [(6–4)PPs] [##REF##23303275##36##]. In addition, these products can hinder DNA and RNA from replicating, as a result of acting as physical obstacles to DNA polymerase and RNA polymerase and facilitating replication fork stagnation formation, leading to chain breakage that can consequently lead to chromosomal abnormalities[##REF##17700065##37##] [##REF##27898391##38##]. UVA-induced photoproducts, of which CPDs are the main products and [(6–4)PP] formation is not significant, are much less than those induced by UVB and were previously considered harmless in many cases [##REF##25700512##39##]. UVB is considered to be the main reason of skin photoaging [##REF##31436008##40##], with [(6–4)PPs] as its main products that trigger cell signaling pathways, activate the defense system, and lead to DNA repair or apoptosis [##REF##19775361##41##]. Reportedly, ROS participate in stem cell self-renewal, which in response to DNA double-strand breaks, by actiating the ataxia-telangiectasia mutated protein kinase [##REF##22901002##42##].</p>", "<p id=\"Par13\">P53, a transcription factor that can activate the inhibitor of the cell cycle and participate in DNA damage response and transcription of apoptosis [##REF##34888311##43##], may also play a role in premature aging by causing reactive damage to DNA [##REF##20729567##44##]. p53 is associated with the two key kinases of ataxia telangiectasia and rad3-related protein-checkpoint kinase 1 pathway [##REF##25962492##45##] that induce the transcription of cell cycle suppressor p21 gene and delay damaged G1 cells from entering S phase, preventing new start events at the beginning of the duplication process and slowing down the branching processes of reproduction of UV-irradiated cells in the S phase. Thus, p53 transiently suppresses DNA synthesis in UV-damaged cells [##REF##19793801##46##]. Consequently, DNA repair can be completed before DNA synthesis to reduce the DNA mutation rate [##REF##28263867##47##]. Studies have confirmed the involvement of the ROS-p38-p53 pathway in UV-induced cell damage, in which p38 mediates p53 phosphorylation after UV irradiation [##REF##26936104##48##]. Moreover, ROS can affect the overexpression of p53 and p21<sup>cip1/waf1</sup>, leading to the failure of DNA or apoptosis repair [##REF##23378590##49##].</p>", "<title>Mitochondrial DNA damage and apoptosis by ROS</title>", "<p id=\"Par14\">Apoptosis is generally induced by the activation of cell surface death receptors (exogenous pathways) that are initiated mainly by the binding of death ligands of the TNF superfamily of cytokines, including TNF, Fas-ligand, and TNF-related apoptosis-inducing ligand with their death receptors TNF receptor 1, Fas (also known as CD95), and DR4 or DR5, respectively. It can also be induced by various signals from the mitochondria and endoplasmic reticulum (endogenous pathways) [##REF##30131615##50##].</p>", "<p id=\"Par15\">UV-induced apoptosis represents a clearing mechanism that eliminates DNA-damaged cells, thereby reducing the risk of malignant transformation. To sum up, ROS can be directly involved in UV-induced apoptosis [##REF##23519108##51##] by not only destroying several key structural and functional proteins and DNA but also inducing the release of cytochrome c (cyt-c) from the mitochondria, thereby accelerating photodamage of the skin [##REF##31861553##52##].</p>", "<title>Apoptosis induced by unrepaired DNA damage</title>", "<p id=\"Par16\">ROS can induce DNA damage, which appears to be a determinant of UV-induced apoptosis [##REF##23519108##51##]. Obstruction of cell cycles provides time for the recognition and repair of UV photoproducts, most of which are removed by DNA excisional repair [##REF##34829793##53##]. However, if DNA damages are extensive and cannot be repaired, and RNA polymerase cannot transcribe the necessary gene products, then apoptosis will be triggered [##REF##28245638##54##].</p>", "<title>Mitochondrial DNA damage and endogenous apoptosis</title>", "<p id=\"Par17\">Large-scale mitochondrial DNA (mtDNA) deletion has been documented in skin tissues exposed to sunlight [##REF##19955330##55##]. mtDNA damage also leads to the maladjustment of oxidative phosphorylation and an increase in ROS production [##REF##31083540##56##]. The activation or inhibition of apoptosis is documented mainly determined by the balance between members of the Bax protein family, for example, pro-apoptotic proteins like Bax, Bak and Bid, and anti-apoptotic members such as Bcl-2 and Bcl-x [##UREF##1##57##]. Bcl-2 inhibits the activation of caspases 3 and 8 and promotes the release of cyt-c, while Bcl-x partially inhibits such release [##REF##30966844##58##]. UV can promote the translocation of Bax to the mitochondria [##REF##17965970##59##], increasing the expression of p53 and Bax, and decreasing the expression of Bcl-2 [##REF##30902393##60##]. A study showed that ROS can affect the transcriptional regulation of the Bax gene to a large extent through p53 [##REF##32619203##61##]. Once released in the cytoplasm, cyt-c works with the apoptotic peptidase activating factor 1 (Apaf-1) to form a kind of apoptotic body that recruits and activates caspase 9 to initiate caspase 3-dependent cell death [##REF##27679857##62##].</p>", "<p id=\"Par18\">Studies on nucleotide excision repair-defective cells have shown that unrepaired UV-induced DNA damage can trigger the degradation of Bcl-2, the activation of caspase 3, and the destruction of mitochondrial membrane potential; thus, leading to the apoptosis of cells [##REF##21852793##63##]. Within 24 h of UV exposure, Bcl-2 protein levels drop by about 90%, and this decrease can be prevented by the treatment with protease inhibitors MG115 or MG132, or partially eliminated by caspase 3 inhibitor DEVD-FMK and caspase 9 inhibitor LEHD-FMK [##REF##17213957##64##].</p>", "<title>Inflammation by ROS and its regulatory mechanism in skin connective tissue</title>", "<p id=\"Par19\">Antioxidant enzymes in the skin that remove ROS can be depleted by prolonged exposure to UV light. Photooxidation activates mitogen-activated protein kinase (MAPK), and regulates the expression of NF-κB and activator protein-1 (AP-1) by increasing ROS [##REF##32402583##65##]. AP-1 and NF-κB activate MMPs in the dermis and epidermis [##REF##22922035##66##]. This reduces the synthesis of types I and III procollagen, thereby damaging the formation of new collagen while also degrading it; thus leading to photodamage [##REF##24946848##67##]. Moreover, ROS can directly trigger collagen fiber collapse through the overexpression of MMP-1 [##REF##25351668##68##].</p>", "<p id=\"Par20\">MAPK is comprised of the ERK, JNK, and P38 kinase, variously involving in cell proliferation, apoptosis, and inflammation [##REF##31861553##52##]. Blocking the MAPK pathway can inhibit the expression of pro-inflammatory cytokines, such as IL-6, Cyclooxygenase-2 (COX-2), and IL-8 [##REF##17334833##69##].</p>", "<p id=\"Par21\">The NF-κB pathway has been widely demonstrated to play a crucial role in mediating skin inflammation, and its activation is crucial to the aging process [##REF##27486771##70##]. When activated, pro-inflammatory factors infiltrate into the skin, leading to clinical syndromes, such as erythema and epidermal hyperplasia [##REF##27995657##71##]. Inactivated NF-κB is present as a cytoplasmic heterodimer, which consists of the P65 and P50 subunits, and can bind to the inhibitory protein IκB. IKK complex can be recruited by the ubiquitin binding protein NF-κB essential modulator (NEMO) on the new ubiquitin chain formed by the receptor. When activated, this NEMO-dependent IKK complex phosphorylates IκB at Ser32 and Ser36, and induces the ubiquitination of protein and the degradation of the proteasome, releasing the inhibitory effect of IκB on NF-κB [##REF##22573609##72##]. Next, the activated NF-κB heterodimer translocates to the nucleus, promoting the transcription of pro-inflammatory genes [##REF##24952939##73##], particularly, the genes that control apoptosis, causing the production of cytokines and interferons that regulate inflammatory responses. These targets include TNF-α, IL-1, IL-6, and IL-8 [##REF##23915189##74##]. IKK also directly phosphorylates the p65/50 dimer at the position 536 of p65, which has been shown to further increase the nuclear transcriptional activity of NF-κB [##REF##30887112##75##].</p>", "<p id=\"Par22\">Photoaging is characterized by matrix metalloproteinase induction and dermal collagen loss [##REF##27935061##35##]. The loss of skin elasticity and the reduction in collagen content leads to the formation of wrinkles [##REF##29128706##76##], since collagen makes up about 70% of the dermis. Regulation of collagen is achieved by promoting the synthesis of cytokine transforming growth factor beta (TGF-β), inhibiting transcription factor AP-1, and activating the degradation of MMPs [##REF##25351668##68##].</p>", "<p id=\"Par23\">ROS can induce AP-1, a heterodimer composed of c-fos and c-jun, which downregulates type I collagen and upregulates MMPs in aging skin [##REF##19914807##77##], and can also inhibit the TGF-β signaling pathway in skin fibroblasts. As a result, reducing synthesis of new collagen and decreasing collagen numbers in the dermis [##REF##31906195##78##].</p>", "<p id=\"Par24\">In human dermal fibroblasts, TGF-β/Smad signaling pathway is key to maintain the integrity of the dermal structure, by increasing the extracellular matrix (ECM) production, and decreasing its degradation [##REF##19641518##79##]. Initially, TGF-β binds to TGF-β receptor II (TβRII), which then recruits and phosphorylates TGF-β receptor I (TβRI), leading to the activation of transcription factors Smad2 and Smad3. And then the activated Smad2 or Smad3 bind to Smad4, forming the heterogeneous Smad complex, which translocates into the nucleus and interacts with Smad-binding element (SBE) of the TGF-β target gene [##REF##25227288##80##]. Therefore, TGF-β/ Smad signaling directly upregulates ECM genes, including those involved in the production of collagen, fibronectin, decorin, and versican [##REF##31906195##78##].</p>", "<p id=\"Par25\">MMPs, which are zinc-dependent enzymes, play key roles in the degradation of collagen in dermal cells. To date, there has been 28 MMPs are identified [##REF##32869366##81##]. Oxidative stress is involved in the degradation of collagen, while inflammation stimulates epidermal thickness, and these two interact with each other in both processes. The expression and activation of MMP-1, MMP-2, MMP-3, MMP-9 and/or MMP-13 are increased by oxidative stress, thereby reducing the collagens in the skin [##REF##23467430##82##]. Among them, MMP-1 and -3 are important mediators for the degradation of ECM and the formation of skin wrinkles during UVB-induced photoaging [##REF##27090187##83##]. MMP-1 (collagenase type I) is a fully functional mesenchymal collagenase-1 that can initiate the degradation of collagenase types I and III into ¾ and ¼ fragments, respectively [##REF##23430258##84##]. Moreover, MMP-3 degrades type IV collagen into non-collagen components and elastic fibers. Overexpression of MMP-1 and MMP-3 are widely believed to result in wrinkle formation and sagging skin [##REF##22573609##72##]. MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) degrade gelatin. MMP-2, MMP-7 and MMP-12 (elastase) degrade elastin [##REF##17334833##69##]. Upregulated MMPs inhibit collagen type I alpha1 (COL1A1), elastin (ELN), and hyaluronan synthase 2 (HAS2), and regulates the synthesis of hyaluronic acid [##REF##17334833##69##].</p>", "<p id=\"Par26\">Recent studies have found that inflammasomes play an important role in UV-induced skin photodamage. Inflammasomes mainly include Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) and NLRP3. And ROS induced by photodamge of the skin can activate inflammasomes. Studies have found that NLRP1 inflammasomes have taken centre stage in skin biology, as mutations in NLRP1 underlie the genetic etiology of dermatological diseases and increase the susceptibility to skin cancer [##REF##29545203##85##]. Other studies have found that the activation of NLRP3 can recruit and activate pro-inflammatory protease caspase-1, and then produce IL-1β, IL-18 and other cytokines, triggering a process of inflammation-relatedcell death named pyroptosis. Indeed, pyroptosis is a rapid and inflammatory form of lytic programmed cell death [##REF##34983566##86##]. Therefore, inflammasomes can be considered significant in UV-induced skin photodamage and may be potential targets for the treatment and improvement of photodamge of the skin.</p>" ]
[ "<title>Author contributions</title>", "<p>MW draft the manuscript and prepared figures. XH and NL edited and revised the manuscript. HD supervised the entire article. All authors approved final version of manuscript.</p>", "<title>Funding</title>", "<p>National Natural Science Foundation of China (numbers 82273521).</p>", "<title>Availability of data and materials</title>", "<p>This is a review article, no data associated in this manuscript.</p>", "<title>Declarations</title>", "<title>Competing interests</title>", "<p id=\"Par28\">The results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration by another publisher. The authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper. There is no conflict of interest among the 4 authors of the article entitled “Role of Reactive Oxygen Species in Ultraviolet-induced Photodamage of the Skin”: Min Wei, Xin He, Na Liu, Hui Deng.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Depths of ultraviolet (UV) radiation (UVR). UVC, with the shortest wavelength (100–290 nm) and highest energy, is mostly blocked by the ozone layer and rarely reaches human skin. Contrastingly, both UVB and UVA can penetrate the ozone layer. UVA, with the longest wavelength (320–400 nm) and lowest energy, can reach deep into the dermis, whereas UVB (wavelength: 290–320 nm) mostly reaches the surface of the dermis</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Effects of reactive oxygen species (ROS) depend on the dose and persistence of the particles. A low level of ROS can lead to mutation, a medium level of ROS can cause senescence, and a high level of ROS usually cause cell death, such as apoptosis and necrosis</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Production and regulation of reactive oxygen species (ROS). Mitochondria and NADPH oxidases (NOXs) are the two main contributors of endogenous ROS. O<sub>2</sub>• − is formed from molecular O<sub>2</sub> following the acceptance of a single electron from the electron transport chain (ETC) in the mitochondria or from NOXs. Superoxide dismutase (SOD) enzymes convert O<sub>2</sub> •− into H<sub>2</sub>O<sub>2</sub>, which can then be reduced and converted by peroxiredoxin (PRX), glutathione peroxidase (GPX), and catalase to form H<sub>2</sub>O and OH•. The latter is extremely reactive and causes damage to DNA, proteins, and lipids</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Signaling pathways of reactive oxygen species (ROS) in ultraviolet (UV)-induced skin photodamage. ROS regulate DNA damage and cell signaling pathways, and cause an imbalance of skin oxidants and antioxidants, which accelerates skin photodamage. Unrepaired damaged DNA, including cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts [(6–4)PPs], can cause cell death. Moreover, ROS signaling regulates transcription factors, such as AP-1 and nuclear factor-kappa B (NF-κB), inducing the expression of matrix metalloproteinases (MMPs), upregulating the expression of inflammatory factors, such as IL-1β, IL-6, and tumor necrosis factor (TNF)-α, and downregulating TGF-β expression and Smad signaling. Thus, extracellular matrix genes and collagen type I alpha1 (COL1A1), elastin (ELN), and hyaluronan synthase 2 (HAS2) are downregulated. Moreover, collagen types I and III are degraded into ¾ and ¼ fragments, respectively, while type IV collagen is degraded into non-collagen components and elastic fibers, resulting in accelerated aging in the form of skin relaxation, deepened wrinkles, and decreased skin elasticity. ROS are also directly or indirectly involved in UV-induced mitochondrial apoptosis by affecting the expression of Bcl-2 and Bcl-x in the mitochondria, thus inducing the release of cytochrome c (cyt-c). Once released, cyt-c functions with the apoptotic peptidase activating factor 1 (Apaf-1) to form a type of apoptotic body that recruits and activates caspase 9 to initiate caspase 3-dependent apoptosis</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Signaling pathway of MAPK in UV-induced skin photodamage affected by ROS. UV irradiation can induce ROS and then has an impact on activating the MAPK pathway within the cells. Then, the downstream factors extracellular signal-regulated kinase (ERK), P38 kinase, and c-Jun N-terminal kinase (JNK) in the MAPK pathway can enter the nucleus and regulate transcription factors, such as AP-1 and NF-κB, inducing the expression of MMPs, upregulating the expression of inflammatory factors, such as IL-1β, IL-6, TNF-α, and downregulating TGF-β expression</p></caption></fig>" ]
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[{"label": ["2."], "surname": ["Honda", "Yamamoto", "Sawada", "Egawa", "Kitoh", "Otsuka", "Dainichi", "Nakajima", "Miyachi", "Kabashima"], "given-names": ["T", "O", "Y", "G", "A", "A", "T", "S", "Y", "K"], "article-title": ["Receptor-interacting protein kinase 3 controls keratinocyte activation in a necroptosis-independent manner and promotes psoriatic dermatitis in mice"], "source": ["J Allergy Clin Immunol"], "year": ["2017"], "volume": ["140"], "issue": ["619\u2013622"], "fpage": ["e616"]}, {"label": ["57."], "mixed-citation": ["Carpenter, R.; Brady, M.F. BAX Gene. In StatPearls, StatPearls Publishing Copyright \u00a9 2020, StatPearls Publishing LLC.: Treasure Island (FL), 2020."]}]
{ "acronym": [], "definition": [] }
86
CC BY
no
2024-01-14 23:43:47
Cell Div. 2024 Jan 12; 19:1
oa_package/0d/d9/PMC10787507.tar.gz
PMC10787508
0
[ "<title>Introduction</title>", "<p id=\"Par5\">Over one billion people worldwide are living with mental illness [##REF##33069297##1##] and this number is continuing to rise [##REF##35026139##2##]. While there has been an increase in mental healthcare expenditure [##UREF##0##3##–##REF##36039126##5##] and access to traditional first-line treatments (i.e., psychotherapy and pharmacotherapy), in countries such as Australia and the US, this has not reduced the burden of mental illness or substantially improved therapeutic outcomes [##REF##28127925##6##]. Mental illness continues to be among the leading causes of disability globally and is estimated to cost the global economy over $6 trillion US by 2030 [##REF##33069297##1##]. A rapidly growing body of evidence indicates that lifestyle medicine approaches can be highly effective for numerous mental health indications [##UREF##2##7##–##REF##35185421##10##] and are an underutilised treatment option that has the potential to offer a flexible, and empowering approach to improving mental health outcomes.</p>", "<p id=\"Par6\">Lifestyle medicine, also known as behavioural medicine, is a branch of medicine that applies environmental, behavioural, medical, and motivational principles to promote the management of healthy lifestyle behaviours [##UREF##4##11##]. Physical exercise, sleep, diet, mindfulness meditation, and strengthening positive relationships are examples of lifestyle medicine activities with demonstrated efficacy to prevent and treat a wide variety of mental illnesses; albeit to varying degrees [##UREF##2##7##, ##UREF##3##9##, ##REF##32931092##12##, ##UREF##5##13##]. For example, physical exercise and mindfulness-based interventions have been shown to be as effective as pharmacotherapy in the treatment of Major Depressive Disorder [##REF##31541410##14##–##REF##36796860##17##] and Generalised Anxiety Disorder [##UREF##6##18##], respectively. Clinical trials have shown that interventions targeting sleep can reduce depression severity [##REF##25867693##19##–##REF##30579141##21##], while the field of nutritional psychiatry [##REF##28942748##22##] has provided early evidence that interventions targeting diet quality can also reduce depressive symptoms [##REF##28137247##23##]. Collectively, the weight of high-quality clinical evidence has led to lifestyle modification being recommended as a first-line treatment for mental illness in international treatment guidelines by organisations such as the National Institute for Health and Care Excellence (i.e. NICE Depression in adults: treatment and management [##REF##19861376##24##]), the Royal Australian and New Zealand College of Psychiatrists (i.e. RANZCP clinical practice guidelines for mood disorders [##UREF##2##7##]), and the World Federation of Societies for Biological Psychiatry (WFSBP) and Australasian Society of Lifestyle Medicine (ASLM; i.e., Clinical guidelines for the use of lifestyle-based mental health care in major depressive disorder [##UREF##5##13##]).</p>", "<p id=\"Par7\">The value of lifestyle medicine comes not only from its capacity to treat mental illness but from its positive concurrent impacts on physical health [##REF##31324560##25##]. This is particularly relevant for people living with mental illness who are twice as likely to be diagnosed with a cardiometabolic condition such as diabetes, obesity and cardiovascular disease [##REF##31324560##25##–##REF##21934057##29##], while those with a ‘severe mental illness’ (e.g., schizophrenia, bipolar disorder, obsessive compulsive disorder) have a 10–20 year reduction in life expectancy [##REF##28684403##30##–##REF##33128044##32##], largely due to these physical comorbidities. It is well established that lifestyle modification (e.g., diet, physical exercise, sleep) plays an important role in both preventing and treating cardiometabolic conditions and increasing life expectancy [##REF##31324560##25##]. That these physical health benefits are unique to lifestyle medicine, as compared to psychotherapy and psychopharmacology, lends further weight to the utility of the widespread implementation of this approach.</p>", "<p id=\"Par8\">Despite the weight of positive evidence, lifestyle-based interventions are rarely prescribed and have not been widely integrated into the mental healthcare system [##UREF##3##9##, ##REF##35185421##10##]. Barriers to the wide-spread implementation of lifestyle medicine for mental illness are complex and include, but are not limited to: a lack of education and training in the prescription of effective lifestyle medicine approaches amongst practitioners [##REF##32286095##33##, ##REF##34398577##34##], limited funding pathways for allied health professionals to treat mental illness, and a scarcity of programs designed to support healthy lifestyle change [##UREF##3##9##]. One key ingredient to encouraging individual-level uptake and system-level implementation of lifestyle medicine is understanding how acceptable this approach is to end-users. Acceptability to end-users is important for all health treatments [##UREF##9##35##, ##REF##28126032##36##], and especially for behavioural approaches which require significant sustained motivation and effort to be effective. Initiating and maintaining the level of lifestyle behaviour required to improve mental health outcomes is difficult and often more so for people living with mental illness [##REF##31324560##25##]. People living with mental illness experience unique barriers to health behaviour change such as low mood, amotivation, reduced social support [##REF##27502153##37##], higher rates of sleep disturbance, poor diet quality, and sedentary behaviour [##REF##31324560##25##]. These distinct barriers make it particularly important to differentiate the perspectives of people with (i.e. patients) and without (i.e. potential future patients) mental illness. Evaluating the acceptability of lifestyle medicine and identifying any reservations people may have will provide insights into the value of continuing efforts to integrate lifestyle medicine alongside pharmacological and psychological approaches.</p>", "<p id=\"Par9\">The Theoretical Framework of Acceptability (TFA [##REF##28126032##36##]) is a gold-standard, empirically derived framework that outlines seven unique component constructs of acceptability: affective attitude, ethicality, burden, intervention coherence, perceived effectiveness, opportunity cost and self-efficacy (see Table ##TAB##0##1## for definitions). It was developed via evidence synthesis to bring clarity to the concept of acceptability and enable a nuanced examination of its many facets. In the context of the current study, the TFA provided a rigorous framework upon which to investigate: 1) the acceptability of lifestyle medicine for the treatment of mental illness, 2) whether lived experience of mental illness influences the acceptability of lifestyle medicine, and 3) compare the acceptability of lifestyle medicine to psychopharmacotherapy and psychotherapy.\n</p>" ]
[ "<title>Methods</title>", "<title>Design</title>", "<p id=\"Par10\">This study employed a cross-sectional survey-based research design.</p>", "<title>Participants</title>", "<p id=\"Par11\">Participants included 899 Australian adults (aged 18 years and above) recruited via advertisements on social media platforms (Facebook and Twitter), online forums (Reddit) and community organisation email lists (i.e., mental health support groups). Inclusion criteria were being aged 18 years or older and residing in Australia, and no exclusion criteria were applied. Upon completion of the study survey, participants were invited to enter a prize draw to win one of three $50 grocery/department store gift vouchers to show appreciation for their effort. The current analyses excluded participants who did not complete all survey items (<italic>n</italic> = 250). Thus, the resulting study sample included 649 participants (62.6% female, <italic>mean age</italic> = 34.8 years, <italic>SD</italic> = 12.7). This study was conducted in accordance with the Declaration of Helsinki, approved by the Monash University Human Research Ethics Committee, and all participants provided written informed consent.</p>", "<title>Measures</title>", "<title>Acceptability survey construction</title>", "<p id=\"Par12\">A survey assessing the acceptability of lifestyle medicine, psychotherapy and pharmacotherapy to treat mental illness across each of the seven constructs of the TFA was developed by the research team. Survey construction and piloting was conducted using previously published methods [##REF##32858300##38##, ##REF##33076881##39##] and comprised the following five steps:<list list-type=\"order\"><list-item><p id=\"Par13\">A literature review was conducted to identify words and phrasing commonly used to describe and assess each of the seven TFA constructs (e.g. Affective Attitude: like, enjoy, feel positive; Intervention Coherence: comprehensible, understand, easy to follow [##REF##32858300##38##–##REF##31948423##41##].</p></list-item><list-item><p id=\"Par14\">The research team discussed these options and achieved consensus on the most grammatically correct and readable phrasing to accurately reflect each TFA construct (see Table ##TAB##1##2##).</p></list-item><list-item><p id=\"Par15\">Two researchers drafted items for each TFA acceptability domain based on the agreed phrasing.</p></list-item><list-item><p id=\"Par16\">Items were reviewed and refined by the broader research team and consensus achieved on the wording that most accurately reflected the core conceptual meaning of the TFA constructs.</p></list-item><list-item><p id=\"Par17\">The survey items were piloted with five non-academic community members who provided feedback on clarity and ease of completion.</p></list-item></list></p>", "<p id=\"Par18\">Shortly after data collection Sekhon and colleagues published a validated TFA-based questionnaire designed to assess the acceptability of healthcare interventions [##REF##35232455##42##]. The items in the current survey align closely with those in the validated scale (e.g. “I understand how engaging in lifestyle medicine activities could treat mental illness” vs “It is clear to me how [intervention] will help [manage/improve] my [behaviour/condition/clinical outcome]” [##REF##35232455##42##]).</p>", "<title>Acceptability survey</title>", "<p id=\"Par19\">The final survey was delivered online in English via Qualtrics, median completion time was 10.2 minutes. The survey comprised three sections as described below (see ##SUPPL##0##Supplementary material## for full survey).</p>", "<p id=\"Par20\">\n<italic>i) Demographics.</italic> This section collected demographic data including age, gender, employment status, income, and past or present mental illness diagnosis.</p>", "<p id=\"Par21\">\n<italic>ii) Information section.</italic> As community awareness of lifestyle medicine and its applications vary widely, a brief information section was included that provided definitions of lifestyle medicine, mental illness, and examples of the use of lifestyle medicine to treat mental illness. The information also noted that lifestyle interventions could be undertaken with or without professional supports (i.e. independent behaviour change vs. with a dietitian, exercise physiologist, health coach). The information section was written by the research team and reviewed by an independent researcher for accuracy and unbiased phrasing.</p>", "<p id=\"Par22\">\n<italic>iii) Acceptability.</italic> This section assessed the acceptability of lifestyle medicine, psychotherapy, and pharmacotherapy to treat mental illness. Each approach was rated across seven items, one for each component construct of the TFA. Participants indicated the extent to which they agreed or disagreed with each statement on a five-point Likert scale (1 = strongly disagree to 5 = strongly agree). As items assessing burden and opportunity cost were phrased negatively, responses to these items were reverse scored (i.e., strongly disagree became strongly agree, disagree became agree and vice versa) to ensure the directionality of responses across component constructs was comparable. Participants were then asked to rank five common lifestyle medicine activities (exercise, diet, sleep, social connection, and meditation) in order according to which they would most, to least likely engage with. Participants were also asked to rank the three treatment modalities according to which they would prefer to be prescribed if they were experiencing a mental illness.</p>", "<title>Data analysis</title>", "<p id=\"Par23\">Data were analysed using Statistical Package for Social Sciences (SPSS) version 25.0. Descriptive statistics were used to document the acceptability of lifestyle medicine for treating mental illness across the seven TFA component constructs and to compare participants’ preferred lifestyle medicine activities and treatment modality (lifestyle medicine, pharmacotherapy, or psychotherapy). Mann-Whitney U tests were used to investigate differences in the acceptability of lifestyle medicine between people with and without a lived experience of mental illness. A series of Friedman’s repeated measures ANOVAs were used to examine differences in acceptability scores across the three treatment modalities for each TFA component construct. Wilcoxon signed-ranks test were used for post hoc analyses. An alpha of 0.05 was applied to all analyses.</p>" ]
[ "<title>Results</title>", "<title>Participant characteristics</title>", "<p id=\"Par24\">Of the 649 participants included in the analysis, 348 (53.6%) reported a past or present mental illness. Affective (47.2%) and anxiety disorders (41.9%) were the most prevalent mental illnesses and 231 participants reported experiencing more than one mental illness. Participants with and without lived experience of mental illness did not differ in age or gender, however those with a lived experience were more likely to be unemployed and have a lower income (Table ##TAB##2##3##). The majority of participants resided in east Australian states (Victoria (<italic>n</italic> = 320), New South Wales or Australian Capital Territory (<italic>n</italic> = 137) and Queensland (<italic>n</italic> = 80)), followed by Western Australia (<italic>n</italic> = 48), South Australia (<italic>n</italic> = 43), Northern Territory (<italic>n</italic> = 15), and Tasmania (<italic>n</italic> = 6).\n</p>", "<title>Acceptability of lifestyle medicine</title>", "<p id=\"Par25\">The majority of participants agreed or strongly agreed that they felt positive about lifestyle medicine (affective attitude = 76.9%), that this treatment approach aligned with their personal values (ethicality = 74.9%), they understood how lifestyle medicine would work (intervention coherence = 86.4%) and thought it would be effective (perceived effectiveness = 69.6%). The burden associated with engaging in lifestyle medicine was acceptable to fewer participants (53%). Less than half of participants reported that what they would have to give up in order to engage was acceptable (opportunity cost = 47.3%) or were confident in their ability to engage in lifestyle medicine activities (self-efficacy = 45.7%; Fig. ##FIG##0##1##). Across common lifestyle medicine activities with demonstrated efficacy for mental illness, participants indicated they would be most likely to engage in physical exercise (30.2%), followed by diet (14.5%), sleep (13.8%), social connection (9.3%), and meditation (3.9%). This pattern of preferences held for participants with lived experience of mental illness, however people without lived experience had a slight preference for sleep interventions (16.3%) over diet modification (15.9%).</p>", "<title>Effect of lived experience on the acceptability of lifestyle medicine</title>", "<p id=\"Par26\">Responses to TFA items assessing burden and self-efficacy to engage in lifestyle medicine activities differed significantly between individuals with and without lived experience of a mental illness (Fig. ##FIG##1##2##, see supplementary materials Table S##SUPPL##0##1## for full frequency statistics). The burden associated with engaging in lifestyle medicine was less acceptable to participants who had experienced mental illness than those who had not (<italic>U =</italic> 60,470<italic>, z =</italic> 3.57<italic>, r =</italic> 0.14<italic>, p</italic> &lt; 0.001). Participants with lived experience also reported lower self-efficacy to engage in lifestyle medicine activities compared to participants without lived experience (<italic>U =</italic> 61,587<italic>, z =</italic> 4.02<italic>, r =</italic> 0.16<italic>, p</italic> &lt; 0.001). No significant differences in responses to items assessing affective attitude, ethicality, intervention coherence, opportunity cost, or perceived effectiveness were observed (all <italic>p</italic> &gt; 0.05).</p>", "<title>The acceptability of lifestyle medicine compared to psychotherapy and pharmacotherapy</title>", "<p id=\"Par27\">Lifestyle medicine (46.8%) was most frequently ranked as participant’s preferred treatment modality, followed by psychotherapy (37.6%) and pharmacotherapy (15.6%). This order of preferences held for participants without lived experience of mental illness, however, people with lived experience preferred psychotherapy (41.7%) followed by lifestyle medicine (34.8%). The distribution of participant responses was significantly different between the three treatment modalities for the TFA component constructs of affective attitude (<italic>X</italic><sup><italic>2</italic></sup> (2) <italic>86.20, = p</italic> &lt; 0.001), burden (<italic>X</italic><sup><italic>2</italic></sup> (2) <italic>86.71, = p</italic> &lt; 0.001), ethicality (<italic>X</italic><sup><italic>2</italic></sup> (2) <italic>= 215.60, = p</italic> &lt; 0.001), intervention coherence (<italic>X</italic><sup><italic>2</italic></sup> (2) <italic>30.45, = p</italic> &lt; 0.001), perceived effectiveness (<italic>X</italic><sup><italic>2</italic></sup> (2) <italic>87.39, = p</italic> &lt; 0.001), and self-efficacy (<italic>X</italic><sup><italic>2</italic></sup> (2) <italic>80.18, = p</italic> &lt; 0.001). There were no significant differences in opportunity cost (Fig. ##FIG##2##3##, see supplementary materials Table S##SUPPL##0##2## for full frequency statistics). Participants felt more positive (affective attitude) towards lifestyle medicine (<italic>M</italic> = 3.92, <italic>Z</italic> = − 6.48, <italic>p</italic> &lt; 0.001) and psychotherapy (<italic>M</italic> = 3.99, <italic>Z</italic> = − 8.44, <italic>p</italic> &lt; 0.001) than pharmacotherapy (<italic>M</italic> = 3.54) and reported these approaches to be more in line with their personal values (ethicality; <italic>Z</italic> = − 10.24, <italic>p</italic> &lt; 0.001 and <italic>Z</italic> = − 12.50, <italic>p</italic> &lt; 0.001, respectively). In contrast pharmacotherapy (<italic>M</italic> = 3.54) was perceived as significantly less burdensome than lifestyle medicine (<italic>M</italic> = 3.54, <italic>Z</italic> = − 8.27, <italic>p</italic> &lt; 0.001) and psychotherapy (<italic>M</italic> = 3.54, <italic>Z</italic> = − 8.44, <italic>p</italic> &lt; 0.001). Intervention coherence ratings were significantly higher for psychotherapy (<italic>M</italic> = 4.27) than lifestyle medicine (<italic>M</italic> = 4.10, <italic>Z</italic> = − 4.97, <italic>p</italic> &lt; 0.001) and pharmacotherapy (<italic>M</italic> = 4.15, <italic>Z</italic> = − 3.84, <italic>p</italic> &lt; 0.001). Pharmacotherapy (<italic>M</italic> = 3.58) was perceived as less effective than lifestyle medicine (<italic>M =</italic> 4.00, <italic>Z</italic> = − 4.03, <italic>p</italic> &lt; 0.001) and psychotherapy (<italic>M =</italic> 4.00, <italic>Z</italic> = − 9.71, <italic>p</italic> &lt; 0.001), while lifestyle medicine was perceived as less effective than psychotherapy (<italic>Z</italic> = − 5.18, <italic>p</italic> &lt; 0.001). Finally, self-efficacy differed significantly between all treatment modalities (all <italic>p</italic> &lt; 0.001) such that participants were most confident in their ability to engage in psychotherapy (<italic>M</italic> = 3.66), followed by pharmacotherapy (<italic>M</italic> = 3.53) and lifestyle medicine (<italic>M</italic> = 3.19).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par28\">The current study is the first to investigate the acceptability of lifestyle medicine for treating mental illness and explore differences in perspectives across people with and without lived experience of mental illness. The broad pattern of acceptability was such that participants typically found the more emotive (affective attitude, ethicality) and cognitive (intervention coherence, perceived effectiveness) component constructs of the TFA to be highly acceptable. In contrast, across component constructs that reflect the practicalities of engaging in lifestyle medicine (burden, opportunity cost, and self-efficacy), acceptability was comparatively lower. Participants with lived experience of mental illness viewed lifestyle medicine as more burdensome and were less confident in their ability to engage, than those who had not experienced mental illness. Across the whole sample, participants felt more positive about (affective attitude) lifestyle medicine and psychotherapy, and that these two approaches were more in line with their values (ethicality) than pharmacotherapy. Pharmacotherapy, however, was perceived as less burdensome than the non-pharmacological treatments, and participants were least confident in their ability to engage in lifestyle medicine.</p>", "<p id=\"Par29\">That participants generally felt positive about lifestyle medicine, believed it aligned with their values, understood how lifestyle medicine could treat mental illness, and perceived it to be an effective treatment option, indicate a favourable attitude towards this approach within the community. However, approximately half of participants felt that adopting lifestyle medicine activities would demand considerable effort, necessitate sacrificing other important priorities, and present challenges due to low self-efficacy, all of which would likely impede engagement to some degree. This highlights the need to support people to overcome barriers to initiating and maintaining health behaviour change. Behavioural science evidence indicates that group-based programs, supervised by an allied health professional (e.g. exercise physiologist or dietitian), alongside individualised motivational support, are most successful in establishing long-term health behaviour change [##REF##26978184##16##, ##REF##28137247##23##, ##REF##12453955##43##] and therefore promoting mental health outcomes. However, referrals to allied health professionals for mental illness are rare, despite lifestyle change being recommended by peak bodies in psychiatry as non-negotiable first-line treatment for numerous mental health indications [##UREF##2##7##]. In Australia for example, 61% of primary health care visits for a mental health indication result in a prescription for medication versus less than 3% for lifestyle modification [##UREF##10##44##]. A lack of education and training is a known contributor to low referral rates by primary health care providers [##UREF##11##45##]. Furthermore, when referrals do occur, there is significant variability in allied health professionals’ knowledge of the best practice approaches for mental health indications and evidence-based programs specifically designed to assist people with mental illness in overcoming barriers to behaviour change are scarce [##REF##35185421##10##].</p>", "<p id=\"Par30\">The acceptability of lifestyle medicine did not differ between people with and without personal experience of mental illness across affective attitude, ethicality, intervention coherence, or perceived effectiveness. People with lived experience, did however, report lower self-efficacy to engage in lifestyle medicine. This is of particular importance as self-efficacy is a known predictor for the adoption and maintenance of healthy lifestyle behaviours [##REF##28918861##46##–##REF##29172808##48##]. To be maximally successful interventions should therefore embed strategies to increase self-efficacy. For example, providing opportunities for people to experience mastery has been shown to improve self-efficacy and adherence to lifestyle interventions [##UREF##13##49##–##REF##25053214##51##]. Participants with lived experienced also felt that engaging in lifestyle medicine would be more burdensome. This may reflect the difficulties people face when attempting lifestyle changes while experiencing mental health symptoms (e.g. low energy, anhedonia, reduced motivation). It suggests that people who have experienced mental illness have a deeper understanding of the challenges associated with making health behaviour change while unwell, and are likely to require greater practical and psychological support to feel empowered and capable of engaging in sustained lifestyle change. Given a long history of researchers developing treatments without consulting end-users, and the potential downstream consequences for adherence, these findings highlight the importance of engaging people with experience of mental illness in the co-design of lifestyle-based interventions to ensure that such programs meet their wants and needs.</p>", "<p id=\"Par31\">When comparing treatment modalities, the was a clear preference for psychotherapy and lifestyle medicine over medication. These findings are consistent with an existing body of research showing widespread preference for non-medication treatments in psychiatry and indicate that this perspective has not changed in over 20 years [##REF##10940143##52##, ##UREF##14##53##]. While previous studies have primarily compared psychotherapy to pharmacotherapy [##UREF##14##53##], the results of the current study extend this work by also demonstrating a preference for lifestyle medicine. It highlights an ongoing clinical discrepancy that, for many, the least desired treatment approach is the most widely available and commonly prescribed [##UREF##10##44##]. While there has been global acknowledgement of the need to increase patient choice [##REF##20135903##54##], this shift has yet to occur at scale despite evidence that receiving a preferred treatment may result in greater treatment compliance, additional therapeutic benefit, and increase cost-effectiveness [##UREF##14##53##, ##REF##20135903##54##].</p>", "<p id=\"Par32\">The use of the TFA enabled insight into which facets of acceptability may be driving treatment preferences. Participants felt more positive about and ethically aligned with the use of lifestyle medicine and psychotherapy than pharmacotherapy. They also perceived psychotherapy, followed by lifestyle medicine, as being more effective than pharmacotherapy in treating mental illness. That lifestyle medicine was widely perceived to be effective is notable, given how recent and evolving the use of behavioural interventions for mental health is. While perceived understanding of <italic>how</italic> each treatment might work (intervention coherence) was highest (91.7%) for psychotherapy, the degree to which this was better understood than lifestyle medicine (86.4%) or pharmacology (90.2%) was modest. In comparison, pharmacotherapy was viewed as being less burdensome than both lifestyle medicine and psychotherapy. This is unsurprising given the substantial time and effort frequently associated with engaging in these activities [##REF##17107299##55##] compared with medication. Finally, participants felt they would need to give up something (opportunity cost) in order to engage in any treatment for mental illness. Time and cost are commonly cited as barriers to engaging in lifestyle medicine and psychotherapy [##REF##17107299##55##–##REF##32613867##57##], and medication use may be associated with side-effects [##UREF##15##58##], possibly resulting in a need to sacrifice other aspects of health. However, additional qualitative research is needed to provide a greater understanding of the factors underlying this finding.</p>", "<p id=\"Par33\">The current findings have implications for the design and implementation of lifestyle medicine interventions into mental healthcare. While a preference for lifestyle medicine for mental illness was observed, and many components of acceptability were high, there are also clear barriers to engaging in lifestyle medicine. Going forward, harnessing gold-standard frameworks for behavioural intervention design and development (e.g., The Behaviour Change Wheel [##REF##21513547##59##], ORBIT [##REF##25642841##60##]) is likely to be particularly valuable in identifying and systematically addressing barriers to change. The sustainability and scalability of lifestyle medicine programs should also be considered. Leveraging the rise in digital health innovations may offer an avenue for greater scalability; however, given the efficacy professional support offers, combined approaches may balance the need for sustainability, scalability and personalisation, and ultimately lead to greater improvements in mental health outcomes. Although understanding acceptability to end-users is essential, documenting how acceptable lifestyle-based therapeutics are to clinicians (general practitioners, psychologists, psychiatrists, allied health clinicians), service managers, and payers (government and private) is also necessary. As gatekeepers to mental healthcare services and policy, the combined perspectives of these key stakeholders will be essential to facilitate system-level change.</p>", "<p id=\"Par34\">The current data should be considered in context of a number of strengths and limitations. The use of the TFA provided a rich multifaceted breakdown of acceptability, enabling a nuanced appreciation of community attitudes to lifestyle medicine for mental illness and how this compares with current first-line treatments. It was, however, restricted to the assessment of <italic>prospective </italic>(before treatment) acceptability in the context of the examples provided, without considering participants’ experience with the various treatment approaches. First-hand experience of these treatments, whether as a patient or clinician, will inevitably alter perspectives. This study also explored the acceptability of lifestyle medicine as a broad category of approaches. Given the observed preference for specific lifestyle activities (i.e., exercise, diet, and sleep), it will be valuable to assess <italic>concurrent</italic> (during treatment) and <italic>retrospective</italic> (after treatment) acceptability of specific lifestyle interventions. Lastly, the current study did not account for potential variations in acceptability based on type of mental health diagnosis due to the diverse nature of the sample. Further research would be necessary to explore potential interactions between diagnosis and the acceptability of different lifestyle medicine approaches.</p>" ]
[ "<title>Conclusion</title>", "<p id=\"Par35\">There is a compelling body of evidence demonstrating the effectiveness of lifestyle medicine approaches in treating mental illness. Given the current growth in the field, now is the time to progress the development of integrated behavioural approaches and systematic implementation into mental health care. Understanding the acceptability of interventions is a crucial initial step towards achieving this. The current findings illustrate a preference for lifestyle medicine for treating mental illness and indicate a need for healthcare infrastructure to support programs that help people initiate and, most importantly, maintain healthy lifestyle change. Designing and implementing interventions specifically for people with mental illness that address the burden and opportunity cost of engaging in lifestyle medicine and increase self-efficacy will be particularly powerful.</p>" ]
[ "<title>Objective</title>", "<p id=\"Par1\">While lifestyle medicine can be highly effective for treating a range of mental illnesses these approaches are grossly underutilised and have not been systematically implemented into health care systems. Understanding the acceptability of lifestyle medicine is a critical first step to remediate this. This study evaluated the acceptability of lifestyle medicine relative to pharmacotherapy and psychotherapy, and explore perspectives of people with and without lived experience of mental illness.</p>", "<title>Methods</title>", "<p id=\"Par2\">Six hundred and forty-nine adult Australian residents (62.6% female; 53.6% with a lifetime diagnosis of mental illness) completed an online survey based on the Theoretical Framework of Acceptability assessing the acceptability of lifestyle medicine, pharmacotherapy and psychotherapy for treating mental illness.</p>", "<title>Results</title>", "<p id=\"Par3\">Most participants felt positive about lifestyle medicine (76.9%) and felt that such approaches aligned with their personal values (74.9%). They understood how lifestyle medicine worked (86.4%) and believed it would be effective (69.6%). Lived experience of mental illness was associated with greater perceived burden and lower self-efficacy to engage in lifestyle medicine activities (both <italic>p</italic> &lt; 0.001). While there was a clear preference for psychotherapy and lifestyle medicine over pharmacotherapy, pharmacotherapy was perceived as least effortful (<italic>p</italic> &lt; .001) and participants were least confident in their ability to engage in lifestyle medicine (<italic>p</italic> &lt; 0.05).</p>", "<title>Conclusion</title>", "<p id=\"Par4\">The findings indicate strong acceptability of lifestyle medicine for mental illness, a preference for non-pharmacological treatment approaches, and an understanding of the challenges associated with making long-term healthy lifestyle modifications amongst people who have lived experience of mental illness.</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s12889-024-17683-y.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>Not applicable.</p>", "<title>Authors’ contributions</title>", "<p>All authors contributed to the conception and planning of the study. KR and RS led the research design. RP was responsible for data collection. RP and KR analysed the data. All authors participated in the interpretation of the data. KR and RP drafted the manuscript. All authors read, critically reviewed and approved the final manuscript.</p>", "<title>Funding</title>", "<p>We are extremely grateful to the Wilson Foundation and David Winston Turner Endowment Fund whose generous philanthropic investment in the BrainPark research team and facility made this research possible. Karyn Richardson and Sam Hughes were supported by the Wilson Foundation. Prof Yücel’s role on this paper was funded through a National Health and Medical Research Council Fellowship (NHMRC; #APP1117188). Prof Yücel also receives funding from: government funding bodies such as the NHMRC, Australian Research Council (ARC), Australian Defence Science and Technology (DST), the Department of Industry, Innovation and Science (DIIS), the National Institutes of Health (NIH, USA); philanthropic donations from the David Winston Turner Endowment Fund, Wilson Foundation; sponsored Investigator-Initiated trials including Incannex Healthcare Ltd. These funding sources had no role in the data analysis, presentation, or interpretation and write-up of the data. Murat Yücel also sits on the Advisory Boards of: Centre of The Urban Mental Health, University of Amsterdam; and Enosis Therapeutics.</p>", "<title>Availability of data and materials</title>", "<p>The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par36\">The study protocol was reviewed by the Monash University Human Research Ethics Committee. Written informed consent was obtained from all individual participants included in the study.</p>", "<title>Consent for publication</title>", "<p id=\"Par37\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par38\">The authors declare no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Acceptability of lifestyle medicine for treating mental illness. Items assessing burden and opportunity cost were reverse scored such that strongly agree and agree indicated that the burden and opportunity cost was acceptable to participants (i.e. lower scores indicate these constructs are less acceptable)</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Acceptability of lifestyle medicine for people with and without lived experience of mental illness. Items assessing burden and opportunity cost were reverse scored such that strongly agree and agree indicated that the burden and opportunity cost was acceptable to participants (i.e. lower scores indicate these constructs are less acceptable). MI+ = participants with lived experience of mental illness; MI- = participants with no lived experience of mental illness. **<italic>p</italic> &lt; 0.001</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Acceptability of lifestyle medicine, psychotherapy and pharmacotherapy. Items assessing burden and opportunity cost were reverse scored such that strongly agree and agree indicated that the burden and opportunity cost was acceptable to participants. (i.e. lower scores indicate these constructs are less acceptable). LM = Lifestyle medicine; Psy = Psychotherapy, Pha = Pharmacotherapy. ** <italic>p</italic> &lt; .001</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>TFA component constructs, adapted from Sekhon et al., 2017 [##REF##28126032##36##]</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>TFA Component Construct</th><th>Definition</th></tr></thead><tbody><tr><td>Affective Attitude</td><td>How an individual feels about the intervention</td></tr><tr><td>Burden</td><td>The perceived amount of effort that is required to participate in the intervention</td></tr><tr><td>Ethicality</td><td>The extent to which the intervention has good fit with an individual’s value system</td></tr><tr><td>Intervention Coherence</td><td>The extent to which the participant understands the intervention and how it works</td></tr><tr><td>Opportunity Cost</td><td>The extent to which benefits, profits, or values must be given up to engage in the intervention</td></tr><tr><td>Perceived Effectiveness</td><td>The extent to which the intervention is perceived as likely to achieve its purpose</td></tr><tr><td>Self-Efficacy</td><td>The participant’s confidence that they can perform the behaviour(s) required to participate in the intervention</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Example survey items</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>TFA Component Construct</th><th>Key words</th><th>Example Item</th></tr></thead><tbody><tr><td>Affective Attitude</td><td>“feel positive”</td><td>I feel positive about the use of lifestyle medicine activities to treat mental illness</td></tr><tr><td>Burden</td><td>“effort”</td><td>I think engaging in lifestyle medicine activities to treat mental illness would require too much effort</td></tr><tr><td>Ethicality</td><td>“my personal values”</td><td>Using lifestyle medicine activities to treat mental illness fits with my personal values</td></tr><tr><td>Intervention Coherence</td><td>“understand”</td><td>I understand how engaging in lifestyle medicine activities could treat mental illness</td></tr><tr><td>Opportunity Cost</td><td>“give up”</td><td>Engaging in lifestyle medicine activities to treat mental illness would come at a cost, it would mean giving up other things that are important to me</td></tr><tr><td>Perceived Effectiveness</td><td>“effective”</td><td>Engaging in regular lifestyle medicine activities would be an effective treatment for mental illness</td></tr><tr><td>Self-Efficacy</td><td>“confident”</td><td>If I had a mental illness, I am confident I could regularly engage in lifestyle medicine activities to treat it</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Participant Characteristics</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Characteristic</th><th>Overall<break/>Sample<break/>(<italic>n = 649)</italic></th><th>Lived<break/>Experience<break/>(<italic>n</italic> = 348)</th><th>No Lived Experience<break/>(<italic>n</italic> = 301)</th><th/></tr><tr><th/><th colspan=\"3\"><italic>N (%) or mean (SD)</italic></th><th><italic>p</italic></th></tr></thead><tbody><tr><td>Age</td><td>34.8 (12.7)</td><td>32.3 (11.1)</td><td>35.6 (13.4)</td><td>0.44</td></tr><tr><td>Gender</td><td/><td/><td/><td>0.08</td></tr><tr><td> Male</td><td>234 (36.1)</td><td>110 (31.6)</td><td>124 (41.2)</td><td/></tr><tr><td> Female</td><td>406 (62.6)</td><td>232 (66.7)</td><td>174 (57.8)</td><td/></tr><tr><td> Non-binary/conforming</td><td>6 (0.9)</td><td>4 (1.1)</td><td>2 (0.7)</td><td/></tr><tr><td> Other</td><td>3 (0.5)</td><td>2 (0.6)</td><td>1 (0.3)</td><td/></tr><tr><td>Education</td><td/><td/><td/><td>0.09</td></tr><tr><td> Primary school</td><td>2 (0.3)</td><td>2 (0.6)</td><td>64 (21.3)</td><td/></tr><tr><td> Secondary school</td><td>140 (21.6)</td><td>76 (21.8)</td><td>29 (9.6)</td><td/></tr><tr><td> TAFE</td><td>84 (12.9)</td><td>55 (15.8)</td><td>6 (2.0)</td><td/></tr><tr><td> Apprenticeship</td><td>17 (2.6)</td><td>11 (3.2)</td><td>133 (44.2)</td><td/></tr><tr><td> Bachelor’s</td><td>279 (43.0)</td><td>146 (42.0)</td><td>56 (18.6)</td><td/></tr><tr><td> Master’s</td><td>101 (15.6)</td><td>45 (12.9)</td><td>10 (3.3)</td><td/></tr><tr><td> Doctoral</td><td>22 (3.4)</td><td>12 (3.4)</td><td>3 (1.0)</td><td/></tr><tr><td> None of the above</td><td>4 (0.6)</td><td>1 (0.3)</td><td>64 (21.3)</td><td/></tr><tr><td>Employment</td><td/><td/><td/><td>0.001</td></tr><tr><td> Employed</td><td>510 (78.6)</td><td>259 (74.4)</td><td>251 (83.4)</td><td/></tr><tr><td> Unemployed</td><td>82 (12.6)</td><td>58 (16.7)</td><td>24 (8.0)</td><td/></tr><tr><td> Retired</td><td>18 (2.8)</td><td>8 (2.3)</td><td>10 (3.3)</td><td/></tr><tr><td> Student</td><td>135 (20.8)</td><td>74 (21.3)</td><td>24 (8.0)</td><td/></tr><tr><td colspan=\"4\">Annual Income</td><td>0.001</td></tr><tr><td> $0 - $18,200</td><td>155 (23.9)</td><td>94 (27.0)</td><td>61 (20.3)</td><td/></tr><tr><td> $18,201 - $37,000</td><td>115 (17.7)</td><td>71 (20.4)</td><td>44 (14.6)</td><td/></tr><tr><td> $37,001 - $90,000</td><td>219 (33.7)</td><td>110 (31.6)</td><td>109 (36.2)</td><td/></tr><tr><td> $90,001 - $180,000</td><td>138 (21.3)</td><td>69 (19.8)</td><td>69 (22.9)</td><td/></tr><tr><td> $180,001 and over</td><td>22 (3.4)</td><td>4 (1.1)</td><td>18 (6.0)</td><td/></tr><tr><td colspan=\"4\">Experienced mental illness</td><td/></tr><tr><td> Yes</td><td>348 (53.6)</td><td/><td/><td/></tr><tr><td> No</td><td>301 (46.4)</td><td/><td/><td/></tr><tr><td colspan=\"4\">Mental Illness</td><td/></tr><tr><td> Affective disorders</td><td>307 (47.2)</td><td/><td/><td/></tr><tr><td> Anxiety disorders</td><td>272 (41.9)</td><td/><td/><td/></tr><tr><td> Obsessive Compulsive Disorder</td><td>31 (4.8)</td><td/><td/><td/></tr><tr><td> Substance Addiction</td><td>40 (6.2)</td><td/><td/><td/></tr><tr><td> Gambling Addiction</td><td>8 (1.2)</td><td/><td/><td/></tr><tr><td> Psychotic disorders</td><td>7 (1.1)</td><td/><td/><td/></tr><tr><td> Eating and Body Image Disorders</td><td>61 (9.5)</td><td/><td/><td/></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p>The same key words and item structure was used for TFA items assessing the acceptability of pharmacotherapy and psychotherapy</p></table-wrap-foot>", "<table-wrap-foot><p><italic>M</italic> = mean, <italic>SD</italic> = standard deviation, <italic>n</italic> = frequency; % = percentage.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Karyn Richardson and Rachel Petukhova are co-first author.</p></fn><fn><p>Murat Yücel and Rebecca Segrave are co-senior author.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"12889_2024_17683_MOESM1_ESM.docx\"><caption><p><bold>Additional file 1.</bold>\n</p></caption></media>" ]
[{"label": ["3."], "mixed-citation": ["Australian Institute of Health and Welfare. Health expenditure Australia 2020\u201321. 2022.\u00a0"], "ext-link": ["https://www.thelancet.com/pdfs/journals/langlo/PIIS2214-109X(20)30432-0.pdf"]}, {"label": ["4."], "mixed-citation": ["Australian Institute of Health and Welfare. Health expenditure Australia 2010\u201311. 2012.\u00a0"], "ext-link": ["https://www.thelancet.com/pdfs/journals/langlo/PIIS2214-109X(20)30432-0.pdf"]}, {"label": ["7."], "surname": ["Malhi", "Bell", "Bassett", "Boyce", "Bryant", "Hazell"], "given-names": ["GS", "E", "D", "P", "R", "P"], "article-title": ["The 2020 Royal Australian and new Zealand College of Psychiatrists clinical practice guidelines for mood disorders"], "source": ["Aust N Z J Psychiat"], "year": ["2021"], "volume": ["55"], "issue": ["1"], "fpage": ["7"], "lpage": ["117"], "pub-id": ["10.1177/0004867420979353"]}, {"label": ["9."], "surname": ["Marx", "Jacka", "O\u2019Neil"], "given-names": ["W", "F", "A"], "article-title": ["Lifestyle-based mental health care in psychiatry: translating evidence into practice"], "source": ["Aust N Z J Psychiat"], "year": ["2021"], "volume": ["55"], "issue": ["7"], "fpage": ["641"], "lpage": ["643"], "pub-id": ["10.1177/00048674211011250"]}, {"label": ["11."], "surname": ["Eggar", "Binns", "Rossner", "Sagner"], "given-names": ["G", "A", "S", "M"], "source": ["Lifestyle medicine: lifestyle, the environment and preventive medicine in health and disease. 3rd ed"], "year": ["2017"], "publisher-name": ["Academic Press"]}, {"label": ["13."], "surname": ["Marx", "Manger", "Blencowe", "Murray", "Ho", "Lawn"], "given-names": ["W", "SH", "M", "G", "FYY", "S"], "article-title": ["Clinical guidelines for the use of lifestyle-based mental health care in major depressive disorder: world Federation of Societies for biological psychiatry (WFSBP) and Australasian Society of Lifestyle Medicine (ASLM) taskforce"], "source": ["World J Biol Psychiat"], "year": ["2023"], "volume": ["24"], "issue": ["5"], "fpage": ["333"], "lpage": ["386"], "pub-id": ["10.1080/15622975.2022.2112074"]}, {"label": ["18."], "surname": ["Hoge", "Bui", "Mete", "Dutton", "Baker", "Simon"], "given-names": ["EA", "E", "M", "MA", "AW", "NM"], "article-title": ["Mindfulness-based stress reduction vs escitalopram for the treatment of adults with anxiety disorders: a randomized clinical trial"], "source": ["JAMA Psychiat"], "year": ["2023"], "volume": ["80"], "issue": ["1"], "fpage": ["13"], "pub-id": ["10.1001/jamapsychiatry.2022.3679"]}, {"label": ["26."], "surname": ["Afzal", "Siddiqi", "Ahmad", "Afsheen", "Aslam", "Ali"], "given-names": ["M", "N", "B", "N", "F", "A"], "article-title": ["Prevalence of overweight and obesity in people with severe mental illness: systematic review and meta-analysis"], "source": ["Front Endocrinol"], "year": ["2021"], "volume": ["12"], "fpage": ["769309"], "pub-id": ["10.3389/fendo.2021.769309"]}, {"label": ["31."], "surname": ["Meier", "Mattheisen", "Mors", "Schendel", "Mortensen", "Plessen"], "given-names": ["SM", "M", "O", "DE", "PB", "KJ"], "article-title": ["Mortality among persons with obsessive-compulsive disorder in Denmark"], "source": ["JAMA Psychiat"], "year": ["2016"], "volume": ["73"], "issue": ["3"], "fpage": ["268"], "pub-id": ["10.1001/jamapsychiatry.2015.3105"]}, {"label": ["35."], "surname": ["Proctor", "Silmere", "Raghavan", "Hovmand", "Aarons", "Bunger"], "given-names": ["E", "H", "R", "P", "G", "A"], "article-title": ["Outcomes for implementation research: conceptual distinctions, measurement challenges, and research agenda"], "source": ["Adm Policy Ment Health Ment Health Serv Res"], "year": ["2011"], "volume": ["38"], "issue": ["2"], "fpage": ["65"], "lpage": ["76"], "pub-id": ["10.1007/s10488-010-0319-7"]}, {"label": ["44."], "mixed-citation": ["Britt H, Miller GC, Henderson J, Bayram C, Harrison C, Valenti L, et al. General practice activity in Australia 2015\u201316. Sydney: Sydney University Press;\u00a02016. (General practice series)."]}, {"label": ["45."], "surname": ["Kleemann", "Bracht", "Stanton", "Schuch"], "given-names": ["E", "CG", "R", "FB"], "article-title": ["Exercise prescription for people with mental illness: an evaluation of mental health professionals\u2019 knowledge, beliefs, barriers, and behaviors"], "source": ["Braz J Psychiat"], "year": ["2020"], "volume": ["42"], "issue": ["3"], "fpage": ["271"], "lpage": ["277"], "pub-id": ["10.1590/1516-4446-2019-0547"]}, {"label": ["47."], "surname": ["Sniehotta", "Scholz", "Schwarzer"], "given-names": ["FF", "U", "R"], "article-title": ["Bridging the intention\u2013behaviour gap: planning, self-efficacy, and action control in the adoption and maintenance of physical exercise"], "source": ["Psychol Health"], "year": ["2005"], "volume": ["20"], "issue": ["2"], "fpage": ["143"], "lpage": ["160"], "pub-id": ["10.1080/08870440512331317670"]}, {"label": ["49."], "surname": ["French", "Olander", "Chisholm", "Mc Sharry"], "given-names": ["DP", "EK", "A", "J"], "article-title": ["Which behaviour change techniques are most effective at increasing older adults\u2019 self-efficacy and physical activity behaviour?"], "source": ["Syst Rev Ann Behav Med"], "year": ["2014"], "volume": ["48"], "issue": ["2"], "fpage": ["225"], "lpage": ["234"], "pub-id": ["10.1007/s12160-014-9593-z"]}, {"label": ["53."], "surname": ["McHugh", "Whitton", "Peckham", "Welge", "Otto"], "given-names": ["RK", "SW", "AD", "JA", "MW"], "article-title": ["Patient preference for psychological vs pharmacologic treatment of psychiatric disorders: a meta-analytic review"], "source": ["J Clin Psychiat"], "year": ["2013"], "volume": ["74"], "issue": ["06"], "fpage": ["595"], "lpage": ["602"], "pub-id": ["10.4088/JCP.12r07757"]}, {"label": ["58."], "surname": ["Velligan", "Sajatovic", "Hatch", "Kramata", "Docherty"], "given-names": ["DI", "M", "A", "P", "J"], "article-title": ["Why do psychiatric patients stop antipsychotic medication? A systematic review of reasons for nonadherence to medication in patients with serious mental illness"], "source": ["Patient Prefer Adher"], "year": ["2017"], "volume": ["11"], "fpage": ["449"], "lpage": ["468"], "pub-id": ["10.2147/PPA.S124658"]}]
{ "acronym": [], "definition": [] }
60
CC BY
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2024-01-14 23:43:47
BMC Public Health. 2024 Jan 13; 24:171
oa_package/39/8a/PMC10787508.tar.gz
PMC10787509
38216997
[ "<title>Introduction</title>", "<p id=\"Par27\">Global warming causes climate variation which is the essential factor in the spread of climate-sensitive infectious [##REF##36742229##1##]. A temperature rise enhances vector and pathogen metabolism allowing faster prevalence, whereas unpredictable rainfall patterns may excess the availability of favorable breeding sites [##REF##29500926##2##, ##REF##34227831##3##]. The combination of warm and moderate humidity provides typical conditions for several types of species <italic>Aedes</italic>, <italic>Culex</italic>, and <italic>Anopheles</italic> mosquitoes to survive transferring diseases like dengue, filariasis, yellow fever, and zika virus [##UREF##0##4##, ##UREF##1##5##]. For instance, lymphatic filariasis and West Nile fever are mainly vectored by <italic>Culex pipiens</italic>. Additionally, controlling mosquito vectors carries several dangers due to the vector resistance to conventional pesticides as well as their negative impact on the environment and human health [##UREF##2##6##, ##UREF##3##7##]. On the other side, microorganisms including bacteria, fungi, and protozoa pose a great danger to individuals, animals, and plants. World Health Organization (WHO) states that the rapid emergence of infectious diseases owing to infectious bacteria threatens all the world's countries causing millions of deaths annually [##UREF##4##8##]. Biofilm formation by pathogens in bacterial communities such as multicellular organisms show significant resistance to antibiotics leading to health and economic problems especially in poor countries because of the high therapy costs [##UREF##5##9##]. Taking these risks seriously, there is an urgent need to uncover economical, biocompatible, competitive, and effective alternative methods to replace traditional treatments.</p>", "<p id=\"Par28\">Nanomaterials have emerged as strong antimicrobial and larvicidal agents depending on their quantum size effect, large surface area to volume ratio, and high chemical reactivity. Metal and metal oxide nanoparticles (NPs) have been applied in various fields comprising medicine, drug delivery, energy, agricultural, catalysis, and biosensors [##REF##33430726##10##]. For example, zinc oxide (ZnO), magnesium oxide (MgO), tin oxide (SnO<sub>2</sub>), silver oxide (Ag<sub>2</sub>O), zirconium oxide (ZrO<sub>2</sub>) and copper oxide (CuO) have been utilized as highly efficient nanomaterials in health care sector [##REF##35517446##11##–##UREF##7##13##]. In particular, ZnO NPs have drawn much attention as an antimicrobial and anticancer agent for inhibiting the microorganism’s growth thanks to their semiconducting nature, safety, and unique physicochemical characteristics. Recent studies have reported the capability to modify the microstructure of nanomaterial by doping for enhancement of its physical and chemical properties. Integration of the ZnO matrix with different metal ions; Mn, Fe, Cr, Ag, Cu, Cd, Sn, Ga, etc., will create intermediate energy levels and hence increase free charge carriers’ mobility [##UREF##7##13##, ##UREF##8##14##]. Tuan Vu et al. have developed gold/zinc oxide (Au/ZnO) nanoflower as a strong photocatalyst and an antimicrobial agent for the degradation of Tartrazine (TA) and killing <italic>Escherichia coli</italic> (E. coli) [##UREF##9##15##]. Also, Abd El-Latef et al. have substituted Cu<sup>2+</sup> into ZnO matrix by hydrothermal assisted co-precipitation method for enhancement of insecticidal activity against <italic>Spodoptera littoralis</italic> [##UREF##10##16##]. Among various transition metals, chromium is a promising dopant owing to the close ionic radius of Cr<sup>3+</sup> (0.063 nm) to Zn<sup>2+</sup> (0.074 nm); besides, the electronegativity of Zn is similar to Cr 1.66. Moreover, chromium oxide (Cr<sub>2</sub>O<sub>3</sub>) is a p-type with an energy gap of 3.10 eV which is useful for increasing active sites and oxygen species [##UREF##11##17##, ##UREF##12##18##]. In this context, Shah et al. have prepared ZnO and chromium doped ZnO nanoparticles using leaf extract of <italic>Citrus reticulata</italic> indicating strong biological activities against <italic>E. coli</italic>, <italic>S. aureus</italic>, <italic>K. pneumonia</italic> and <italic>P. aeruginosa</italic> with zone of inhibition of 22.4 ± 1.98, 15.5 ± 0.78, 10.2 ± 0.55 and 7.5 ± 1.55 mm, respectively dependence of high generation of reactive oxygen species [##UREF##13##19##]. In medical applications, Cr doped ZnO has been applied as a therapy for estimating the cytotoxic effects using human fetal lung fibroblast cells (WI38 cells) based on the morphological nature of the nanoparticles [##REF##38016358##20##]. To date, several techniques are employed for fabricating nanoscale materials including hydrothermal, co-precipitation, chemical vapor deposition, spray pyrolysis, solution combustion, sol–gel, and magnetron sputtering. Among them, a simple, scalable, and controllable chemical co-precipitation technique was performed to obtain high quality nanoparticles [##REF##38016358##20##, ##UREF##14##21##]. The current work aims to modify the topological nature of ZnO NPs by Cr<sup>3+</sup> replacements to one dimensional nanocomb structure for biomedical applications.</p>" ]
[]
[ "<title>Results and discussion</title>", "<title>Structural analysis</title>", "<p id=\"Par36\">The XRD profile of Cr/ZnO nanostructures was illustrated in Fig. ##FIG##0##1##a. As can be seen, the prepared nanopowder of polycrystalline nature and well-defined peaks located at 31.85°, 34.49°, 36.30°, 47.72°, 56.79°, 62.98°, 66.59°, 68.17°, 69.24° corresponding to the reflection planes 100, 002, 101, 102, 110, 103, 200, 112, 201 ascribed to the hexagonal wurtzite structure and coincide with the standard card JCPDS No. 36–1451, space group (P63mc). The sample has a relatively intense peak however the observed broadening clearly reveals the small crystal size of the nanocomposite. No phases related to impurities or unreacted ions were observed in the pattern, otherwise the weak peak located at 2 54.25° is attributed to chromium oxide (Cr<sub>2</sub>O<sub>3</sub>). Despite the close ionic radius of Cr<sup>3+</sup> to Zn<sup>2+</sup>, the large amount of chromium ions concentrations (Cr<sup>3+</sup>, 5 wt.%) inside the host framework led to phase segregation [##UREF##17##25##–##UREF##19##27##]. Moreover, the crystallographic parameters including crystalline size and lattice strain were determined from the Williamson-Hall (W–H) plot expressed as:where, is the full width at half maximum (FWHM), is the Bragg’s diffraction angle, is the X-ray wavelength 1.5418 Å, and k is a constant 0.94. The internal strain was given from the slope and the crystallite size was defined from the intercept , Fig. ##FIG##0##1##b. Scherrer equation was also applied to evaluate the size of crystals by the following equation:</p>", "<p id=\"Par37\">Lattice strain , dislocation density and degree of crystallinity were estimated by the formulas:</p>", "<p id=\"Par38\">\n , and, respectively. As presented in Table ##TAB##0##1##, the nanocomposite shows a crystallite size range from 17–19 nm. In addition to, large strain and dislocation density associated with the presence of Cr contents and crystal defects that prevented the crystal growth result in crystal size reduction [##UREF##19##27##, ##UREF##20##28##].</p>", "<title>Composition and functional group identification</title>", "<p id=\"Par39\">The chemical composition and purity of Cr/ZnO NPs were investigated from EDX spectroscopy. The spectrum has detected the compositional peaks of Zn, O, Cr elements of weight percentages (wt.%) 62.16, 29.72, and 8.12 respectively. The presence of chromium peak in EDX spectrum reveals the incorporation of Cr<sup>3+</sup> into the host matrix, Fig. ##FIG##1##2##a [##UREF##21##29##, ##UREF##22##30##]. The absence of impurities related to unreacted ions emphasizes the high purity of the nanocomposite. The functional group and chemical bonding were defined from FTIR through wavenumber range from 400 to 3500 cm<sup>−1</sup> as depicted in Fig. ##FIG##1##2##b. The vibrational bands at 729.43 cm<sup>−1</sup> and 860.52 cm<sup>−1</sup> are attributed to the stretching mode the Zn O bond. Besides, the weak absorption bands at 438.40 cm<sup>−1</sup> is assigned to chromium oxide (Cr<sub>2</sub>O<sub>3</sub>). The two weak bands at 447.67 cm<sup>−1</sup> and 462.23 cm<sup>−1</sup> are associated with metal oxide Zn O Cr and Cr O bonds, respectively. In general, nanometal oxides exhibit vibrational frequencies at wavenumber less than 1000 cm<sup>−1</sup>. The observed weak IR bands at wavenumber varied between 1265 and 1470 cm<sup>−1</sup> are assigned to O-H stretching vibrations which arises from absorbing atmospheric moisture [##UREF##23##31##, ##UREF##24##32##]. The FTIR spectrum clearly approves that chromium ions have occupied zinc oxide framework as demonstrated in the XRD result.</p>", "<title>Morphological characteristics</title>", "<p id=\"Par40\">The surface morphology of the synthesized nanocomposite was visualized by SEM micrograph executed at different magnifications. As described in Fig. ##FIG##2##3##(a,b), the particles appear in nanocomb-like shapes, aggregated together with high density. The surface appears not uniform and has different shapes; some particles take thin flake shape [##UREF##25##33##]. Sharp needles linked to nanocomb were observed in the SEM image, Fig. ##FIG##2##3##b. These needles have a strong impact on killing microbes and mosquitoes. Further, average size and particles distribution were determined by TEM micrograph. Figure ##FIG##2##3##c, shows the particles in thin sheet, needle, and spherical shape with mean size 20 nm. Little needles are assembled in nanorods of diameter 6 nm, length 80 nm, and aspect ratio 13 [##UREF##26##34##, ##UREF##27##35##]. The microstructure analysis has approved that the nanocombs possess superior architecture topological features. Where, the dimensions confinement and small size enable them to be used as antimicrobial and larvicidal agent for human health protection.</p>", "<title>Optical analysis</title>", "<p id=\"Par41\">The optical characteristics of Cr/ZnO coated thin film were analyzed from transmittance (T%) and reflectance (R%) spectra through the wavelength varied between 300 800 nm. The thin film exhibited high optical transmittance greater than 82% in the visible region. Besides, a strong absorption edge was detected at 390 nm attributed to electron transition from valence band (V.B) to conduction band (C.B), Fig. ##FIG##3##4##a. The optical reflectance displayed peaks and valleys ascribed to electron-photon interaction or electron scattering, Fig. ##FIG##3##4##b [##UREF##28##36##, ##REF##36803540##37##]. The optical absorption coefficient ( was calculated from T% and R% using the following relation:</p>", "<p id=\"Par42\">Figure ##FIG##4##5##a illustrates the absorption coefficient as a function of photon energy . The thin film exhibits an optical absorption peak at 3.20 eV in which the electron absorbs incident photon energy and jumps to the conduction band leading to increased free charge carriers [##UREF##28##36##]. For further optical analysis, the optical band gap was determined from Tauc’s equation expressed as [##REF##36803540##37##]:</p>", "<p id=\"Par43\">Here, A is an independent constant, and n is constant value determines the electron transition behavior. here, n was taken by the value 0.5 refers to allowed direct transition. The of Cr doped ZnO was evaluated from versus plot by extending the straight part to zero photon energy axis equals 3.31 eV, Fig. ##FIG##4##5##b. The wide energy gap emphasizes the quantum size effect of the nanocomposite which is attributed to the large surface area and reactive surface sites [##REF##36803540##37##, ##UREF##29##38##]. Cr/ZnO nanocombs show a wide energy gap that is attributed to the small particle size. The substitution of Cr in the host lattice results in broadening of the energy gap, which also generates more free charge carriers according to Moss-Burstein effect. Both reactive oxygen species (ROS) in addition to metal ions production have a strong impact on harmful internal components comprising proteins and DNA causing bacterial and larvicidal death [##UREF##30##39##].</p>", "<title>Mosquito larvicidal activity</title>", "<p id=\"Par44\">Different concentrations of Cr/ZnO nanocomb were applied on the third larval instars of <italic>C. pipiens</italic> for 24 h to evaluate their efficacy. The findings refer to an increase in nanoparticle concentrations leading to high larval mortality. As presented in Table ##TAB##1##2##, the death rate was 100% at the ratio of 200 ppm and 35 ± 0.629% at 25 ppm. The LC<sub>50</sub> and LC<sub>90</sub> values were 30.15 and 100.22 ppm, respectively while the control did not show any toxicity to mosquito larvae and Chi-square (χ2) value shows a significant difference at p &lt; 0.01 level. The probability analysis of <italic>C. pipiens</italic> larval mortality utilizing the nanocomposites was shown in Fig. ##FIG##5##6##. The results of this work indicated the mortality of mosquito larvae caused by the internal toxic power of small particles inside the cuticle and the accumulation of nanoparticles in the alimentary canal particularly in the midgut which represents a defense region against the toxicity of insecticides. Also, comb and needle particles can penetrate the larval midgut, damaging DNA, overlapping with organelles, and interrupting physiological processes, which prevent mosquito larvae growth and cause death [##REF##29173489##40##, ##UREF##31##41##]. Related results were reported by Gunathilaka et al. and Ibrahim and Ali, ZnO NPs were effective against mosquito larvae caused mortality to the first larval instar of <italic>C. pipiens</italic> and star-shaped ZnO NPs were more effective against larvae than needle, round, and cubical-shapes [##REF##35423367##42##, ##UREF##32##43##]<italic>.</italic> Moreover, metal and metal oxide nanoparticles have strong toxicity and physiological impact on insect pests [##UREF##32##43##–##REF##36320778##45##].</p>", "<title>Light and SEM images</title>", "<p id=\"Par45\">The synthesized Cr/ZnO nanoparticles impacting <italic>C. pipiens</italic> larvae caused a change in their morphology features as indicated by histopathological studies after 48 h of treatment, as illustrated in (Figs. ##FIG##6##7##, ##FIG##7##8##). Light microscope images of treated larvae with fabricated samples showed the deformation in head and abdominal segments with an accumulation of nanoparticles in the alimentary canal (Fig. ##FIG##6##7##b, c) compared with the normal structure of the controlled one (Fig. ##FIG##6##7##a). Moreover, SEM microscopy images of the control larval instar observed normal morphological structures of the head and abdominal structure without any malformation (Fig. ##FIG##7##8##a, c), while (Fig. ##FIG##7##8##b, d) illustrated the destroyed head and mouth parts and deformation of the abdominal segments. This observation is similar to the works of literature [##UREF##34##46##, ##UREF##35##47##].</p>", "<title>Ultrastructural changes in the midgut of <italic>C. pipiens</italic> larvae by Cr/ZnO treatment</title>", "<p id=\"Par46\">Transmission electron micrographs of the midgut of control and LC<sub>50</sub> treated <italic>C. pipiens</italic> larvae illustrated how the particles interact with the larval body cells compared to the control larvae. The ultrastructure of the normal epithelial cell of <italic>C. pipiens</italic> larvae midgut has uniform microvilli, and the nucleus with scattered chromatin (Fig. ##FIG##8##9##a, b) was depicted previously by literature [##UREF##36##48##]. The cytoplasm of the control larvae showed regular structure and normal distribution of the cell organelles and normal nuclei are central and rounded as shown in (Fig. ##FIG##8##9##c, d). Conversely, Cr/ZnO nanocomb treated midgut epithelial cells display complete destruction and rupture near the basement membrane. There was a clear abnormal nuclei shape with heterochromatin and the aggregation of a high amount of needle-shape nanocomposites in the cytoplasm destroyed cell structure and entered the hemocoel resulting in insect death. (Fig. ##FIG##9##10##a, b, c). Moreover, the treated larvae showed several forms of vacuolation in the cytoplasm, and a clear condensation appeared in the nucleus and vacuolated nuclei (Fig. ##FIG##9##10##d, e, f). Similar changes have been reported by Ibrahim et al. about the abnormal appearance of the midgut epithelia as condensation of the nuclear chromatin, abnormal shape of nuclei, absence of microvilli, and appearance of numerous vacuoles of <italic>C. pipens</italic> immature stages exposed to ZnO NPs [##REF##37105619##49##]. The gut in insects has a key role in regulating different mechanisms as a vital organ in ecotoxicological investigations since the epithelial midgut is the first organ exposed to a large amount of toxins ingested by an insect [##REF##29560839##50##]. Additionally, the midgut represented a physical and chemical barrier to toxic substances ingested during feeding and ZnO NPs treated larvae showed that the microvilli of the midgut cells lining were very short compared to control as well as many vacuolations appeared in the cytoplasm and a clear condensation appeared in the nucleus [##UREF##37##51##]. Mohamed et al. reported that MnCoO nanostructures induce significant damage in the midgut structure of <italic>C. pipiens</italic> which showed the destruction of columnar epithelial cells causing the widening of intercellular spaces, and cytoplasmic vacuolization [##REF##36984973##52##]. The mode action of metal oxide nanoparticles in the larval midgut via its influence on digestive tract enzymes, DNA malformation, production of reactive oxygen species, causing cellular leakage, and disorder of the organelle functions causing larval death [##UREF##38##53##].</p>", "<title>Antimicrobial activity</title>", "<p id=\"Par47\">Antimicrobial results revealed that the Cr doped zinc oxide nanoparticles are more efficient against pathogenic bacteria at high concentrations. As shown in (Fig. ##FIG##10##11##, ##FIG##11##12##) and Table ##TAB##2##3##, <italic>Bacillus subtilis</italic> was the most sensitive among the tested microbes with 21.9 mm inhibition zone at 30 µg. Moreover, the inhibition zones of <italic>Staphylococcus aureus</italic> and <italic>Bacillus cereus</italic> were 16 and 16.93 mm, respectively at the high concentration<italic>. Escherichia coli</italic> and <italic>Proteus vulgaris</italic> have an inhibition zone of 17.93 and 19 mm, respectively while <italic>Pseudomonas aeruginosa</italic> showed non sensitivity at the same concentration. However, the control drug exhibited higher activity compared to Cr/ZnO nanocomb as demonstrated in the Table ##TAB##2##3##. From the results, the antibacterial activity of the nanoparticles was concentration dependent. The comb shape of Cr doped ZnO increased the penetration and harmed both gram-positive and gram-negative bacteria as the needles produced from it enhanced the potential activity of nanocomposite in destroying the pathogenic bacteria although most bacterial structures are resistant to major antibiotics. Besides that, Gram-negative bacteria have a great attraction towards positively charged nanoparticles, which result in cellular destruction [##UREF##39##54##]. In the present work, the antimicrobial activity of the nanocomb is efficacious against pathogenic microorganisms. These findings agree with earlier studies that demonstrated the antibacterial potential of Cr doped ZnO nanostructure against pathogenic gram- positive and gram-negative bacteria such as <italic>Escherichia coli</italic>, <italic>Staphylococcus aureus</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Bacillus cereus</italic> [##UREF##40##55##, ##REF##35514831##56##]. Also, Vijayalakshmi and Sivaraj investigated the antibacterial activity of Cr doped ZnO nanocomb results indicated enhanced bactericidal activities against <italic>E. coli</italic> and <italic>S. aureus</italic> compared to undoped ZnO–NPs [##UREF##41##57##]. The enhanced bactericidal activity of Cr-doped ZnO was associated with an increase in ROS production, and a reduction in particle size [##REF##33989295##58##, ##UREF##42##59##].</p>", "<p id=\"Par48\">Furthermore, the mechanisms of metal oxide nanomaterials as anti-bacterial agents show through interrupting the microbial cell directly or producing reactive oxygen species (ROS) that cause DNA damage, lipid peroxidation, and protein oxidation, also can destroy bacterial cells via the penetration and leakage of the bacterial membranes [##REF##37474922##60##–##UREF##44##62##]. These intracellular functional disorders are initiated by the oxidative stress manipulated by ROS leading to cell death as illustrated in Fig. ##FIG##12##13## according to Shah et al. [##UREF##39##54##, ##REF##37474922##60##]. These results coincided with Olejnik et al. (2021) who reported that the ZnO nanocomb recorded higher toxicity as compared with spherical particles underlining the significance of particle size and shape for cytotoxicity [##REF##33205376##63##]. Also, Babayevska et al. (2022) have shown that zinc oxide rods have the ability to penetrate through bacteria more easily than spheres [##REF##35581357##64##]. Moreover, the Cr doped ZnO provides a more effective anti-bacterial agent compared to the pristine ZnO [##UREF##41##57##].</p>" ]
[ "<title>Results and discussion</title>", "<title>Structural analysis</title>", "<p id=\"Par36\">The XRD profile of Cr/ZnO nanostructures was illustrated in Fig. ##FIG##0##1##a. As can be seen, the prepared nanopowder of polycrystalline nature and well-defined peaks located at 31.85°, 34.49°, 36.30°, 47.72°, 56.79°, 62.98°, 66.59°, 68.17°, 69.24° corresponding to the reflection planes 100, 002, 101, 102, 110, 103, 200, 112, 201 ascribed to the hexagonal wurtzite structure and coincide with the standard card JCPDS No. 36–1451, space group (P63mc). The sample has a relatively intense peak however the observed broadening clearly reveals the small crystal size of the nanocomposite. No phases related to impurities or unreacted ions were observed in the pattern, otherwise the weak peak located at 2 54.25° is attributed to chromium oxide (Cr<sub>2</sub>O<sub>3</sub>). Despite the close ionic radius of Cr<sup>3+</sup> to Zn<sup>2+</sup>, the large amount of chromium ions concentrations (Cr<sup>3+</sup>, 5 wt.%) inside the host framework led to phase segregation [##UREF##17##25##–##UREF##19##27##]. Moreover, the crystallographic parameters including crystalline size and lattice strain were determined from the Williamson-Hall (W–H) plot expressed as:where, is the full width at half maximum (FWHM), is the Bragg’s diffraction angle, is the X-ray wavelength 1.5418 Å, and k is a constant 0.94. The internal strain was given from the slope and the crystallite size was defined from the intercept , Fig. ##FIG##0##1##b. Scherrer equation was also applied to evaluate the size of crystals by the following equation:</p>", "<p id=\"Par37\">Lattice strain , dislocation density and degree of crystallinity were estimated by the formulas:</p>", "<p id=\"Par38\">\n , and, respectively. As presented in Table ##TAB##0##1##, the nanocomposite shows a crystallite size range from 17–19 nm. In addition to, large strain and dislocation density associated with the presence of Cr contents and crystal defects that prevented the crystal growth result in crystal size reduction [##UREF##19##27##, ##UREF##20##28##].</p>", "<title>Composition and functional group identification</title>", "<p id=\"Par39\">The chemical composition and purity of Cr/ZnO NPs were investigated from EDX spectroscopy. The spectrum has detected the compositional peaks of Zn, O, Cr elements of weight percentages (wt.%) 62.16, 29.72, and 8.12 respectively. The presence of chromium peak in EDX spectrum reveals the incorporation of Cr<sup>3+</sup> into the host matrix, Fig. ##FIG##1##2##a [##UREF##21##29##, ##UREF##22##30##]. The absence of impurities related to unreacted ions emphasizes the high purity of the nanocomposite. The functional group and chemical bonding were defined from FTIR through wavenumber range from 400 to 3500 cm<sup>−1</sup> as depicted in Fig. ##FIG##1##2##b. The vibrational bands at 729.43 cm<sup>−1</sup> and 860.52 cm<sup>−1</sup> are attributed to the stretching mode the Zn O bond. Besides, the weak absorption bands at 438.40 cm<sup>−1</sup> is assigned to chromium oxide (Cr<sub>2</sub>O<sub>3</sub>). The two weak bands at 447.67 cm<sup>−1</sup> and 462.23 cm<sup>−1</sup> are associated with metal oxide Zn O Cr and Cr O bonds, respectively. In general, nanometal oxides exhibit vibrational frequencies at wavenumber less than 1000 cm<sup>−1</sup>. The observed weak IR bands at wavenumber varied between 1265 and 1470 cm<sup>−1</sup> are assigned to O-H stretching vibrations which arises from absorbing atmospheric moisture [##UREF##23##31##, ##UREF##24##32##]. The FTIR spectrum clearly approves that chromium ions have occupied zinc oxide framework as demonstrated in the XRD result.</p>", "<title>Morphological characteristics</title>", "<p id=\"Par40\">The surface morphology of the synthesized nanocomposite was visualized by SEM micrograph executed at different magnifications. As described in Fig. ##FIG##2##3##(a,b), the particles appear in nanocomb-like shapes, aggregated together with high density. The surface appears not uniform and has different shapes; some particles take thin flake shape [##UREF##25##33##]. Sharp needles linked to nanocomb were observed in the SEM image, Fig. ##FIG##2##3##b. These needles have a strong impact on killing microbes and mosquitoes. Further, average size and particles distribution were determined by TEM micrograph. Figure ##FIG##2##3##c, shows the particles in thin sheet, needle, and spherical shape with mean size 20 nm. Little needles are assembled in nanorods of diameter 6 nm, length 80 nm, and aspect ratio 13 [##UREF##26##34##, ##UREF##27##35##]. The microstructure analysis has approved that the nanocombs possess superior architecture topological features. Where, the dimensions confinement and small size enable them to be used as antimicrobial and larvicidal agent for human health protection.</p>", "<title>Optical analysis</title>", "<p id=\"Par41\">The optical characteristics of Cr/ZnO coated thin film were analyzed from transmittance (T%) and reflectance (R%) spectra through the wavelength varied between 300 800 nm. The thin film exhibited high optical transmittance greater than 82% in the visible region. Besides, a strong absorption edge was detected at 390 nm attributed to electron transition from valence band (V.B) to conduction band (C.B), Fig. ##FIG##3##4##a. The optical reflectance displayed peaks and valleys ascribed to electron-photon interaction or electron scattering, Fig. ##FIG##3##4##b [##UREF##28##36##, ##REF##36803540##37##]. The optical absorption coefficient ( was calculated from T% and R% using the following relation:</p>", "<p id=\"Par42\">Figure ##FIG##4##5##a illustrates the absorption coefficient as a function of photon energy . The thin film exhibits an optical absorption peak at 3.20 eV in which the electron absorbs incident photon energy and jumps to the conduction band leading to increased free charge carriers [##UREF##28##36##]. For further optical analysis, the optical band gap was determined from Tauc’s equation expressed as [##REF##36803540##37##]:</p>", "<p id=\"Par43\">Here, A is an independent constant, and n is constant value determines the electron transition behavior. here, n was taken by the value 0.5 refers to allowed direct transition. The of Cr doped ZnO was evaluated from versus plot by extending the straight part to zero photon energy axis equals 3.31 eV, Fig. ##FIG##4##5##b. The wide energy gap emphasizes the quantum size effect of the nanocomposite which is attributed to the large surface area and reactive surface sites [##REF##36803540##37##, ##UREF##29##38##]. Cr/ZnO nanocombs show a wide energy gap that is attributed to the small particle size. The substitution of Cr in the host lattice results in broadening of the energy gap, which also generates more free charge carriers according to Moss-Burstein effect. Both reactive oxygen species (ROS) in addition to metal ions production have a strong impact on harmful internal components comprising proteins and DNA causing bacterial and larvicidal death [##UREF##30##39##].</p>", "<title>Mosquito larvicidal activity</title>", "<p id=\"Par44\">Different concentrations of Cr/ZnO nanocomb were applied on the third larval instars of <italic>C. pipiens</italic> for 24 h to evaluate their efficacy. The findings refer to an increase in nanoparticle concentrations leading to high larval mortality. As presented in Table ##TAB##1##2##, the death rate was 100% at the ratio of 200 ppm and 35 ± 0.629% at 25 ppm. The LC<sub>50</sub> and LC<sub>90</sub> values were 30.15 and 100.22 ppm, respectively while the control did not show any toxicity to mosquito larvae and Chi-square (χ2) value shows a significant difference at p &lt; 0.01 level. The probability analysis of <italic>C. pipiens</italic> larval mortality utilizing the nanocomposites was shown in Fig. ##FIG##5##6##. The results of this work indicated the mortality of mosquito larvae caused by the internal toxic power of small particles inside the cuticle and the accumulation of nanoparticles in the alimentary canal particularly in the midgut which represents a defense region against the toxicity of insecticides. Also, comb and needle particles can penetrate the larval midgut, damaging DNA, overlapping with organelles, and interrupting physiological processes, which prevent mosquito larvae growth and cause death [##REF##29173489##40##, ##UREF##31##41##]. Related results were reported by Gunathilaka et al. and Ibrahim and Ali, ZnO NPs were effective against mosquito larvae caused mortality to the first larval instar of <italic>C. pipiens</italic> and star-shaped ZnO NPs were more effective against larvae than needle, round, and cubical-shapes [##REF##35423367##42##, ##UREF##32##43##]<italic>.</italic> Moreover, metal and metal oxide nanoparticles have strong toxicity and physiological impact on insect pests [##UREF##32##43##–##REF##36320778##45##].</p>", "<title>Light and SEM images</title>", "<p id=\"Par45\">The synthesized Cr/ZnO nanoparticles impacting <italic>C. pipiens</italic> larvae caused a change in their morphology features as indicated by histopathological studies after 48 h of treatment, as illustrated in (Figs. ##FIG##6##7##, ##FIG##7##8##). Light microscope images of treated larvae with fabricated samples showed the deformation in head and abdominal segments with an accumulation of nanoparticles in the alimentary canal (Fig. ##FIG##6##7##b, c) compared with the normal structure of the controlled one (Fig. ##FIG##6##7##a). Moreover, SEM microscopy images of the control larval instar observed normal morphological structures of the head and abdominal structure without any malformation (Fig. ##FIG##7##8##a, c), while (Fig. ##FIG##7##8##b, d) illustrated the destroyed head and mouth parts and deformation of the abdominal segments. This observation is similar to the works of literature [##UREF##34##46##, ##UREF##35##47##].</p>", "<title>Ultrastructural changes in the midgut of <italic>C. pipiens</italic> larvae by Cr/ZnO treatment</title>", "<p id=\"Par46\">Transmission electron micrographs of the midgut of control and LC<sub>50</sub> treated <italic>C. pipiens</italic> larvae illustrated how the particles interact with the larval body cells compared to the control larvae. The ultrastructure of the normal epithelial cell of <italic>C. pipiens</italic> larvae midgut has uniform microvilli, and the nucleus with scattered chromatin (Fig. ##FIG##8##9##a, b) was depicted previously by literature [##UREF##36##48##]. The cytoplasm of the control larvae showed regular structure and normal distribution of the cell organelles and normal nuclei are central and rounded as shown in (Fig. ##FIG##8##9##c, d). Conversely, Cr/ZnO nanocomb treated midgut epithelial cells display complete destruction and rupture near the basement membrane. There was a clear abnormal nuclei shape with heterochromatin and the aggregation of a high amount of needle-shape nanocomposites in the cytoplasm destroyed cell structure and entered the hemocoel resulting in insect death. (Fig. ##FIG##9##10##a, b, c). Moreover, the treated larvae showed several forms of vacuolation in the cytoplasm, and a clear condensation appeared in the nucleus and vacuolated nuclei (Fig. ##FIG##9##10##d, e, f). Similar changes have been reported by Ibrahim et al. about the abnormal appearance of the midgut epithelia as condensation of the nuclear chromatin, abnormal shape of nuclei, absence of microvilli, and appearance of numerous vacuoles of <italic>C. pipens</italic> immature stages exposed to ZnO NPs [##REF##37105619##49##]. The gut in insects has a key role in regulating different mechanisms as a vital organ in ecotoxicological investigations since the epithelial midgut is the first organ exposed to a large amount of toxins ingested by an insect [##REF##29560839##50##]. Additionally, the midgut represented a physical and chemical barrier to toxic substances ingested during feeding and ZnO NPs treated larvae showed that the microvilli of the midgut cells lining were very short compared to control as well as many vacuolations appeared in the cytoplasm and a clear condensation appeared in the nucleus [##UREF##37##51##]. Mohamed et al. reported that MnCoO nanostructures induce significant damage in the midgut structure of <italic>C. pipiens</italic> which showed the destruction of columnar epithelial cells causing the widening of intercellular spaces, and cytoplasmic vacuolization [##REF##36984973##52##]. The mode action of metal oxide nanoparticles in the larval midgut via its influence on digestive tract enzymes, DNA malformation, production of reactive oxygen species, causing cellular leakage, and disorder of the organelle functions causing larval death [##UREF##38##53##].</p>", "<title>Antimicrobial activity</title>", "<p id=\"Par47\">Antimicrobial results revealed that the Cr doped zinc oxide nanoparticles are more efficient against pathogenic bacteria at high concentrations. As shown in (Fig. ##FIG##10##11##, ##FIG##11##12##) and Table ##TAB##2##3##, <italic>Bacillus subtilis</italic> was the most sensitive among the tested microbes with 21.9 mm inhibition zone at 30 µg. Moreover, the inhibition zones of <italic>Staphylococcus aureus</italic> and <italic>Bacillus cereus</italic> were 16 and 16.93 mm, respectively at the high concentration<italic>. Escherichia coli</italic> and <italic>Proteus vulgaris</italic> have an inhibition zone of 17.93 and 19 mm, respectively while <italic>Pseudomonas aeruginosa</italic> showed non sensitivity at the same concentration. However, the control drug exhibited higher activity compared to Cr/ZnO nanocomb as demonstrated in the Table ##TAB##2##3##. From the results, the antibacterial activity of the nanoparticles was concentration dependent. The comb shape of Cr doped ZnO increased the penetration and harmed both gram-positive and gram-negative bacteria as the needles produced from it enhanced the potential activity of nanocomposite in destroying the pathogenic bacteria although most bacterial structures are resistant to major antibiotics. Besides that, Gram-negative bacteria have a great attraction towards positively charged nanoparticles, which result in cellular destruction [##UREF##39##54##]. In the present work, the antimicrobial activity of the nanocomb is efficacious against pathogenic microorganisms. These findings agree with earlier studies that demonstrated the antibacterial potential of Cr doped ZnO nanostructure against pathogenic gram- positive and gram-negative bacteria such as <italic>Escherichia coli</italic>, <italic>Staphylococcus aureus</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Bacillus cereus</italic> [##UREF##40##55##, ##REF##35514831##56##]. Also, Vijayalakshmi and Sivaraj investigated the antibacterial activity of Cr doped ZnO nanocomb results indicated enhanced bactericidal activities against <italic>E. coli</italic> and <italic>S. aureus</italic> compared to undoped ZnO–NPs [##UREF##41##57##]. The enhanced bactericidal activity of Cr-doped ZnO was associated with an increase in ROS production, and a reduction in particle size [##REF##33989295##58##, ##UREF##42##59##].</p>", "<p id=\"Par48\">Furthermore, the mechanisms of metal oxide nanomaterials as anti-bacterial agents show through interrupting the microbial cell directly or producing reactive oxygen species (ROS) that cause DNA damage, lipid peroxidation, and protein oxidation, also can destroy bacterial cells via the penetration and leakage of the bacterial membranes [##REF##37474922##60##–##UREF##44##62##]. These intracellular functional disorders are initiated by the oxidative stress manipulated by ROS leading to cell death as illustrated in Fig. ##FIG##12##13## according to Shah et al. [##UREF##39##54##, ##REF##37474922##60##]. These results coincided with Olejnik et al. (2021) who reported that the ZnO nanocomb recorded higher toxicity as compared with spherical particles underlining the significance of particle size and shape for cytotoxicity [##REF##33205376##63##]. Also, Babayevska et al. (2022) have shown that zinc oxide rods have the ability to penetrate through bacteria more easily than spheres [##REF##35581357##64##]. Moreover, the Cr doped ZnO provides a more effective anti-bacterial agent compared to the pristine ZnO [##UREF##41##57##].</p>" ]
[ "<title>Conclusions</title>", "<p id=\"Par49\">Cr doped ZnO nanocomposite was successfully synthesized by simple co-precipitation method. The prepared sample was characterized using various spectroscopic techniques suggesting small crystallite size, large internal strain, and dislocation density of . Integration of Cr<sup>3+</sup> inside the host ZnO matrix was confirmed using EDX and FTIR spectra. The SEM images revealed the nanocomb structure of Cr/ZnO with aspect ratio 13 and large surface area. Moreover, the TEM image showed the particles in nanoflakes linked with sharp needles. The morphological analysis emphasized the special shape and structure of the nanocomposite which can enable it to be used as an efficient larvicidal and anti-bacterial agent. The fabricated thin film exhibited a wide band gap of 3.31 eV close to the visible region. In larvicidal study, the nanoparticles result in 100% mortality at concentration 200 ppm beside that the treated larvae showed destruction in midgut epithelium cells and malformation of their organelles. In antimicrobial activity, the nanocomb strongly caused DNA damage to bacterial cells and cell membrane leakage which results in cell death. In conclusion, the synthesized Cr/ZnO nanocomposite exhibited a significant impact on eradicating microbes and mosquitoes anchored to their superior structural-morphological and optical characteristics.</p>" ]
[ "<p id=\"Par1\">Zinc chromium oxide (Cr/ZnO, 5wt.%) was prepared by a facile chemical co-precipitation route. The structure, composition, and chemical bonding were analyzed using X-ray diffraction (XRD), Energy dispersive X-ray spectroscopy (EDX), and Fourier-transform infrared spectroscopy (FTIR) indicating that chromium ions were integrated the host framework to form Cr/ZnO nanocomposite. Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) micrographs showed comb-shaped nanoparticles with an average size 20 nm and large surface area. The energy gap of the thin films was estimated from T% and R% measurements which exhibit a strong optical absorption edge close to the visible spectrum. The insecticidal activity of the synthesized nanocombs against <italic>C. pipiens</italic> larvae was evaluated with LC<sub>50</sub> (30.15 ppm) and LC<sub>90</sub> (100.22 ppm). Besides, the nanocomposite showed high antibacterial performance against gram-positive bacteria (<italic>Bacillus subtilis</italic>) and gram-negative bacteria (<italic>Proteus vulgaris</italic>) with inhibition zones 21.9 and 19 mm, respectively.</p>", "<title>Keywords</title>", "<p>Open access funding provided by The Science, Technology &amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).</p>" ]
[ "<title>Experimental section</title>", "<title>Chemicals and reagents</title>", "<p id=\"Par29\">Zinc chloride dihydrate, ZnCl<sub>2</sub>.2H<sub>2</sub>O, 98.0%, Sodium hydroxide (NaOH ≥ 98.0%), Chromium nitrate nonahydrate Cr(NO<sub>3</sub>)0.9H<sub>2</sub>O, ≥ 97.0%), Cacodylate buffer, Glutaraldehyde, Osmium tetroxide (OsO<sub>4</sub>), Ethanol, and Acetone were purchased from Alfa Aesar, All reagents were used without any purification,</p>", "<title>Preparation and characterization of zinc chromium oxide nanocomposite</title>", "<p id=\"Par30\">Zn<sub>x</sub>Cr<sub>1-x</sub>O, x 5 wt% was prepared by chemical co-precipitation approach. Firstly, 8.35 g zinc chloride dihydrate was dissolved in 50 ml double distilled water and 1.28 g chromium nitrate nanohydrate in 25 ml under a magnetic stirrer. Chromium salt solution was carefully added to zinc chloride with continuous stirring for 3h at room temperature. 2.92 g sodium hydroxide (NaOH) was dissolved in 65 ml double distilled water and then slowly added drop by drop to aqueous solutions under stirring at a constant speed for 4h without heating. A homogeneous white gray precipitate was formed at pH ~ 9. Then separated using filter paper and washed many times with distilled water. The resulting powder was dried in a furnace at 70 °C overnight and eventually annealed at 400 °C for 2h.</p>", "<title>Characterization of the nanomaterial</title>", "<p id=\"Par31\">The phase and crystallographic features of Cr/ZnO nanocomposite were recognized by XRD (Bruker D8 Advance Eco) operated at λ = 1.54 Å(Cu Kα radiation) at 2θ angle varied from 25° to 75°. Morphological nature and elemental composition were recorded using scanning electron micrograph (SEM; Helios Nanolab. 400) attached with energy dispersive X-ray analysis (EDX). The functional group was identified by Fourier transform infrared (FT-IR spectrum 100, Perkin Elmer) at a resolution of 400–2000 cm<sup>−1</sup> with 16 scan speed. Transmission electron microscopy (TEM; Model: JEOL JEM-2100) was carried out to visualize the average size and particle’s shape. A spin coater sputter coater (Model, EMS 150T ES) was utilized to fabricate the thin films. Transmittance (T%) and reflectance (R%) spectra were measured via UV–Vis spectrophotometer JASCO (V-570) to investigate the energy gap of the fabricated thin films.</p>", "<title>Mosquito rearing</title>", "<p id=\"Par32\">The egg rafts of <italic>C. pipiens</italic> were kept and hatched at a controlled temperature of 27 30° and 80% humidity. The larvae fed on fish food and grew in plastic trays that measured 30 25 5 cm. As for the adult mosquitoes, they were kept in cages that measured 30 30 30 cm and fed with a solution that contained 10% glucose.</p>", "<title>Larvicidal activity</title>", "<p id=\"Par33\">The insecticidal activity was investigated by placing 25 mosquito larvae into 200 mL of sterilized double–distilled water with Cr/ZnO at concentrations (25, 50, 100, 150, and 200 ppm) in a 250 mL beaker. Pure water was applied as a control for each individual concentration. The dead larvae were recorded after 24h of treatment, and the death percentage was calculated from the average of replicates [##UREF##15##22##]. Further, this formula was used to determine the fatal rates.</p>", "<title>Ultrastructure examination</title>", "<p id=\"Par34\">The midgut ultra-structural alteration of <italic>C. pipiens</italic> larvae was investigated after exposure to Cr/ZnO NPs according to El-Samad et al. with a little modification [##REF##36087814##23##] and the tissues were examined with a JEOL 1200 EX II transmission electron microscope at the central laboratory, Faculty of Science, Zagazig University.</p>", "<title>Antibacterial activity</title>", "<p id=\"Par35\">The antibacterial performance of the prepared specimen was investigated by the disc diffusion method against pathogenic bacteria such as <italic>Staphylococcus aureus</italic>, <italic>Bacillus subtilis</italic> and <italic>Bacillus cereus</italic> (gram-positive), and <italic>Escherichia coli</italic>, <italic>Proteus vulgaris</italic> and <italic>Pseudomonas aeruginosa</italic> (gram-negative). Whatman No.1 filter paper discs of 6 mm dia. were immersed with 10 and 30 μg of Cr/ZnO NPs synthesized nanocomposites for each strain and determined the zones of inhibition around the disks after incubating the plates at 37 °C for 24 h [##UREF##16##24##]. Each examination was repeated three times, then the data were analyzed using statistical methods and using Gentamycin (4 µg/ml) as a positive control of bacteria. All findings were statistically calculated as the mean ± standard error (SE) of the average values. The chi-square method was utilized to show the significant difference, the obtained data was compared with the control group using regression co-efficient. Also, Probit transformation analysis was applied to determine the LC<sub>50</sub> and LC<sub>90</sub> values.</p>" ]
[ "<title>Author contributions</title>", "<p>EE raising the idea, nanomaterials synthesis and physical characterization, and writing the original draft. WAM supervised the biological tests. SAE performed the biological tests and wrote the manuscript. KDO and MK revised the manuscript and supervised the research. EE and WAM reviewed the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>Open access funding is provided by The Science, Technology &amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This work was supported by funds from Zagazig University, Egypt.</p>", "<title>Availability of data and materials</title>", "<p>The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par50\">Not applicable.</p>", "<title>Consent for publication</title>", "<p id=\"Par51\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par52\">The authors declare that they have no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p><bold>a</bold> XRD pattern and <bold>b</bold> W–H plot of Cr/ZnO nanocomposite</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p><bold>a</bold> EDX spectrum and <bold>b</bold> FTIR of Cr/ZnO nanocomposite</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p><bold>a, b</bold> SEM micrograph and <bold>c</bold> TEM micrograph of Cr/ZnO nanocomb</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p><bold>a</bold> Optical transmittance and <bold>b</bold> Reflectance of Cr/ZnO thin film</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p><bold>a</bold> Optical absorption and <bold>b</bold> Energy gap of Cr/ZnO thin film</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>The probability analysis of mortality of <italic>C. pipiens</italic> mosquito larvae exposed to Cr/ZnO nanocomb</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig.7</label><caption><p>Light microscope photographs of body parts (head, abdomen, and respiratory opening) of the 3rd larval instars of <italic>Cx. pipiens</italic> control <bold>a</bold> and larvae treated with Cr/ZnO NPs <bold>b</bold>, <bold>c</bold> (10 × 40) (Abd: abdomen; G: gills; H: head: Siph: siphon: Th; thorax)</p></caption></fig>", "<fig id=\"Fig8\"><label>Fig. 8</label><caption><p>The SEM micrographs of third larval instar of control <italic>C. pipiens</italic> larvae <bold>a</bold>, <bold>c</bold> showed normal head capsule, mouth parts and abdominal segments, <bold>b</bold>, <bold>d</bold> larvae treated by Cr/ZnO NPs at 48 h post treatment showed deformed head capsule, mouth parts and abdominal segments (<italic>Abd</italic> abdomen, <italic>dAbd</italic> deformed abdominal segments, <italic>H</italic> head: d H: deformed head: Mp; mouthparts)</p></caption></fig>", "<fig id=\"Fig9\"><label>Fig. 9</label><caption><p>Transmission electron micrographs showing the ultrastructural in the midgut of the normal third larval instar of <italic>C. pipiens</italic> mosquito <bold>a</bold>, <bold>b</bold> The normal structure of the epithelium showing the nucleus having scattered chromatin. <bold>c</bold>, <bold>d</bold> the gut cytoplasm has mitochondria and microvilli. Cy(cytoplasm), M (mitochondria), N (nucleus), Ne (nuclear envelope), and MV (microvilli)</p></caption></fig>", "<fig id=\"Fig10\"><label>Fig. 10</label><caption><p>Transmission electron micrographs showing the ultrastructural changes in the midgut of the third larval instar of <italic>C. pipiens</italic> mosquito in response to LC<sub>50</sub> treatment with Cr/ZnO NPs <bold>a</bold>, <bold>b</bold> An epithelial cell in treated larvae showing missed microvilli and clear condensation of the nucleus with clear vacuolation in the cytoplasm near the basement membrane. <bold>c</bold>, <bold>d</bold> the cells have vacuolated nuclei and nucleus with heterochromatin. <bold>e</bold>, <bold>f</bold> The epithelium in the treated larva vacuolated and have abnormal nucleus shapes. Cy (cytoplasm), dMV (microvilli), HC (heterochromatin), M (mitochondria), Ab N (abnormal nucleus) and VN (vacuole nucleus)</p></caption></fig>", "<fig id=\"Fig11\"><label>Fig. 11</label><caption><p>The inhibition zones indicating the antimicrobial activity of Cr/ZnO against gram positive and gram-negative bacteria</p></caption></fig>", "<fig id=\"Fig12\"><label>Fig. 12</label><caption><p>A chart depicting zones of inhibition of Cr/ZnO NPs against the test bacteria</p></caption></fig>", "<fig id=\"Fig13\"><label>Fig. 13</label><caption><p>Antibacterial activity mechanism of Cr/ZnO NPs</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Crystallographic parameters of Cr doped ZnO nanocomb obtained from W–H plot and Scherrer formula</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"7\">Crystallographic parameters</th></tr><tr><th align=\"left\" colspan=\"3\">W–H plot</th><th align=\"left\" colspan=\"4\">Scherrer equation</th></tr></thead><tbody><tr><td align=\"left\">Nanocomb</td><td align=\"left\">(nm)</td><td align=\"left\"></td><td align=\"left\">(nm)</td><td align=\"left\"></td><td align=\"left\"></td><td align=\"left\"></td></tr><tr><td align=\"left\">Cr/ZnO</td><td align=\"left\">18.97</td><td align=\"left\">7.84</td><td align=\"left\">16.74</td><td align=\"left\">69.00</td><td align=\"left\">35.70</td><td align=\"left\">26.73</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Toxic effect of nanostructure Cr/ZnO against third larval instar of <italic>C. pipiens</italic></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Concentrations (ppm)</th><th align=\"left\" rowspan=\"2\">Mean ± S.E</th><th align=\"left\">LC<sub>50</sub></th><th align=\"left\">LC<sub>90</sub></th><th align=\"left\" rowspan=\"2\">Regression equation</th><th align=\"left\" rowspan=\"2\">X<sup>2</sup></th></tr><tr><th align=\"left\">(LCL)</th><th align=\"left\">(UCL)</th></tr></thead><tbody><tr><td align=\"left\">25</td><td char=\"±\" align=\"char\">35 ± 0.629<sup>a</sup></td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">50</td><td char=\"±\" align=\"char\">73.75 ± 0.47<sup>ab</sup></td><td align=\"left\">30.15</td><td align=\"left\">100.22</td><td char=\".\" align=\"char\">Y = -4.07 + (X × 1.19)</td><td char=\".\" align=\"char\">9.7</td></tr><tr><td align=\"left\">100</td><td char=\"±\" align=\"char\">80 ± 0.64<sup>b</sup></td><td align=\"left\">(24.12: 35.71)</td><td align=\"left\">(82.55:131.66)</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">150</td><td char=\"±\" align=\"char\">92.5 ± 0.288<sup>c</sup></td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">200</td><td char=\"±\" align=\"char\">100 ± 0.00<sup>d</sup></td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Antibacterial activity of Cr/ZnO nanocomb</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Inhibition zone diameter in mm</th><th align=\"left\" colspan=\"2\">Cr/ZnO (Mean ± SE)</th><th align=\"left\" rowspan=\"2\">Positive control</th></tr><tr><th align=\"left\">Tested bacteria</th><th align=\"left\">10 µg</th><th align=\"left\">30 µg</th></tr></thead><tbody><tr><td align=\"left\">Gram positive bacteria</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">Gentamycin</td></tr><tr><td align=\"left\"><italic> Staphylococcus aureus</italic></td><td align=\"left\">Non sensitive</td><td align=\"left\">16 ± 0.45</td><td align=\"left\">24 ± 1.07</td></tr><tr><td align=\"left\"><italic> Bacillus subtilis</italic></td><td align=\"left\">18.13 ± 0.41</td><td align=\"left\">21.9 ± 0.85</td><td align=\"left\">26 ± 0.64</td></tr><tr><td align=\"left\"><italic> Bacillus cereus</italic></td><td align=\"left\">11 ± 1.oo</td><td align=\"left\">16.93 ± 0.60</td><td align=\"left\">25 ± 1.04</td></tr><tr><td align=\"left\" colspan=\"4\">Gram negative bacteria</td></tr><tr><td align=\"left\"><italic> Escherichia coli</italic></td><td align=\"left\">16.96 ± 0.25</td><td align=\"left\">17.93 ± 0.60</td><td align=\"left\">30 ± 0.20</td></tr><tr><td align=\"left\"><italic> Proteus vulgaris</italic></td><td align=\"left\">15.96 ± 0.45</td><td align=\"left\">19 ± 1.00</td><td align=\"left\">25 ± 1.09</td></tr><tr><td align=\"left\"><italic> Pseudomonas aeruginosa</italic></td><td align=\"left\">Non sensitive</td><td align=\"left\">Non sensitive</td><td align=\"left\">27 ± 0.86</td></tr></tbody></table></table-wrap>" ]
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\n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\uptheta $$\\end{document}</tex-math><mml:math id=\"M28\"><mml:mi mathvariant=\"normal\">θ</mml:mi></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\uplambda $$\\end{document}</tex-math><mml:math id=\"M30\"><mml:mi mathvariant=\"normal\">λ</mml:mi></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$=$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:mo>=</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M33\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$=$$\\end{document}</tex-math><mml:math id=\"M34\"><mml:mo>=</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M35\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$=\\frac{\\mathrm{k\\lambda }}{{\\text{D}}}$$\\end{document}</tex-math><mml:math id=\"M36\"><mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">k</mml:mi><mml:mi>λ</mml:mi></mml:mrow><mml:mtext>D</mml:mtext></mml:mfrac></mml:mrow></mml:math></alternatives></inline-formula>", "<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M37\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{D}}=\\frac{\\mathrm{K\\lambda }}{\\mathrm{\\beta cos\\theta }}$$\\end{document}</tex-math><mml:math id=\"M38\" display=\"block\"><mml:mrow><mml:mtext>D</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">K</mml:mi><mml:mi>λ</mml:mi></mml:mrow><mml:mrow><mml:mi>β</mml:mi><mml:mi mathvariant=\"normal\">cos</mml:mi><mml:mi>θ</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></disp-formula>", "<inline-formula id=\"IEq17\"><alternatives><tex-math id=\"M39\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$(\\upvarepsilon )$$\\end{document}</tex-math><mml:math id=\"M40\"><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi mathvariant=\"normal\">ε</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq18\"><alternatives><tex-math id=\"M41\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$(\\updelta )$$\\end{document}</tex-math><mml:math id=\"M42\"><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi mathvariant=\"normal\">δ</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq19\"><alternatives><tex-math id=\"M43\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\left({{\\text{X}}}_{{\\text{c}}}\\right)$$\\end{document}</tex-math><mml:math id=\"M44\"><mml:mfenced close=\")\" open=\"(\"><mml:msub><mml:mtext>X</mml:mtext><mml:mtext>c</mml:mtext></mml:msub></mml:mfenced></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq20\"><alternatives><tex-math id=\"M45\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\upvarepsilon =\\frac{\\upbeta }{4\\mathrm{ tan\\theta }}$$\\end{document}</tex-math><mml:math id=\"M46\"><mml:mrow><mml:mi mathvariant=\"normal\">ε</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mi mathvariant=\"normal\">β</mml:mi><mml:mrow><mml:mn>4</mml:mn><mml:mrow><mml:mi mathvariant=\"normal\">tan</mml:mi><mml:mi>θ</mml:mi></mml:mrow></mml:mrow></mml:mfrac></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq21\"><alternatives><tex-math id=\"M47\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\updelta =\\frac{1}{{{\\text{D}}}^{2}}$$\\end{document}</tex-math><mml:math id=\"M48\"><mml:mrow><mml:mi mathvariant=\"normal\">δ</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:msup><mml:mrow><mml:mtext>D</mml:mtext></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mfrac></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq22\"><alternatives><tex-math id=\"M49\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${{\\text{X}}}_{{\\text{c}}}=\\frac{0.24}{\\beta }$$\\end{document}</tex-math><mml:math id=\"M50\"><mml:mrow><mml:msub><mml:mtext>X</mml:mtext><mml:mtext>c</mml:mtext></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>0.24</mml:mn></mml:mrow><mml:mi>β</mml:mi></mml:mfrac></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq40\"><alternatives><tex-math id=\"M51\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{D}}$$\\end{document}</tex-math><mml:math id=\"M52\"><mml:mtext>D</mml:mtext></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq41\"><alternatives><tex-math id=\"M53\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\upvarepsilon \\times {10}^{-4}$$\\end{document}</tex-math><mml:math id=\"M54\"><mml:mrow><mml:mi mathvariant=\"normal\">ε</mml:mi><mml:mo>×</mml:mo><mml:msup><mml:mrow><mml:mn>10</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>4</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq42\"><alternatives><tex-math id=\"M55\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\text{D}}$$\\end{document}</tex-math><mml:math id=\"M56\"><mml:mtext>D</mml:mtext></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq43\"><alternatives><tex-math id=\"M57\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\upvarepsilon \\times {10}^{-4}$$\\end{document}</tex-math><mml:math id=\"M58\"><mml:mrow><mml:mi mathvariant=\"normal\">ε</mml:mi><mml:mo>×</mml:mo><mml:msup><mml:mrow><mml:mn>10</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>4</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq44\"><alternatives><tex-math id=\"M59\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\updelta \\times {10}^{-4}{\\mathrm{ nm}}^{-2}$$\\end{document}</tex-math><mml:math id=\"M60\"><mml:mrow><mml:mi mathvariant=\"normal\">δ</mml:mi><mml:mo>×</mml:mo><mml:msup><mml:mrow><mml:mn>10</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>4</mml:mn></mml:mrow></mml:msup><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">nm</mml:mi></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq45\"><alternatives><tex-math id=\"M61\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${{\\text{X}}}_{{\\text{c}}}$$\\end{document}</tex-math><mml:math id=\"M62\"><mml:msub><mml:mtext>X</mml:mtext><mml:mtext>c</mml:mtext></mml:msub></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq23\"><alternatives><tex-math id=\"M63\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-$$\\end{document}</tex-math><mml:math id=\"M64\"><mml:mo>-</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq24\"><alternatives><tex-math id=\"M65\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-$$\\end{document}</tex-math><mml:math id=\"M66\"><mml:mo>-</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq25\"><alternatives><tex-math id=\"M67\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-$$\\end{document}</tex-math><mml:math id=\"M68\"><mml:mo>-</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq26\"><alternatives><tex-math id=\"M69\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-$$\\end{document}</tex-math><mml:math id=\"M70\"><mml:mo>-</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq27\"><alternatives><tex-math id=\"M71\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim $$\\end{document}</tex-math><mml:math id=\"M72\"><mml:mo>∼</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq28\"><alternatives><tex-math id=\"M73\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim $$\\end{document}</tex-math><mml:math id=\"M74\"><mml:mo>∼</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq29\"><alternatives><tex-math id=\"M75\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim $$\\end{document}</tex-math><mml:math id=\"M76\"><mml:mo>∼</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq30\"><alternatives><tex-math id=\"M77\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-$$\\end{document}</tex-math><mml:math id=\"M78\"><mml:mo>-</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq31\"><alternatives><tex-math id=\"M79\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathrm{\\alpha })$$\\end{document}</tex-math><mml:math id=\"M80\"><mml:mrow><mml:mi>α</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math></alternatives></inline-formula>", "<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M81\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\alpha = \\frac{1}{d}Ln\\left[ {\\frac{{(1 - R^{2} )}}{2T}\\,\\, + \\sqrt {\\frac{(1 - R)}{{4T^{2} }}\\, + \\,R^{2} } } \\right] $$\\end{document}</tex-math><mml:math id=\"M82\" display=\"block\"><mml:mrow><mml:mi>α</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mi>d</mml:mi></mml:mfrac><mml:mi>L</mml:mi><mml:mi>n</mml:mi><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mfrac><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:msup><mml:mi>R</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mi>T</mml:mi></mml:mrow></mml:mfrac><mml:mspace width=\"0.166667em\"/><mml:mspace width=\"0.166667em\"/><mml:mo>+</mml:mo><mml:msqrt><mml:mrow><mml:mfrac><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:mi>R</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mn>4</mml:mn><mml:msup><mml:mi>T</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac><mml:mspace width=\"0.166667em\"/><mml:mo>+</mml:mo><mml:mspace width=\"0.166667em\"/><mml:msup><mml:mi>R</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:msqrt></mml:mrow></mml:mfenced></mml:mrow></mml:math></alternatives></disp-formula>", "<inline-formula id=\"IEq32\"><alternatives><tex-math id=\"M83\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$(\\mathrm{h\\nu })$$\\end{document}</tex-math><mml:math id=\"M84\"><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi mathvariant=\"normal\">h</mml:mi><mml:mi>ν</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq33\"><alternatives><tex-math id=\"M85\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim $$\\end{document}</tex-math><mml:math id=\"M86\"><mml:mo>∼</mml:mo></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq34\"><alternatives><tex-math id=\"M87\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$({{\\text{E}}}_{{\\text{g}}})$$\\end{document}</tex-math><mml:math id=\"M88\"><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mtext>E</mml:mtext><mml:mtext>g</mml:mtext></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math></alternatives></inline-formula>", "<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M89\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathrm{\\alpha }=\\frac{{\\text{A}}}{\\mathrm{h\\nu }} (\\mathrm{h\\nu }-{{\\text{E}}}_{{\\text{g}}}{)}^{{\\text{n}}}$$\\end{document}</tex-math><mml:math id=\"M90\" display=\"block\"><mml:mrow><mml:mi>α</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mtext>A</mml:mtext><mml:mrow><mml:mi mathvariant=\"normal\">h</mml:mi><mml:mi>ν</mml:mi></mml:mrow></mml:mfrac><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi mathvariant=\"normal\">h</mml:mi><mml:mi>ν</mml:mi></mml:mrow><mml:mo>-</mml:mo></mml:mrow><mml:msub><mml:mtext>E</mml:mtext><mml:mtext>g</mml:mtext></mml:msub><mml:msup><mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mtext>n</mml:mtext></mml:msup></mml:mrow></mml:math></alternatives></disp-formula>", "<inline-formula id=\"IEq35\"><alternatives><tex-math id=\"M91\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${{\\text{E}}}_{{\\text{g}}}$$\\end{document}</tex-math><mml:math id=\"M92\"><mml:msub><mml:mtext>E</mml:mtext><mml:mtext>g</mml:mtext></mml:msub></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq36\"><alternatives><tex-math id=\"M93\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${(\\mathrm{\\alpha h\\nu })}^{2}$$\\end{document}</tex-math><mml:math id=\"M94\"><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>α</mml:mi><mml:mi mathvariant=\"normal\">h</mml:mi><mml:mi>ν</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq37\"><alternatives><tex-math id=\"M95\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$(\\mathrm{h\\nu })$$\\end{document}</tex-math><mml:math id=\"M96\"><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi mathvariant=\"normal\">h</mml:mi><mml:mi>ν</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq38\"><alternatives><tex-math id=\"M97\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim 36\\times {10}^{-4} {{\\text{nm}}}^{-2}$$\\end{document}</tex-math><mml:math id=\"M98\"><mml:mrow><mml:mo>∼</mml:mo><mml:mn>36</mml:mn><mml:mo>×</mml:mo><mml:msup><mml:mrow><mml:mn>10</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>4</mml:mn></mml:mrow></mml:msup><mml:msup><mml:mrow><mml:mtext>nm</mml:mtext></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></alternatives></inline-formula>", "<inline-formula id=\"IEq39\"><alternatives><tex-math id=\"M99\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$=$$\\end{document}</tex-math><mml:math id=\"M100\"><mml:mo>=</mml:mo></mml:math></alternatives></inline-formula>" ]
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[ "<table-wrap-foot><p>LC<sub>50</sub> and LC<sub>90</sub>: lethal concentration that kills 50 and 90% of the exposed <italic>C. pipiens</italic> larvae; UCL: upper confidence limit; LCL: lower confidence limit; X<sup>2</sup> = Chi-square, p &lt; 0.05, significance level, Mean value of five replicates, (SE) standard error. Control (distilled water), nil mortality. Letters indicate degree of significant based on Tukey’s HSD tests between different treatments and control. Letter (a) mean high significant, and letter (c) mean low significant, while the same letters are not significantly different</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[{"label": ["4."], "surname": ["Leibovici", "Bylund", "Bjorkman", "Tokarevich", "Thierfelder", "Evengard", "Quegan"], "given-names": ["DG", "H", "C", "N", "T", "B", "S"], "article-title": ["Associating land cover changes with patterns of incidences of climate-sensitive infections: an example on tick-borne diseases in the nordic area"], "source": ["Int J Environ Res Public Health"], "year": ["2021"], "volume": ["20"], "fpage": ["10963"], "pub-id": ["10.3390/ijerph182010963"]}, {"label": ["5."], "mixed-citation": ["World Health Organization (WHO). Dengue and severe dengue. 2022; "], "ext-link": ["https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue"]}, {"label": ["6."], "surname": ["Rashed", "Beall", "Rashad", "Mostafa"], "given-names": ["SS", "GW", "EM", "WA"], "article-title": ["Control of human filarial vector, "], "italic": ["C. quinquefasciatus", "Diptera: Cuicidae"], "source": ["J Egypt Soc Parasitol (JESP)"], "year": ["2020"], "volume": ["50"], "issue": ["1"], "fpage": ["221"], "lpage": ["227"], "pub-id": ["10.21608/jesp.2020.112999"]}, {"label": ["7."], "surname": ["Aziz", "Alshehri", "Alanazi", "Panneerselvam", "Trivedi", "Maggi", "Sut", "Dall\u2019Acqua S."], "given-names": ["A", "MA", "NA", "C", "S", "F", "S"], "article-title": ["Phytochemical analysis of "], "italic": ["Rhazya stricta"], "source": ["Sci Total Enviro"], "year": ["2020"], "volume": ["700"], "fpage": ["134443"], "pub-id": ["10.1016/j.scitotenv.2019.134443"]}, {"label": ["8."], "surname": ["Khana", "Musarrat", "Al-Khedhairy"], "given-names": ["ST", "J", "AA"], "article-title": ["Countering drug resistance, infectious diseases, and sepsis using metal and metal oxides nanoparticles: Current status"], "source": ["Colloids Surf, B"], "year": ["2016"], "volume": ["146"], "fpage": ["70"], "lpage": ["83"], "pub-id": ["10.1016/j.colsurfb.2016.05.046"]}, {"label": ["9."], "surname": ["Lalabadi", "Hashemi", "Feng", "Jafari"], "given-names": ["MA", "H", "J", "SM"], "article-title": ["Carbon nanomaterials against pathogens; the antimicrobial activity of carbon nanotubes, graphene/graphene oxide, fullerenes, and their nanocomposites"], "source": ["Adva in Colloid and Interface Sci"], "year": ["2020"], "volume": ["284"], "fpage": ["102250"], "pub-id": ["10.1016/j.cis.2020.102250"]}, {"label": ["12."], "surname": ["Han", "Xiang", "Meng", "Dong", "Ying"], "given-names": ["W", "W", "Z", "S", "L"], "article-title": ["A novel Cr-doped CdS/ZnO nanocomposite for efficient photocatalytic hydroxylation of benzene to phenol"], "source": ["Coll Surfaces A Physicochem Engin Aspects."], "year": ["2023"], "volume": ["670"], "fpage": ["131529"], "pub-id": ["10.1016/j.colsurfa.2023.131529"]}, {"label": ["13."], "surname": ["Umashankar", "Devi", "Gurushantha", "Manjunatha", "Al-Dossari", "Shobha", "Kumar", "Kumar", "Keshavamurthy", "NS EL-Gawaad, Madhu A, Reddy SS, Srinatha N."], "given-names": ["R", "SH", "K", "SO", "M", "G", "KJ", "MR", "K"], "article-title": ["Improved photocatalytic, antimicrobial and photoelectrochemical properties of nanocrystalline Cu"], "sup": ["2+"], "source": ["Ceramics Intern"], "year": ["2023"], "volume": ["49"], "fpage": ["22449"], "lpage": ["22459"], "pub-id": ["10.1016/j.ceramint.2023.04.077"]}, {"label": ["14."], "surname": ["Mirzapoor", "Ghashghaee"], "given-names": ["A", "E"], "article-title": ["Nano engineered synthesis layer by layer and transparent synergistic antimicrobial coating based on Se/Ag/TiO"], "sub": ["2"], "source": ["Inorg Chem Comm"], "year": ["2023"], "volume": ["153"], "fpage": ["110840"], "pub-id": ["10.1016/j.inoche.2023.110840"]}, {"label": ["15."], "surname": ["Vu", "Tuyet Pham"], "given-names": ["AT", "TA"], "article-title": ["Study on method of doping au nanoparticles on ZnO stratified microstructure to enhance photocatalytic ability and antibacterial activity"], "source": ["Bull of Chem Reac Engin Cata."], "year": ["2023"], "volume": ["18"], "fpage": ["131"], "lpage": ["150"], "pub-id": ["10.9767/bcrec.17566"]}, {"label": ["16."], "surname": ["El-Latef", "Wahba", "Mousa", "El-Bassyouni", "El-Shamy"], "given-names": ["EA", "MN", "S", "GT", "AM"], "article-title": ["Cu-doped ZnO-nanoparticles as a novel eco-friendly insecticide for controlling "], "italic": ["Spodoptera littoralis"], "source": ["Biocata and Agric Biotech"], "year": ["2023"], "volume": ["52"], "fpage": ["102823"], "pub-id": ["10.1016/j.bcab.2023.102823"]}, {"label": ["17."], "surname": ["Singh", "Dotiyal", "Bhardwaj", "Kumar", "Bhandari", "Verma"], "given-names": ["J", "M", "P", "K", "P", "V"], "article-title": ["Influence of anionic surfactant (SDS) on the structural properties of chromium oxide (Cr"], "sub": ["2", "3"], "source": ["Mater Today Proc"], "year": ["2022"], "volume": ["69"], "fpage": ["193"], "lpage": ["198"], "pub-id": ["10.1016/j.matpr.2022.08.340"]}, {"label": ["18."], "surname": ["Debnath", "Chakraborty", "Bandyopadhyay", "Sharma", "Mahapatra", "Das", "Sutradhar"], "given-names": ["T", "T", "A", "S", "AS", "S", "S"], "article-title": ["Modulation of structural, morphological and electrical charge transport property of Cr-doped ZnO nanomaterials prepared by chemical process"], "source": ["Mater Sci Eng, B"], "year": ["2022"], "volume": ["280"], "fpage": ["15688"], "pub-id": ["10.1016/j.mseb.2022.115688"]}, {"label": ["19."], "surname": ["Imam Shah", "Jabeen", "Irum", "Shafique Ahmad", "Tauseef", "Farooq Khan", "Anwaar", "Shafique", "Kashif Haleem", "Mehmood", "Zaheer Hussain"], "given-names": ["N", "N", "S", "K", "I", "T", "S", "S", "S", "A", "S"], "article-title": ["Environmentally benign and economical bio-fabrication of ZnO and Cr-doped ZnO nanoparticles using leaf extract of "], "italic": ["Citrus reticulata"], "source": ["Mat Today Commun"], "year": ["2021"], "volume": ["27"], "fpage": ["102383"], "pub-id": ["10.1016/j.mtcomm.2021.102383"]}, {"label": ["21."], "surname": ["Panchal", "Sharma", "Reddy", "Nehra", "Sharma", "Nehra"], "given-names": ["P", "R", "AS", "K", "A", "SP"], "article-title": ["Eco-friendly synthesis of Ag-doped ZnO/MgO as a potential photocatalyst for antimicrobial and dye degradation applications"], "source": ["Coordination Chem Rev"], "year": ["2023"], "volume": ["493"], "fpage": ["215283"], "pub-id": ["10.1016/j.ccr.2023.215283"]}, {"label": ["22."], "surname": ["Mostafa", "Abdel-Raoof", "Attala", "Elgazza"], "given-names": ["WA", "AM", "K", "E"], "article-title": ["Enhancement the larvicidal activity of nanostructure copper oxide against "], "italic": ["Culex pipiens"], "source": ["Mater Res Express"], "year": ["2021"], "volume": ["8"], "fpage": ["115006"], "pub-id": ["10.1088/2053-1591/ac343d"]}, {"label": ["24."], "surname": ["Bharathi", "Boopathyraja", "Nachimuthu", "Kannan"], "given-names": ["DS", "A", "S", "K"], "article-title": ["Green synthesis, characterization and antibacterial activity of SiO2\u2013 ZnO nanocomposite by dictyota bartayresiana extract and its cytotoxic effect on HT29 cell line"], "source": ["J Cluster Sci"], "year": ["2022"], "volume": ["33"], "fpage": ["2499"], "lpage": ["2515"], "pub-id": ["10.1007/s10876-021-02170-w"]}, {"label": ["25."], "surname": ["Debnath", "Chakraborty", "Bandyopadhyay", "Sharma", "Mahapatra", "Das", "Sutradhar"], "given-names": ["T", "T", "A", "S", "AS", "S", "S"], "article-title": ["Modulation of structural, morphological and electrical charge transport property of Cr-doped ZnO nanomaterials prepared by chemical process"], "source": ["Mater Sci and Engin B"], "year": ["2022"], "volume": ["280"], "fpage": ["115688"], "pub-id": ["10.1016/j.mseb.2022.115688"]}, {"label": ["26."], "surname": ["Lokesha", "Prinsloo", "Mohanty", "Sheppard"], "given-names": ["HS", "ARE", "P", "CJ"], "article-title": ["Impact of Cr doping on the structure, optical and magnetic properties of nanocrystalline ZnO particles"], "source": ["J Alloys Comp"], "year": ["2023"], "volume": ["960"], "fpage": ["170815"], "pub-id": ["10.1016/j.jallcom.2023.170815"]}, {"label": ["27."], "surname": ["Nadeem", "Munawar", "Mukhtar", "Rahman", "Riaz", "Iqbal"], "given-names": ["MS", "T", "F", "MN", "M", "F"], "article-title": ["Enhancement in the photocatalytic and antimicrobial properties of ZnO nanoparticles by structural variations and energy bandgap tuning through Fe and Co co-doping"], "source": ["Ceramics Inter"], "year": ["2021"], "volume": ["47"], "fpage": ["11109"], "lpage": ["11121"], "pub-id": ["10.1016/j.ceramint.2020.12.234"]}, {"label": ["28."], "surname": ["David", "Doss", "Pole"], "given-names": ["SA", "A", "RPP"], "article-title": ["Biosynthesis of chromium oxide nanoparticles by "], "italic": ["Momordica charantia"], "source": ["Res Surfaces Interfaces"], "year": ["2023"], "volume": ["11"], "fpage": ["100120"], "pub-id": ["10.1016/j.rsurfi.2023.100120"]}, {"label": ["29."], "surname": ["Ashokkumar", "Muthusamy"], "given-names": ["M", "C"], "article-title": ["Role of ionic radii and electronegativity of co-dopants (Co, Ni and Cr) on properties of Cu doped ZnO and evaluation of In-vitro cytotoxicity"], "source": ["Surfaces Interfaces"], "year": ["2022"], "volume": ["30"], "fpage": ["101968"], "pub-id": ["10.1016/j.surfin.2022.101968"]}, {"label": ["30."], "surname": ["Mathai", "Jose", "Anjana", "Aleena", "Kunjumon", "Ittyachan", "Nair", "Vinitha", "Sajan"], "given-names": ["J", "AK", "MP", "PA", "J", "R", "SS", "G", "D"], "article-title": ["Substantial effect of Cr doping on the third-order nonlinear optical properties of ZnO nanostructures"], "source": ["Opt Mater"], "year": ["2023"], "volume": ["142"], "fpage": ["114128"], "pub-id": ["10.1016/j.optmat.2023.114128"]}, {"label": ["31."], "surname": ["Umashankar", "Devi", "Gurushantha", "Manjunatha", "Al-Dossari", "Shobha", "Kumar", "Kumar", "Keshavamurthy", "Abd EL-Gawaad NS, Madhu A, Reddy SS, Srinatha N."], "given-names": ["R", "SH", "K", "SO", "M", "G", "KJR", "MRS", "K"], "article-title": ["Improved photocatalytic, antimicrobial and photoelectrochemical properties of nanocrystalline Cu"], "sup": ["2+"], "source": ["Ceram Inter."], "year": ["2023"], "volume": ["49"], "fpage": ["22449"], "lpage": ["22459"], "pub-id": ["10.1016/j.ceramint.2023.04.077"]}, {"label": ["32."], "surname": ["Rajivgandhi", "Ramachandran", "Alharbi", "Kadaikunnan", "Khaleed", "Manokaran", "Li"], "given-names": ["GN", "G", "NS", "S", "JM", "N", "W"], "article-title": ["Substantial effect of Cr doping on the antimicrobial activity of ZnO nanoparticles prepared by ultrasonication process"], "source": ["Mate Sci and Engin B"], "year": ["2021"], "volume": ["263"], "fpage": ["114817"], "pub-id": ["10.1016/j.mseb.2020.114817"]}, {"label": ["33."], "surname": ["Mathai", "Anjana", "Aleena", "Thomas", "Jose", "Kunjumon", "Nair", "Isaac", "Sajan"], "given-names": ["J", "MP", "PA", "SA", "AK", "J", "SS", "RS", "D"], "article-title": ["Cr doped ZnO nanoparticles as photocatalyst for the degradation of methylene blue dye"], "source": ["J of the Indian Chem Soc"], "year": ["2023"], "volume": ["100"], "fpage": ["101067"], "pub-id": ["10.1016/j.jics.2023.101067"]}, {"label": ["34."], "surname": ["PrakasamA", "Raj", "Ragupathi", "Kumar"], "given-names": ["APM", "RA", "C", "R"], "article-title": ["Morphologies, crystal structure and antibacterial properties of chromium substituted zinc oxide nanoparticles synthesized by microwave method"], "source": ["Mate Today Proc"], "year": ["2022"], "volume": ["49"], "fpage": ["2928"], "lpage": ["2933"], "pub-id": ["10.1016/j.matpr.2021.11.100"]}, {"label": ["35."], "surname": ["El-sayed", "Abdel-Raoof", "Abdellatef", "Hendawy", "El-Abassy", "Ibrahim"], "given-names": ["HM", "AM", "HE", "HAM", "OM", "H"], "article-title": ["Versatile eco-friendly electrochemical sensor based on chromium-doped zinc oxide nanoparticles for determination of safinamide aided by green assessment criteria"], "source": ["Microchem J"], "year": ["2022"], "volume": ["182"], "fpage": ["107900"], "pub-id": ["10.1016/j.microc.2022.107900"]}, {"label": ["36."], "surname": ["Mary", "Manonmoni", "Balanc", "Karthik", "Malliappan"], "given-names": ["KL", "JV", "AMR", "PS", "SP"], "article-title": ["Phytochemical assisted synthesis of Ni doped ZnO nanoparticles using aloe vera extract for enhanced photocatalytic and antibacterial activities"], "source": ["Digest J of Nanomater and Biostruc"], "year": ["2022"], "volume": ["17"], "fpage": ["634"], "lpage": ["648"], "pub-id": ["10.15251/DJNB.2022.172.634"]}, {"label": ["38."], "surname": ["Boulahlib", "Dib", "Ozacar", "Bessekhouad"], "given-names": ["S", "K", "M", "Y"], "article-title": ["Optical, dielectric, and transport properties of Ag-doped ZnO prepared by Aloe Vera assisted method"], "source": ["Optical Mater"], "year": ["2021"], "volume": ["113"], "fpage": ["110889"], "pub-id": ["10.1016/j.optmat.2021.110889"]}, {"label": ["39."], "surname": ["Debnath", "Chakraborty", "Bandyopadhyay", "Sharma", "Mahapatra", "Das", "Sutradhar"], "given-names": ["T", "T", "A", "S", "AS", "S", "S"], "article-title": ["Modulation of structural, morphological and electrical charge transport property of Cr-doped ZnO nanomaterials prepared by chemical process"], "source": ["Mater Sci Eng B."], "year": ["2022"], "volume": ["280"], "fpage": ["115688"], "pub-id": ["10.1016/j.mseb.2022.115688"]}, {"label": ["41."], "surname": ["Nivetha", "Sakthivel", "Hemalatha", "Senthamil", "Prabha"], "given-names": ["A", "C", "J", "C", "I"], "article-title": ["Fabrication of a bifunctionalyzed Calotropis gigantea inspired Ag\u2013Cu\u2013Co trimetal oxide for the remediation of methylene blue, and its larvicidal and antibacterial applications"], "source": ["New J Chem"], "year": ["2023"], "volume": ["47"], "fpage": ["12375"], "lpage": ["12392"], "pub-id": ["10.1039/D3NJ01706K"]}, {"label": ["43."], "surname": ["Ibrahim", "Ali"], "given-names": ["AMA", "AM"], "article-title": ["Silver and zinc oxide nanoparticles induce developmental and physiological changes in the larval and pupal stages of "], "italic": ["Spodoptera littoralis"], "source": ["J Asia Pac Entomol"], "year": ["2018"], "volume": ["21"], "fpage": ["1373"], "lpage": ["1378"], "pub-id": ["10.1016/j.aspen.2018.10.018"]}, {"label": ["44."], "surname": ["Athanassiou", "Kavallieratos", "Benelli", "Losic", "Rani", "Desneux"], "given-names": ["CG", "NG", "G", "D", "UP", "N"], "article-title": ["Nanoparticles for pest control: current status and future perspectives"], "source": ["J Pest Sci"], "year": ["2018"], "volume": ["91"], "fpage": ["1"], "lpage": ["15"], "pub-id": ["10.1007/s10340-017-0898-0"]}, {"label": ["46."], "surname": ["Ishwarya", "Vaseeharan", "Kalyani", "Banumathi", "Govindarajan", "Alharbi", "Kadaikunnan", "Al-anbr", "Khaled", "Benelli"], "given-names": ["R", "B", "S", "B", "M", "NS", "S", "MN", "JM", "G"], "article-title": ["Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and its evaluation of photocatalytic, antibiofilm and larvicidal activity: impact on mosquito morphology and biofilm architecture"], "source": ["J Photochem Photobiol"], "year": ["2017"], "volume": ["178"], "fpage": ["249"], "lpage": ["258"], "pub-id": ["10.1016/j.jphotobiol.2017.11.006"]}, {"label": ["47."], "surname": ["Benelli"], "given-names": ["G"], "article-title": ["Mode of action of nanoparticles against insects Environ"], "source": ["Sci Pollut Res"], "year": ["2018"], "volume": ["25"], "fpage": ["12329"], "lpage": ["12341"], "pub-id": ["10.1007/s11356-018-1850-4"]}, {"label": ["48."], "surname": ["Al-Doaiss", "Al-Mekhlafi", "Abutaha", "Al-Keridis", "Shati", "Al-Kahtani", "Alfaifi"], "given-names": ["A", "F", "N", "LA", "AA", "MA", "MY"], "article-title": ["Morphological, histological and ultrastructural characterization of "], "italic": ["Culex pipien"], "source": ["African Entomol"], "year": ["2021"], "volume": ["29"], "fpage": ["274"], "lpage": ["288"]}, {"label": ["51."], "surname": ["Stojanovic", "Milosevic", "Vitorovic", "Savic-Zdravkovic", "Stankovic", "Stankovic", "Vasiljevic"], "given-names": ["J", "D", "J", "D", "N", "J", "P"], "article-title": ["Histopathology of "], "italic": ["Chironomus riparius", "Diptera, Chironomidae"], "source": ["Arch Biol Sci"], "year": ["2021"], "pub-id": ["10.2298/ABS210515025S"]}, {"label": ["53."], "surname": ["Shamseldean", "Attia", "Korany", "Othamn", "Allam"], "given-names": ["MSM", "MM", "RMS", "NA", "SFM"], "year": ["2022"], "data-title": ["Insecticidal efficacy of nanomaterials used to control mosquito, Culex quinquefasciatus Say, 1823 with special reference to their hepatotoxicity in rats"], "source": ["Biosci Reports"], "pub-id": ["10.1042/BSR20220630"]}, {"label": ["54."], "surname": ["Shah", "Jabeen", "Irum", "Ahmad", "Tauseef", "Khan", "Anwaar", "Shafique", "Haleem", "Mehmood", "Hussain"], "given-names": ["NI", "N", "S", "KS", "T", "TF", "S", "S", "SK", "N", "SZ"], "article-title": ["Environmentally benign and economical bio-fabrication of ZnO and Cr-doped ZnO nanoparticles using leaf extract of "], "italic": ["Citrus reticulata"], "source": ["Mater Today Commun"], "year": ["2021"], "volume": ["27"], "fpage": ["102383"], "pub-id": ["10.1016/j.mtcomm.2021.102383"]}, {"label": ["55."], "surname": ["Kannana", "Radhika", "Kasai", "Gnanasangeethac", "Palani", "Gurushankar", "Koutavarapug", "Lee", "Shim"], "given-names": ["K", "D", "RD", "D", "G", "K", "R", "D", "J"], "article-title": ["Facile fabrication of novel ceria-based nanocomposite (CYO-CSO) via co-precipitation: electrochemical, photocatalytic and antibacterial performances"], "source": ["J Mol Struct"], "year": ["2022"], "volume": ["1256"], "fpage": ["132519"], "pub-id": ["10.1016/j.molstruc.2022.132519"]}, {"label": ["57."], "surname": ["Vijayalakshmi", "Sivaraj"], "given-names": ["K", "D"], "article-title": ["Enhanced antibacterial activity of Cr doped ZnO nanorods synthesized using microwave processing"], "source": ["RSC Adv"], "year": ["2015"], "volume": ["5"], "fpage": ["68461"], "pub-id": ["10.1039/C5RA13375K"]}, {"label": ["59."], "surname": ["Kannan", "Adhika", "Gnanasangeetha", "Lakkaboyana", "Sadasivuni", "Gurushankar", "Hanafiah"], "given-names": ["KR", "D", "D", "SK", "KK", "K", "MM"], "article-title": ["Photocatalytic and antimicrobial properties of microwave synthesized mixed metal oxide nanocomposite"], "source": ["Inorganic Chem Commun"], "year": ["2021"], "volume": ["125"], "fpage": ["108429"], "pub-id": ["10.1016/j.inoche.2020.108429"]}, {"label": ["61."], "surname": ["Lakshmanana", "Surendrana", "Priyaa", "Balakrishnana", "Geethab", "Rameshkumara", "Hegdec", "Vinithac", "Kannand"], "given-names": ["A", "B", "SS", "K", "P", "P", "TA", "G", "K"], "article-title": ["Investigations on structural, optical, dielectric, electronic polarizability, Z-scan and antibacterial properties of Ni/Zn/Fe"], "sub": ["2", "4"], "source": ["J Photochem Photobio A: Chemi"], "year": ["2020"], "volume": ["402"], "fpage": ["112794"], "pub-id": ["10.1016/j.jphotochem.2020.112794"]}, {"label": ["62."], "surname": ["Kannan", "Radhika", "Nesaraj", "Sadasivuni", "Krishna"], "given-names": ["K", "D", "AS", "KK", "LS"], "article-title": ["Facile synthesis of NiO-CYSO nanocomposite for photocatalytic and antibacterial applications"], "source": ["Inorg Chem Commun"], "year": ["2020"], "volume": ["122"], "fpage": ["108307"], "pub-id": ["10.1016/j.inoche.2020.108307"]}]
{ "acronym": [ "NPs", "XRD", "EDX", "FTIR", "SEM", "TEM", "Abd", "G", "H", "Siph", "Th", "dAbd", "dH", "Mp", "Cy", "M", "N", "Ne", "MV", "Nu", "Ch", "dMV", "HC", "Ab N", "VN" ], "definition": [ "Nanoparticles", "X-ray diffraction", "Energy dispersive X-ray spectroscopy", "Fourier-transform infrared spectroscopy", "Scane electron microscopy", "Transmission electron microscopy", "Abdomen", "Gills", "Head", "Siphon", "Thorax", "Deformed abdomin", "Deformed head", "Mouthparts", "Cytoplasm", "Mitochondria", "Nucleus", "Nuclear envelope", "Microvilli.", "Nucleus", "Chromatin clumps", "Microvilli", "Heterochromatin", "Abnormal nucleus", "Vacuole nucleus" ] }
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2024-01-14 23:43:47
BMC Chem. 2024 Jan 12; 18(1):11
oa_package/5b/b7/PMC10787509.tar.gz
PMC10787510
38216965
[ "<title>Introduction</title>", "<p id=\"Par48\">Micro-RNA (miRNA), as a non-coding small RNA composed of 18–25 nucleotides, which is one of the main elements involved in intracellular post-transcriptional regulation [##REF##29570994##1##, ##REF##25565026##2##]. MiRNA are mainly derived from the tissue cells, exosomes, microenvironment and body fluids. The exosomal miRNAs are ubiquitous and important factors that have a systemic and holistic impact on the body [##REF##21609964##3##–##REF##34496230##6##]. Therefore, circulating exosomal miRNAs provides them with a condition to involve in the connection and regulation between different diseases. Based on numerous studies, the circulating miRNAs have a significant intrinsic correlation with certain diseases, such as CVD [##REF##27931616##7##], diabetes [##REF##31638474##8##], obesity [##REF##31611648##9##] and tumors [##REF##32792861##10##]. The detection of humoral biomarkers for disease prediction, prevention and personalized treatment is a major development in medicine, so the combination of exosome miRNAs and 3PM can help in the diagnosis of different diseases [##REF##31737933##11##–##REF##35821883##13##]. Therefore, we need to conform the principles of 3PM to consider the overall impact between different diseases based on the perspective of miRNAs [##REF##32843909##14##], which may be better to improve the outcomes of intervention and treatment.</p>", "<p id=\"Par49\">CVD-related metabolic dysfunction and chronic stress damage caused by adverse factors (such as high cholesterol, oxidized low-density lipoprotein and hyperglycemia in circulation), which further leads to dysfunction of the circulating compositions [##REF##29022571##15##–##REF##29213140##18##]. Meanwhile, the CVD progression can lead to changes of miRNAs expression profile in the internal environment of blood, thereby affecting the function of other tissues and organs [##REF##29022571##15##, ##REF##30467329##16##]. CVD and tumor are generally considered to be two chronic diseases with the aging process of tissues and organs, both of which could be linked through the blood circulation system and have close material exchange via the peripheral environment for all times [##REF##31345432##19##–##REF##31504421##22##] (Fig. ##FIG##0##1##). Based on this particularity, the detection of biomarkers is particularly important in the prediction and prevention of systemic diseases. The mechanism of the CVD-related diseases impact on tumors belongs to the field of reverse Cardio-Oncology, which can help us understand and recognize the systemic and holistic effects of CVD on distant cancers via circulation [##REF##31960736##23##–##REF##33533026##26##].</p>", "<p id=\"Par50\">Tumor is a relatively heterogeneous tissue in the body that is affected by the distribution or density of blood circulation, which is characterized by frequent material exchange with the environment in the blood. For example, the regulation of tumor microenvironment, tumor immune infiltration and metabolic reprogramming can be affected by cardiovascular disease-related miRNAs (Cardio-miRNAs) [##REF##31171526##27##–##REF##33169503##29##]. Meanwhile, the appreciable effects of exosomes-miRNAs on tumor lesions via the circulatory system is a necessary condition for the adaptive survival and progression of tumor cells [##REF##33169503##29##]. Based on these facts, Cardio-miRNA may adversely affect the treatment of tumors and act as a factor to promote tumor miRNAs (Onco-miRNAs), which may lead to poor prognosis of patients with concomitant tumors [##REF##25364765##30##] (Fig. ##FIG##1##2## and Table ##TAB##0##1##).</p>", "<p id=\"Par51\">The cellular regulatory processes and mechanisms between miRNAs and CVD have been well studied, but the role of miRNAs as a systemic influence in the synthesis of cross-talk between different diseases is still less. Especially, some typical biomarkers reflected by changes of expression profile for Cardio-miRNAs in circulation. We sorted out the relevant mechanism of Cardio/Onco-miRNAs involved in CVD phenotype on tumor regulation according to the objective background of reverse Cardio-Oncology. These mechanisms will be key to revealing the systemic or holistic effects of CVD on tumors, which is also an important value for the application of precision medicine in the diagnosis and treatment of systemic diseases. These mechanisms might serve as evidence to supplement the importance of predictable diagnosis and personalized treatment between CVD and tumors, and it also provides a reference for developing systemic principles of improve individual outcomes.</p>" ]
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[ "<title>Conclusions</title>", "<p id=\"Par68\">Our review concludes that CVD and tumors can be linked through miRNAs, and these miRNAs may have a dual role (Cardio-/Onco-miRNAs). However, with aging, the dysfunctions of cardiovascular system may appear and changes of systematic phenotypes of circulating miRNAs showed the adverse effects of Cardio-miRNAs for middle-aged and elderly or obese tumor patients. This connection and regulatory mechanism may further demonstrate the necessity and foresight of the 3PM principles between diagnosing and treating for different diseases. Furthermore, the dual properties of Cardio-/Onco-miRNAs suggest that CVD is systemic and holistic problem or risk factor affect distant tumor cells via the circulation, which may be a potential target for treatment and intervention. Therefore, based on the perspective of CVD phenotyping in oncologic disorders, we need a systemic evaluation, prediction and diagnosis of the patients with concomitant tumors, which may provide a reference for avoiding poor prognosis.</p>" ]
[ "<p id=\"Par1\">With the increase of aging population and prevalence of obesity, the incidence of cardiovascular disease (CVD) and cancer has also presented an increasing tendency. These two different diseases, which share some common risk factors. Relevant studies in the field of reversing Cardio-Oncology have shown that the phenotype of CVD has a significant adverse effect on tumor prognosis, which is mainly manifested by a positive correlation between CVD and malignant progression of concomitant tumors. This distal crosstalk and the link between different diseases makes us aware of the importance of diagnosis, prediction, management and personalized treatment of systemic diseases. The circulatory system bridges the interaction between CVD and cancer, which suggests that we need to fully consider the systemic and holistic characteristics of these two diseases in the process of clinical treatment. The circulating exosome-miRNAs has been intrinsically associated with CVD -related regulation, which has become one of the focuses on clinical and basic research (as biomarker). The changes in the expression profiles of cardiovascular disease-associated miRNAs (Cardio-miRNAs) may adversely affect concomitant tumors. In this article, we sorted and screened CVD and tumor-related miRNA data based on literature, then summarized their commonalities and characteristics (several important pathways), and further discussed the conclusions of Cardio-Oncology related experimental studies. We take a holistic approach to considering CVD as a risk factor for tumor malignancy, which provides an in-depth analysis of the various regulatory mechanisms or pathways involved in the dual attribute miRNAs (Cardio-/Onco-miRNAs). These mechanisms will be key to revealing the systemic effects of CVD on tumors and highlight the holistic nature of different diseases. Therefore, the Cardio-miRNAs should be given great attention from researchers in the field of CVD and tumors, which might become new targets for tumor treatment. Meanwhile, based on the principles of precision medicine (such as the predictive preventive personalized medicine, 3PM) and reverse Cardio-oncology to better improve individual outcomes, we should consider developing personalized medicine and systemic therapy for cancer from the perspective of protecting cardiovascular function.</p>", "<title>Keywords</title>" ]
[ "<title>The outness of cardio-miRNAs affecting tumor progression via crosstalk</title>", "<p id=\"Par52\">Clinical or basic studies associated with reverse Cardio-Oncology have shown that CVD (e.g. hypertension, heart failure (HF), arteriosclerosis (AS) and myocardial infarction (MI) etc.) can influence distant tumor development by secreting circulating factors, such as the noncoding RNA, cytokines and proteins [##REF##31960736##23##, ##REF##33533026##26##, ##REF##34251889##31##]. The Cardio-miRNAs of cardiovascular dysfunction (e.g., pro-inflammatory and cellular senescence) is an important inducing factor for cardiovascular diseases. [##REF##27193456##32##–##REF##31576661##34##] In addition to heart, the function and key role of blood vessels is manifested by vascular endothelial cells [##REF##27989505##35##–##REF##29547400##37##]. Since the blood vessels are structures that interact directly with tumors, the vascular endothelial cells are involved in secreting exosome miRNAs when under the condition of the stress of various factors. This mechanism is also an important way for self-regulation of CVD via paracrine [##REF##27837398##33##], which will affects distal disease progression (such as cancer or tumorigenesis) [##REF##25364765##30##, ##REF##22258631##38##]. However, CVD and cancer share many common risk factors and disease mechanisms, and evidence from some clinical studies suggests that CVD is strongly associated with an increased risk of tumorigenesis [such as, colorectal cancer, liver cancer, lung cancer, melanoma, kidney cancer, lymphoma and breast cancer etc. HR (95% CI) &gt; 1.2, <italic>P</italic> &lt; 0.05] [##REF##30715247##39##–##REF##32661390##43##]. We need to focus on the predictive value of clinical detection of circulating factors in CVD and tumor progression, then develop effective tumor treatment based on the perspective of protecting cardiovascular function.</p>", "<p id=\"Par53\">Evidence from experiments have shown that HF can promote the malignant progression of distal colorectal cancer via circulation [##REF##29459363##44##]. In addition, studies have confirmed that miRNA are important mediators of CVD affecting distant tumor progression from the circulation system [##REF##36967385##45##, ##REF##35656866##46##]. Ye Yuan et al. found that exosome miR-22-3p secreted by cardiomyocytes after myocardial infarction (MI) can promote the malignant progression of distal lung cancer and osteosarcoma, and the main mechanism is the tolerance of tumor cells to ferroptosis [##REF##36967385##45##]. According to these effects of CVD phenotype on tumor, when tumor cells acquire the systemic regulation of relevant signaling pathway from circulating exosome Cardio-miRNAs, it can impact on the malignant process of tumors (Fig. ##FIG##0##1##) [##UREF##0##28##]. Therefore, clarifying the physiological regulatory mechanism of tumor cells involved in Cardio-miRNAs is an important basis for the treatment of CVD-associated tumors.</p>", "<title>The cardio-miRNAs regulate adaptive survival of tumor cells via four major pathways</title>", "<p id=\"Par54\">Based on the reported contribution of related miRNAs to the regulatory mechanism of malignancy process of tumor cells, combining with the fact that exosome-derived cardio-miRNAs can regulate tumors via circulation, we have deeply analyzed and summarized the different signaling pathways involved in Cardio-miRNAs. The aim is to predict and evaluate the impact on adaptive survival of tumor cells based on the regulatory mechanisms of different pathways and related Cardio-miRNAs as markers. Different expression levels of circulating cardio-miRNAs predict the progression of concomitant tumors and can be used to develop personalized treatment options.</p>", "<title>PTEN/PI3K/AKT pathway in tumor</title>", "<p id=\"Par55\">PTEN (Phosphatase and Tensin Homolog), as a tumor suppressor, which has been found inactive in different types of tumor cells. Therefore, the function of PTEN has considered to be one of the important factors affecting the human tumorigenesis [##REF##9090379##47##]. In normal cells, the tumor inhibitory effect of PTEN is mainly achieved by inhibiting the activity of the PI3K/AKT signaling pathway [##REF##26284192##48##]. Meanwhile, the PTEN/PI3K/AKT pathway is involved in regulating important pathways for tumor cell cycle, proliferation and malignant progression, and the expression level and functional activity of PTEN are key to the adaptive survival of tumor cells [##REF##33166764##49##]. Various non-coding RNAs, such as miRNA, lncRNA and cirRNA. etc., have been reported to be involved in the post-transcriptional regulation of PTEN [##REF##26284192##48##]. Exosomes, as a main carrier for miRNAs, is one of the main ways in which the external environment affects tumor cells [##UREF##1##50##]. MiRNAs affect the function of various organs in the body via blood circulation [##REF##31635843##4##, ##UREF##2##51##, ##REF##31504144##52##]. Aging and obesity are major factors contributing to the prevalence and high incidence of cardiovascular disease in the population [##REF##31611648##9##, ##REF##31331491##53##]. Therefore, the upregulation of Cardio-miRNAs from circulation may become an important factor affecting middle-aged and elderly or obese tumor patients [##REF##25364765##30##].</p>", "<p id=\"Par56\">Based on the results of the clinical research and public database (TCGA), we have summarized 9 Cardio-miRNAs that are reported targeting at PTEN (Fig. ##FIG##1##2##A, Table ##TAB##0##1##), such as miR-19a (GC, HCC, RCC) [##REF##30793479##54##–##REF##27779660##56##], miR-21 (UC, HCC, NSCLC, CRC, GBM, ccRCC, RCC, PC) [##REF##24122582##57##–##REF##23792563##62##], miR-25 (BC, HCC) [##REF##31336343##63##, ##REF##27840896##64##], miR-92a/b (HCC, OC, GBM) [##REF##32917956##65##–##REF##34136482##67##], miR-106b (BC, CRC) [##REF##28518139##68##, ##REF##26238857##69##], miR-130b (BC, NSCLC, OS, RCC) [##REF##31228937##70##–##REF##29653464##73##], miR-146b (TC) [##REF##29353884##74##], miR-210 (NSCLC) [##REF##32446904##75##] (Fig. ##FIG##2##3##). In tumor cells, these miRNAs attenuate the inhibitory effect on the PI3K–AKT signaling pathway by directly targeting PTEN. Therefore, PTEN can be regarded as multiple circulating Cardio-miRNA targets and has a wide range of effects on the development of different tumor types, which reflects the role in cross-disease linkage of cardio-miRNAs.</p>", "<p id=\"Par57\">In addition, there are several other miRNAs indirectly involved in regulating and activating PI3K–AKT signaling axis. For example, miR-27a, by targeting PHLPP2, attenuates the inhibition of PDK1/AKT pathway, thereby promoting the malignancy of GC cells [##REF##28327189##76##]. Similarly, miR-25 attenuates the inhibitory effect on the BTG2/AKT pathway and promotes the proliferation of TNBC cells by targeting BTG2 [##REF##29310680##77##]. In GC cells, miR-92b indirectly attenuates the activation inhibition of PI3K–AKT signaling pathway by targeting Dab2IP, and ultimately promotes tumor progression [##REF##31713929##78##]. MiR146b attenuates the activity inhibition of the PI3K–AKT pathway by targeting TRAF6 and TRIM2, respectively, and promotes the course of RCC patients [##REF##35342411##79##, ##REF##30206978##80##]. The M2 macrophage-derived exosome miR-155 eliminates transduction inhibition of IGF1R by targeting HuR (Human antigen R), after which activating the PI3K–AKT pathway and promoting the development of ccRCC [##REF##34131104##81##]. MiR-106b from hepatoma cells activates the AKT/ERK pathway via targeting GPM6A, resulting in upregulation of DYNC1I1 expression and ultimately promoting HCC cell proliferation [##REF##35685464##82##]. In summary, among the various Cardio-miRNAs associated with the PTEN/PI3K/AKT pathway, the one that directly target PTEN may be important factors resulting in the poor prognosis of concomitant tumors.</p>", "<title>Wnt/β-catenin pathway in tumor</title>", "<p id=\"Par58\">Wnt is a ligand protein containing 19 glycoprotein families in mammalian cells [##REF##6297757##83##], which is involved in regulating cell proliferation, adhesion, migration and differentiation through β-catenin-dependent or non-dependent forms [##REF##23258168##84##]. Based on the proliferation pattern and adaptive survival phenotype of tumor cells, it also reflects the abnormal regulatory mechanism of the Wnt/β-Catenin pathway [##REF##23258168##84##]. β-catenin is an adaptor protein that coordinates signal transduction in the Wnt signaling pathway, and it is capable of nuclear translocation to participate in the transcription of EMT-related genes in tumor cells [##REF##22007144##85##]. There are important noncoding RNAs for post-transcriptional regulation of Wnt/β-Catenin pathway, among which miRNAs are ones that involved in regulating malignant progression or adaptive survival of tumors during the Wnt/β-Catenin signal transduction of tumor cells [##REF##24598126##86##–##REF##20041844##89##].</p>", "<p id=\"Par59\">Among the Cardio-miRNAs, 6 miRNAs were associated with AMI, CAD, HF, HCM, CHD, CAV, MI/R, RCVS and ACS, and were upregulated in circulation (Table ##TAB##0##1##) [##REF##25383678##90##–##REF##24053180##102##]. These Cardio-miRNAs can promote the progression of tumor cell malignancy by targeting relevant proteins in the Wnt/β-Catenin pathway (Fig. ##FIG##1##2##B, Table ##TAB##0##1##). To be specific, miR-19a can affect the competitive binding of SMAD2 to β-Catenin and promote the EMT process of GC cells via targeting SMAD2 [##REF##31186404##103##]. MiR-27a can promote the malignant metastasis of TNBC cells by targeting GSK-3β to cause more release of β-Catenin and nuclear transposition [##REF##32801869##104##]. MiR-92a and miR-133a targeted DKK1 and DYGB, respectively, which attenuate the inhibitory effect of these two proteins in the process of Wnt signal transduction, thereby promoting the progression and cell metastasis of OC [##REF##28209618##105##, ##REF##31627092##106##]. MiR-130b can target at down regulating the level of PTEN, following by an attenuation of the inhibition in PI3K/AKT/GSK-3β/β-Catenin pathway, and ultimately resulting in the malignant metastasis of NSCLC [##REF##29653464##73##] (Fig. ##FIG##3##4##). To summarize, Cardio-miRNAs from circulation are involved in the signal transduction of the Wnt/β-Catenin pathway by targeting other cytokines, thereby promoting malignant metastasis or progression of tumors.</p>", "<title>NF-κB pathway in tumor</title>", "<p id=\"Par60\">The NF-κB pathway is one of the most important pathways involved in the regulation of cell physiology and pathometabolism, which includes inflammatory response, apoptosis, differentiation, immune response and cell migration [##REF##32309561##107##, ##REF##33838282##108##]. However, in most cases, NF-κB pathway is related to regulating cellular pro-inflammatory responses and survival, such as the adaptive survival regulation of tumor cells. This typical mechanism was proved by the association between low-level pro-inflammatory response and energy metabolism in tumor cells [##REF##25101566##109##, ##REF##12124178##110##]. In most tumor cells, the NF-κB pathway is highly activated and it mediates the malignant proliferation or survival of cells via nuclear metastasis, and ultimately promotes the metastasis and angiogenesis of tumor [##REF##33838282##108##, ##REF##20066113##111##–##REF##29616037##113##]. Therefore, the post-transcriptional regulation of NF-κB signaling pathway by tumor cells is a key process for the adaptive survival of tumor cells [##REF##29616037##113##, ##UREF##3##114##].</p>", "<p id=\"Par61\">Circulation in patients with cardiovascular diseases, such as ICM, CAD, ASO, HF, AMI, ACS, PH, DCM, provides a way for the transmission of inflammatory response [##REF##27357355##91##, ##REF##31092195##94##, ##REF##26198441##97##, ##REF##35162959##101##, ##REF##35370493##115##–##REF##33783502##130##], and the cardio-miRNAs spread through this way may adversely affect the treatment of concomitant tumors during circulating process (Fig. ##FIG##1##2##C, Table ##TAB##0##1##). For example, miR-16, miR-150 and miR-423 indirectly activated the NF-κB signaling pathway by targeting LDH-A, FOXO4 and TNIP2, respectively, and promoted the progression of NSCLC and BC [##REF##35714705##131##–##REF##32606763##133##]. MiR-21, as a typical noncoding RNA targeting at PTEN, indirectly activates NF-κB signaling in CRC cells via the PI3K–AKT pathway, and ultimately promotes the proliferation of tumor cells [##REF##30811964##60##]. Therefore, it can be speculated that multiple Cardio-miRNA can target PTEN/PI3K/AKT pathway, which may indirectly activate the NF-κB signaling pathway for multi tumor cell types. In addition, miR-155 promotes tumor progression by targeting PPP2CA and indirectly activating the NF-κB signaling pathway via AKT in CRC cells [##REF##21806946##134##]. In the upstream of the NF-κB pathway, miR-146a activates the NF-κB/p65 axis by targeting ligands IRAK1 and TRAF6, ultimately promotes the proliferation of NSCLC cells [##REF##33015045##135##]. As a bidirectional regulator, miR-210 promotes EMT and cell metastasis of PRAD via indirectly activating the NF-κB pathway after targeting SOCS1 and TNIP1 [##REF##33783502##130##] (Fig. ##FIG##4##5##). In summary, Cardio-miRNAs that targeting PTEN/PI3K/AKT and NF-κB pathway may be typical Onco-miRNAs that promote the progression of different types of tumors. Thus, these Cardio-miRNAs became the keys to the adaptive survival of tumor cells via the cross-combination of these two pathways.</p>", "<title>Apoptosis pathway in tumor</title>", "<p id=\"Par62\">Apoptosis is also one of the most important ways for cells to regulate self-physiology and metabolism, and the function is to maintain the tissues in a physiological state and to remove damaged cells, such as cells with DNA damage and high oncogenes expression [##REF##21943236##136##–##REF##27019364##138##]. In order to achieve adaptive proliferation or survival, tumor cells need to implement inhibition of various signaling pathways involved in apoptosis [##REF##21036469##139##]. Therefore, compared with normal cells, apoptosis signaling activity is deregulated during tumorigenesis, which can be achieved by miRNAs targeting at genes that take parts in pro-apoptotic pathway [##REF##17562483##140##]. In general, miRNAs involved in apoptosis regulation significantly affect the expression levels of pro/anti-apoptotic genes, such as oncogenes, endoplasmic reticulum (ER) stress, and apoptosis-related genes from mitochondrial extramembrane [##REF##22409455##141##].</p>", "<p id=\"Par63\">Studies have reported that the up or down regulation of some Cardio-miRNAs in circulation can correspondingly target at pro-/ anti-apoptotic proteins [##REF##25383678##90##–##REF##31092195##94##, ##REF##26198441##97##, ##REF##35162959##101##, ##REF##31660076##116##, ##REF##31033484##117##, ##REF##26916800##126##, ##REF##34612058##142##–##REF##24260372##151##] (Fig. ##FIG##1##2##D, Table ##TAB##0##1##). For example, miR-19a and miR-19b can target Bim on mitochondria in UC and CRC cells, respectively, and promote tumor cell survival and progression [##REF##32591507##152##, ##REF##29455644##153##]. MiR-21 and miR-25 attenuate the transduction process of apoptosis signaling via targeting exogenous apoptosis-inducing receptors FASL/TRAIL in PRAD and CCA cells, respectively, and ultimately result in chemotherapy resistance of tumor [##REF##23177026##154##, ##REF##21953056##155##]. In addition, miR-21 and miR-126 target p53 in RCC and BALL cells respectively. Besides, miR-106b targeted p21, which ultimately deregulated the activity of p53/p21-cyclinE2-Bax/Casp3 signaling pathway and resulted in chemotherapy resistance or poor prognosis [##REF##26238857##69##, ##REF##28184919##156##–##REF##28327152##158##]. When the expression level of miR-133a is downregulated in circulation, it may attenuate the targeted regulation of Bcl-xL/Mcl-1 and enhance the proliferative activity of OS cells [##REF##23756231##159##] (Fig. ##FIG##5##6##). To sum up, changes in the expression level of some Cardio-miRNAs may affect the therapeutic effect of patients with concomitant tumors via targeting the genes related to the apoptotic pathway.</p>", "<title>Other cardio-miRNAs in tumor</title>", "<p id=\"Par64\">In addition to the 4 typical regulatory pathways mentioned above, there are also some Cardio-miRNAs that indirectly promote the malignancy progression of tumor cells. We mainly take 3 targets from different pathway as examples to demonstrate the regulatory effect of these Cardio-miRNAs on tumors (Fig. ##FIG##6##7##, Table ##TAB##0##1##). MiR-16 and miR-27a regulate the activity of the TGF-β signaling pathway by targeting TFAP2A and SMAD2/SMAD4 respectively, which promote tumor EMT and cell cycle [##REF##33824285##160##, ##REF##28370334##161##]. Furthermore, miR-423 attenuates the inhibition of the TGF-β pathway by targeting GREM2 and results in chemotherapy resistance of patients with PRAD [##REF##33144675##162##]. Meanwhile, TGF-β can promote the expression of miR-208 in HCC cells, which attenuates the inhibitory effect on IFITM1 activity by targeting ARID2, and ultimately promote tumor progression [##REF##26169693##163##] (Fig. ##FIG##6##7##A). Due to its dual properties and pleiotropy for tumor cells, the TGF-β pathway is a potential target that needs precise control. The mutation, deletion, amplification, methylation of TGF-β and changes of miRNA levels have been proved to have significant effects on TGF-β signaling activity for different cancer types. Therefore, post-transcriptional survival regulation of TGF-β mediated cancer pathways, which provides important molecular perspectives for treatment or research [##REF##28718426##164##, ##REF##30268436##165##].</p>", "<p id=\"Par65\">The level of miR-19b is upregulated by the activation of the EGFR/AKT pathway, which inhibits the apoptosis pathway in NSCLC cells via targeting Bcl2L11 and PPP2R5E, thereby promoting the proliferation of tumor cells [##REF##29455644##153##]. MiR-423 indirectly attenuates the inhibition of AKT via targeting ING-4 and CREBZF, and it promotes chemotherapy resistance in GBM and BC respectively [##REF##27471108##166##, ##REF##35094653##167##] (Fig. ##FIG##6##7##B). MiR-30d and miR-130b attenuate the inhibition of AP-1 via targeting MYDT1 and ARHGAP1 respectively, which promote the progression of PRAD and ES [##REF##28241827##168##, ##REF##28748534##169##] (Fig. ##FIG##6##7##C). These reported Cardio-miRNAs can indirectly promote the malignant progression of tumors through the cross-targeting of some other pathways, which reflects the diversity of regulatory mechanisms of Cardio-miRNAs. The regulatory mechanisms involved in this minority Cardio-miRNAs only supplement the four major pathways mentioned above. In fact, there are some other regulatory mechanisms that need to be further improved according to the latest studies reports in the future.</p>", "<title>Perspectives and challenges</title>", "<p id=\"Par66\">The cellular regulatory processes and mechanisms between miRNAs and CVD have been well studied [##REF##30354259##170##–##REF##29877320##172##]. However, the role of miRNAs as a systemic influence in the synthesis of crosstalk between different diseases is still less. Especially, some typical biomarkers reflected by changes of expression profile for Cardio-miRNAs in circulation, which may become an important factor that diseases associated with age [##REF##33533026##26##, ##REF##25364765##30##, ##REF##32642841##173##]. Therefore, Cardio-miRNAs may be keys to potential targets that treating chronic complications and malignant progression of tumor [##REF##25364765##30##, ##REF##35261178##174##]. We should also pay more attention to these adverse effects that reverse Cardio-Oncology in the clinical treatment of cancer based on the principles of precision medicine. In this way, a holistic approach to multiple diseases, classification and multi-level diagnosis is carried out to evaluate the regulatory mechanism of reverse cardio-oncology [##REF##33144898##12##–##REF##32843909##14##]. Such as miR-21 is a representation that targets multiple signaling pathways, including PTEN/PI3K/AKT, NF-κB and apoptosis signaling pathways etc., and it also exhibits typical characteristics of Onco-miRNAs (Fig. ##FIG##1##2##) [##REF##21909994##175##–##REF##32244168##177##]. We should predict and evaluate the possible adverse consequences of miR-21 due to underlying metabolic disease, work life and diet according to the 3PM principle. In addition, it cannot be ignored that the upregulation of Cardio-miRNA expression levels may be as a phenotype of the toxic stress damage of chemotherapy drugs on the cardiovascular system during tumor treatment [##REF##32642841##173##]. Particularly, chemotherapy for middle-aged and elderly tumor patients should reduce cardiovascular damage at the same time, because it may be a disadvantage for tumor treatment [##REF##27974396##178##]. Based on the special phenotypes of CVD, we need take a systematic and holistic approach to consider CVD as an important risk factor for tumor malignancy.</p>", "<p id=\"Par67\">However, the adverse effects of Cardio-miRNAs may be a persistent problem for concomitant tumors of aged and obese patients [##REF##31918577##99##]. In middle-aged and older patients, a systemic treatment (3PM) may be more benefit to improving outcomes for concomitant tumors. For example, statins protect cardiovascular by lowering blood lipids or cholesterol, and a combination of drugs can be taken into consideration to treat tumor patients with arteriosclerosis and coronary heart disease. In addition, cardiotoxic chemotherapy drugs (such as anthracyclines), which are often used in chemotherapy for a variety of clinical tumors, and they can be considered to combined with cardioprotective drugs for tumor treatment, which is more likely to achieve a good prognosis (Table ##TAB##1##2##) [##REF##32192047##179##–##REF##34157723##182##]. Furthermore, molecular therapy has gradually become an important method for tumor treatment, such as the use of inhibitors (reverse complementary mimics) targeting Cardio-miRNAs to reduce their adverse effects during treatment process. However, experimental studies are needed to ensure its safety and efficacy before it can be applied clinically [##REF##35637964##183##].</p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors are grateful for Hongbo Ma and Yun Xiong for the suggestions on the revision in this manuscript and work. Sincere thanks to the relevant personnel of the National Geriatric Clinical Research Center for their platform and help.</p>", "<title>Author contributions</title>", "<p>Study concept and design: MY, KS, TL and SG; Literature collection and integration: MY, KS and NH; Analysis and interpretation of data: SG, WX, HG, MY; Statistical analysis: KS, HC, WX, CG; Drafting of the manuscript: MY, KS, SG; Critical revision and final approval of the manuscript: HX. All authors contributed to the article and approved the submitted version.</p>", "<title>Funding</title>", "<p>This study is supported by the National Natural Science Foundation of China Grant (81771511, 82071589 and 81700044); National Key R&amp;D Program of China Grant (2018YFC2000400); National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Grant (Z2018B04); Grant 2021–2025 from Sichuan Provincial Cadre Health Committee, Grant 2022YFS0108 from Science and Technology Department of Sichuan Province and Grant 2021LY22 from Sichuan Provincial People’s Hospital.</p>", "<title>Availability of data and materials</title>", "<p>All datasets generated and analyzed during the current study are available from the corresponding author on request.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par69\">This review article does not contain relevant ethical matters, and all data in this review are based on relevant published studies.</p>", "<title>Consent for publication</title>", "<p id=\"Par70\">All authors agreed to the publication of the review article.</p>", "<title>Competing interests</title>", "<p id=\"Par71\">The authors declare that they have not any competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Effects of Cardio-miRNA on tumors via circulation. The Cardio-miRNAs derived from plasma exosomes from obese or aging populations can influence adaptive survival or progression of tumor cells via circulation</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Cardio-/Onco-miRNA is involved in regulating four signaling pathways for adaptive survival of tumor cells. <bold>A</bold> Cardio-miRNAs that regulating the PTEN/PI3K/AKT signaling pathway; <bold>B</bold> cardio-miRNAs that regulating the Wnt/β-Catein signaling pathway; <bold>C</bold> cardio-miRNAs that regulating the NF-κB signaling pathway; <bold>D</bold> cardio-miRNAs that regulating the apoptosis signaling pathway</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Cardio-miRNAs regulate tumor cells adaptive progression through the PTEN/PI3K/AKT signaling pathway. Bold black indicates key node protein factors involved in the pathway; Bold red letters indicate Cardio-miRNAs. (The red bold represents circulation-derived exosomes Cardio-miRNAs, which may be upregulated in tumor cells; solid arrows indicate promotion or activation; Line segment indicate inhibition)</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Cardio-miRNAs regulate tumor cells adaptive progression through the Wnt/β-Catein signaling pathway. Bold white indicates key node protein factors involved in the pathway; Bold red letters indicate Cardio-miRNAs. (The red bold represents circulation-derived exosomes Cardio-miRNAs, which may be upregulated in tumor cells; solid black arrows indicate promotion or activation; Line segment indicate inhibition; dashed arrows indicate multi-step transfers; White corner arrows indicate gene transcription expression)</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Cardio-miRNAs regulate tumor cells adaptive progression through the NF-κB signaling pathway. Bold black indicates key node protein factors involved in the pathway; bold red letters indicate Cardio-miRNAs. (The red bold represents circulation-derived exosomes Cardio-miRNAs, which may be upregulated in tumor cells; solid black arrows indicate promotion or activation; line segment indicate inhibition; dashed arrows indicate multi-step transfers or activation; white corner arrows indicate gene transcription expression)</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Cardio-miRNAs regulate tumor cells adaptive progression through the apoptosis signaling pathway. Bold black indicates key node protein factors involved in the pathway; Bold red letters indicate Cardio-miRNAs. (The red bold represents circulation-derived exosomes Cardio-miRNAs, which may be upregulated in tumor cells; solid black arrows indicate promotion or activation; Line segment indicate inhibition; Dashed arrows indicate multi-step activation)</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>Regulation of tumor cells adaptive progression by other cardio-miRNAs. <bold>A</bold> Cardio-miRNA regulates the TGF-β signaling pathway; <bold>B</bold> cardio-miRNA regulates the AKT signaling pathway; <bold>C</bold> cardio-miRNA regulates the AP-1 signaling pathway. (The red bold represents circulation-derived exosomes Cardio-miRNAs, which may be upregulated in tumor cells; the arrows/segments corresponding to solid/dashed lines of the same color represent the same pathway)</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Regulation of tumor malignancy corresponding to Cardio-miRNAs</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"5\">Cardiovascular disease (CVD)-miRNAs</th><th align=\"left\" colspan=\"3\">Tumor-promoting effect</th></tr><tr><th align=\"left\">miRNA</th><th align=\"left\">Source<sup>a</sup></th><th align=\"left\">Expression in Change</th><th align=\"left\">Pathology</th><th align=\"left\">References</th><th align=\"left\">Tumor</th><th align=\"left\">Target in tumor tissue</th><th align=\"left\">References</th></tr></thead><tbody><tr><td align=\"left\">miR-16</td><td align=\"left\">Circulation</td><td align=\"left\">↑,↓</td><td align=\"left\">ICM, AS, CAM</td><td align=\"left\">[##REF##35162959##101##, ##REF##35370493##115##]</td><td align=\"left\"><p>LUAD, HCC</p><p>NSCLC</p></td><td align=\"left\"><p>TFAP2A/PSG9/TGF-β</p><p>LDH-A/lactate/NF-κB</p></td><td align=\"left\">[##REF##35714705##131##, ##REF##33824285##160##, ##REF##35666610##184##]</td></tr><tr><td align=\"left\">miR-19a</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">AMI, CAD</td><td align=\"left\">[##REF##25383678##90##, ##REF##27357355##91##]</td><td align=\"left\"><p>GC, HCC</p><p>GC</p><p>UC</p><p>ccRCC</p></td><td align=\"left\"><p>PTEN/PI3K/AKT</p><p>SMAD2/Wnt/β-catenin</p><p>Bim—apoptosis</p><p>PTEN/SMAD4</p></td><td align=\"left\">[##REF##30793479##54##–##REF##24122582##57##, ##REF##31186404##103##, ##REF##32591507##152##]</td></tr><tr><td align=\"left\">miR-19b</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">HF, AMI, AS</td><td align=\"left\">[##REF##34612058##142##–##REF##33756273##144##]</td><td align=\"left\"><p>NSCLC</p><p>CRC</p></td><td align=\"left\"><p>BCL2L11/PPP2R5E</p><p>SMAD4, Bim—apoptosis</p></td><td align=\"left\">[##REF##31186404##103##, ##REF##32591507##152##, ##REF##29455644##153##, ##REF##28938919##185##]</td></tr><tr><td align=\"left\">miR-21</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">ASO, CAD, HF, AMI</td><td align=\"left\">[##REF##26198441##97##, ##REF##31660076##116##, ##REF##31033484##117##, ##REF##31782762##186##]</td><td align=\"left\"><p>UC, HCC, NSCLC, CRC, GBM, ccRCC</p><p>CRC</p><p>RCC</p><p>PC</p></td><td align=\"left\"><p>PTEN/PI3K/AKT</p><p>PTEN/Akt/IKKβ</p><p>PTEN/Akt/NF-ĸB</p><p>p53/p21-cyclin E2-Bax/caspase-3</p><p>FASL</p></td><td align=\"left\">[##REF##24122582##57##–##REF##23792563##62##, ##REF##21806946##134##, ##REF##23177026##154##, ##REF##28184919##156##, ##REF##35205607##187##]</td></tr><tr><td align=\"left\">miR-25</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">HF</td><td align=\"left\">[##REF##25214573##188##, ##REF##24670661##189##]</td><td align=\"left\"><p>TNBC, RBM,</p><p>BC, HCC</p><p>CCA</p></td><td align=\"left\"><p>BTG2/AKT/ERK/MAPK</p><p>PTEN/AKT, MEK4/JNK1</p><p>TRAIL</p></td><td align=\"left\">[##REF##31336343##63##, ##REF##27840896##64##, ##REF##29310680##77##, ##REF##21953056##155##, ##REF##33494806##190##]</td></tr><tr><td align=\"left\">miR-27a</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">CAD, HF, AMI</td><td align=\"left\">[##REF##35553331##92##–##REF##31092195##94##]</td><td align=\"left\"><p>RCC</p><p>NSCLC</p><p>GC</p><p>TNBC</p></td><td align=\"left\"><p>TXNIP</p><p>SMAD2/SMAD4</p><p>PHLPP2/AKT</p><p>GSK-3β/Wnt/β-catenin</p></td><td align=\"left\">[##REF##28327189##76##, ##REF##32801869##104##, ##REF##28370334##161##, ##REF##31629934##191##]</td></tr><tr><td align=\"left\">miR-29a</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">HCM, CHD</td><td align=\"left\">[##REF##24161319##95##, ##REF##31971908##96##]</td><td align=\"left\">AB</td><td align=\"left\">CTNNBIP1/Wnt/β-catenin</td><td align=\"left\">[##REF##34636093##192##]</td></tr><tr><td align=\"left\">miR-30d</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">HF, AMI</td><td align=\"left\">[##REF##31092195##94##, ##REF##25995320##193##]</td><td align=\"left\">PC</td><td align=\"left\">MYPT1/c-JUN/VEGFA</td><td align=\"left\">[##REF##28241827##168##]</td></tr><tr><td align=\"left\">miR-92a</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">CAD, CAV</td><td align=\"left\">[##REF##26198441##97##, ##REF##30582459##98##]</td><td align=\"left\"><p>HCC, NSCLC</p><p>SCCOT</p><p>OC</p><p>CRC</p></td><td align=\"left\"><p>PTEN/PI3K/AKT</p><p>DKK1/ Wnt/β-catenin</p><p>PTEN</p></td><td align=\"left\">[##REF##32917956##65##, ##REF##28534966##66##, ##REF##28209618##105##, ##REF##28779483##194##]</td></tr><tr><td align=\"left\">miR-92b</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">HF, CAD, PH</td><td align=\"left\">[##REF##22120965##195##–##REF##32829668##197##]</td><td align=\"left\"><p>GC</p><p>SCLC, GBM</p></td><td align=\"left\"><p>DAB2IP/PI3K/AKT</p><p>PTEN/AKT</p></td><td align=\"left\">[##REF##34136482##67##, ##REF##31713929##78##, ##REF##26893028##198##]</td></tr><tr><td align=\"left\">miR-106b</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">CAD, HF</td><td align=\"left\">[##REF##25656948##145##, ##REF##30090932##146##]</td><td align=\"left\"><p>BC, CRC</p><p>HCC</p><p>CRC</p><p>ccRCC</p></td><td align=\"left\"><p>PTEN/PI3K/AKT</p><p>GPM6A/DYNC1I1/AKT/ERK</p><p>p21</p><p>TRIM8/p21</p></td><td align=\"left\">[##REF##28518139##68##, ##REF##26238857##69##, ##REF##35685464##82##, ##REF##28327152##158##, ##REF##35002514##199##]</td></tr><tr><td align=\"left\">miR-126</td><td align=\"left\">Circulation</td><td align=\"left\">↓,↑</td><td align=\"left\"><p>AMI, AS,</p><p>IHD, SA, UA</p></td><td align=\"left\">[##REF##26916800##126##, ##REF##30833907##147##–##REF##24260372##151##]</td><td align=\"left\"><p>ccRCC</p><p>BALL</p></td><td align=\"left\"><p>SLC7A5/mTOR-HIF</p><p>p53</p></td><td align=\"left\">[##REF##27300437##157##, ##REF##28257806##200##, ##REF##25572155##201##]</td></tr><tr><td align=\"left\">miR-130b</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">MI/R, RCVS</td><td align=\"left\">[##REF##31918577##99##, ##REF##33314303##100##]</td><td align=\"left\"><p>BC, OS, RCC</p><p>ES</p><p>ccRCC</p><p>NSCLC</p></td><td align=\"left\"><p>PTEN, PTEN/AKT</p><p>ARHGAP1/CDC42/PAK1/AP1</p><p>WNK, TCF4</p><p>PTEN/Wnt-β-catenin</p></td><td align=\"left\">[##REF##31228937##70##–##REF##29653464##73##, ##REF##28748534##169##, ##REF##35689211##202##]</td></tr><tr><td align=\"left\">miR-133a</td><td align=\"left\">Circulation</td><td align=\"left\">↓,↑</td><td align=\"left\">CAD, ACS, AMI</td><td align=\"left\">[##REF##24053180##102##, ##REF##31782762##186##]</td><td align=\"left\"><p>OS</p><p>OC</p></td><td align=\"left\"><p>Bcl-xL/Mcl-1</p><p>PYGB/Wnt-β-catenin</p></td><td align=\"left\">[##REF##31627092##106##, ##REF##23756231##159##]</td></tr><tr><td align=\"left\">miR-146a</td><td align=\"left\">Circulation</td><td align=\"left\">↑,↓</td><td align=\"left\">AMI, ACS, HF, CAD</td><td align=\"left\">[##REF##31092195##94##, ##REF##34209965##118##–##REF##23559634##120##]</td><td align=\"left\">NSCLC</td><td align=\"left\">TRAF6/NF-ĸB/p65</td><td align=\"left\">[##REF##33015045##135##]</td></tr><tr><td align=\"left\">miR-146b</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">MI, PH, HF</td><td align=\"left\">[##REF##32845445##121##, ##REF##31338525##122##]</td><td align=\"left\"><p>TC</p><p>BC</p><p>ccRCC</p></td><td align=\"left\"><p>PTEN/PI3K/AKT</p><p>AUF1/ETS2/MMP2</p><p>TRIM2, TRAF6</p></td><td align=\"left\">[##REF##29353884##74##, ##REF##35342411##79##, ##REF##30206978##80##, ##REF##31071529##203##]</td></tr><tr><td align=\"left\">miR-150</td><td align=\"left\">Circulation</td><td align=\"left\">↓,↑</td><td align=\"left\">PH, HF, AMI</td><td align=\"left\">[##REF##23220912##123##–##REF##31179325##125##]</td><td align=\"left\">NSCLC</td><td align=\"left\">FOXO4/NF-ĸB/Snail</td><td align=\"left\">[##REF##27976702##132##]</td></tr><tr><td align=\"left\">miR-155</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">AS, DCM</td><td align=\"left\">[##REF##26916800##126##, ##REF##25840506##127##]</td><td align=\"left\"><p>MM</p><p>ccRCC</p><p>BC</p><p>CRC</p></td><td align=\"left\"><p>SOCS1/JAK2/STAT3</p><p>IGF1R/PI3K/AKT</p><p>RKIP</p><p>PPP2CA/AKT/NF-κB</p></td><td align=\"left\">[##REF##34131104##81##, ##REF##21806946##134##, ##REF##30285793##204##, ##REF##32580736##205##]</td></tr><tr><td align=\"left\">miR-208</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">CAD, ACS, AMI</td><td align=\"left\">[##REF##31782762##186##, ##REF##26526403##206##, ##REF##21900086##207##]</td><td align=\"left\"><p>PC</p><p>HCC</p></td><td align=\"left\"><p>E-Cadherin/PI3K/AKT/ GSK-3β</p><p>ARID2/IFITM1</p></td><td align=\"left\">[##REF##26169693##163##, ##REF##24604208##208##]</td></tr><tr><td align=\"left\">miR-200a</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">HCM, SCA</td><td align=\"left\">[##REF##26404540##209##, ##REF##27423422##210##]</td><td align=\"left\">BC</td><td align=\"left\">Dicer/miR-16/JNK2/MMP-2 axis</td><td align=\"left\">[##REF##31772330##211##]</td></tr><tr><td align=\"left\">miR-210</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">PH, HF, CAD</td><td align=\"left\">[##REF##27357355##91##, ##REF##32493166##129##, ##REF##33783502##130##]</td><td align=\"left\"><p>PC</p><p>NSCLC</p><p>OC</p></td><td align=\"left\"><p>OCS1/TNIP1/p65/NF-κB</p><p>UPF1/PTEN/PI3K/AKT</p><p>EphrinA3-PI3K/AKT</p></td><td align=\"left\">[##REF##32446904##75##, ##REF##28693582##212##, ##REF##32180353##213##]</td></tr><tr><td align=\"left\">miR-223</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">AS, AMI, SC, AD</td><td align=\"left\">[##REF##30623425##143##, ##REF##26065992##214##, ##REF##27099265##215##]</td><td align=\"left\">ccRCC</td><td align=\"left\">SLC4A4, HIF-2α</td><td align=\"left\">[##REF##30668544##216##, ##REF##34205829##217##]</td></tr><tr><td align=\"left\">miR-423</td><td align=\"left\">Circulation</td><td align=\"left\">↑</td><td align=\"left\">AMI, HF</td><td align=\"left\">[##REF##31092195##94##, ##REF##23388090##128##]</td><td align=\"left\"><p>GBM</p><p>PC</p><p>BC</p></td><td align=\"left\"><p>ING-4/AKT/ERK</p><p>GREM2/TGF-β, CREBZF</p><p>TNIP2/NF-ĸB</p></td><td align=\"left\">[##REF##32606763##133##, ##REF##33144675##162##, ##REF##27471108##166##, ##REF##35094653##167##]</td></tr><tr><td align=\"left\">miR-451</td><td align=\"left\">Circulation</td><td align=\"left\">↓</td><td align=\"left\">PH, AMI</td><td align=\"left\">[##REF##30623425##143##, ##REF##29795360##218##]</td><td align=\"left\">ccRCC</td><td align=\"left\">PSMB8</td><td align=\"left\">[##REF##22440013##219##, ##REF##26779781##220##]</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Potential drugs for joint pharmacologic prevention of cardiovascular disease and cancer (Masoudkabir et al. [##REF##28624099##181##])</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Drug</th><th align=\"left\">Direct target</th><th align=\"left\">Indirect targets</th><th align=\"left\">Action on CVD</th><th align=\"left\">Action on cancer</th></tr></thead><tbody><tr><td align=\"left\">Statins</td><td align=\"left\">HMG-CoAreductase inhibition</td><td align=\"left\"><p>• AMPK activation</p><p>• Inhibition of Cyclines &amp; cycline-dependent kinases</p><p>• Up-regulation of tumor-suppressors (p53, p27, p21)</p><p>• Inhibition of PI3K, serineethreonine kinases, NF-κB, and MAPKs signaling pathways</p></td><td align=\"left\"><p>Improving endothelial function Plaque stabilization</p><p><bold>↓</bold> Atherosclerosis progression</p><p>↓ Myocardial infarction and stroke</p><p>↓ Cardiovascular mortality</p></td><td align=\"left\"><p>Tumor-suppressor and anti-cancer role through:</p><p>↑ Apoptosis</p><p>↓ Proliferation</p><p>↓ Invasion</p><p>↑ Radiosensitization</p><p>↓ DNA damage</p></td></tr><tr><td align=\"left\">ASA</td><td align=\"left\">Inhibition of COX1</td><td align=\"left\">• AMPK activation?</td><td align=\"left\"><p>↓ Myocardial infarction and stroke</p><p>↓ Cardiovascular mortality</p></td><td align=\"left\"><p>↓ Cancer incidence</p><p>↓ Cancer death</p></td></tr><tr><td align=\"left\">ACEIs/ARBs</td><td align=\"left\">ACE inhibition/angiotensin II receptor antagonism</td><td align=\"left\"><p>• ↓ VEGF expression</p><p>• PPAR-γ activation</p></td><td align=\"left\"><p>Improving endothelial function Plaque stabilization</p><p>↓ Atherosclerosis progression</p><p>↓ Myocardial infarction and stroke</p><p>↓ Cardiovascular mortality</p></td><td align=\"left\"><p>↓ Cancer incidence</p><p>Tumor-suppressor and anti-cancer role through:</p><p>↓ DNA damage</p><p>↑ Apoptosis</p><p>↑ Differentiation</p><p>↓ Angiogenesis</p><p>↓ Cell growth</p></td></tr><tr><td align=\"left\">Metformin</td><td align=\"left\">Unknown</td><td align=\"left\">• AMPK activation</td><td align=\"left\"/><td align=\"left\"><p>↓ Cancer incidence</p><p>Tumor suppression by regulating cellular proliferation, cell cycle progression and cellular survival</p></td></tr><tr><td align=\"left\">TZDs</td><td align=\"left\">PPAR-γ agonism</td><td align=\"left\"><p>• AMPK activation</p><p>• Wnt/β-catenin signaling pathway inhibition</p><p>• IGF-1 inhibition</p><p>• Inhibition of leptin gene expression</p></td><td align=\"left\"><p>↓ Coronary and carotid atherosclerosis</p><p>↓ Thrombus formation and acute myocardial infarction and stroke</p><p>↓ Blood pressure</p></td><td align=\"left\"><p>Tumor suppression through:</p><p>↓ Angiogenesis</p><p>↑ Apoptosis</p><p>↓ Self-renewal of cancer cells</p><p>↑ Differentiation</p></td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p><sup>a</sup>Circulation refers to clinical samples derived from serum or plasma</p></table-wrap-foot>", "<table-wrap-foot><p><italic>HMG-CoA-reductase</italic> 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, <italic>AMPK</italic> Adenosine 50 monophosphate -activated protein kinase, <italic>PI3K</italic> phosphoinositide 3-kinase, <italic>NFekB</italic> nuclear factor kappa-B, <italic>MAPK</italic> mitogen-activated kinases, <italic>CVD</italic> cardiovascular disease, <italic>COX1</italic> cyclooxygenase 1, <italic>ACEIs/ARBs</italic> angiotensin-converting enzyme inhibitors/angiotensin II receptor antagonists, <italic>ACE</italic> angiotensin-converting enzyme, <italic>VEGF</italic> vascular endothelial growth factor, <italic>PPAR-g</italic> peroxisome proliferator-activated receptor-g, <italic>TZDs</italic> thiazolidinediones</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Ming Yang and Tiepeng Li contributed equally to this work.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"12967_2023_4680_Fig1_HTML\" id=\"MO1\"/>", "<graphic xlink:href=\"12967_2023_4680_Fig2_HTML\" id=\"MO2\"/>", "<graphic xlink:href=\"12967_2023_4680_Fig3_HTML\" id=\"MO3\"/>", "<graphic xlink:href=\"12967_2023_4680_Fig4_HTML\" id=\"MO4\"/>", "<graphic xlink:href=\"12967_2023_4680_Fig5_HTML\" id=\"MO5\"/>", "<graphic xlink:href=\"12967_2023_4680_Fig6_HTML\" id=\"MO6\"/>", "<graphic xlink:href=\"12967_2023_4680_Fig7_HTML\" id=\"MO7\"/>" ]
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[{"label": ["28."], "surname": ["Zhang", "Yu"], "given-names": ["L", "D"], "article-title": ["Exosomes in cancer development, metastasis, and immunity"], "source": ["Biochim Biophys Acta Rev Cancer"], "year": ["1871"], "volume": ["2019"], "fpage": ["455"], "lpage": ["468"]}, {"label": ["50."], "surname": ["Wu", "Xing", "Wu", "Watabe"], "given-names": ["K", "F", "SY", "K"], "article-title": ["Extracellular vesicles as emerging targets in cancer: recent development from bench to bedside"], "source": ["Biochim Biophys Acta Rev Cancer"], "year": ["1868"], "volume": ["2017"], "fpage": ["538"], "lpage": ["563"]}, {"label": ["51."], "surname": ["Zhang", "Li", "Li", "Li", "Guo", "Yao", "Mi"], "given-names": ["J", "S", "L", "M", "C", "J", "S"], "article-title": ["Exosome and exosomal microRNA: trafficking, sorting, and function"], "source": ["Genomics Proteomics Bioinform"], "year": ["2015"], "volume": ["13"], "fpage": ["17"], "lpage": ["24"], "pub-id": ["10.1016/j.gpb.2015.02.001"]}, {"label": ["114."], "mixed-citation": ["Correction for Jeon et al. A set of NF-kappaB-regulated microRNAs induces acquired TRAIL resistance in Lung cancer, Proc Natl Acad Sci U S A. 2021. 118."]}]
{ "acronym": [ "AMI", "MI", "ASO", "CAD", "ACS", "HF", "AS", "PH", "DCM", "ACAS", "MI/R", "RCVS", "SCAD", "ICM", "HCM", "CHD", "CAV", "IHD", "SA", "UA", "CAM", "LC", "AB", "BALL", "CCA", "CRC", "PRADC", "GC", "UC", "HCC", "NSCLC", "GBM", "BC", "TNBC", "RBM", "RCC", "ccRCC", "SCLC", "ES", "OS", "OC", "MM", "TC", "PC" ], "definition": [ "Acute myocardial infarction", "Myocardial infarction", "Atherosclerosis obliterans", "Coronary artery disease", "Acute coronary syndrome", "Heart failure", "Atherosclerosis", "Pulmonary hypertension", "Dilated cardiomyopathy", "Asymptomatic carotid artery stenosis", "Myocardial ischemia/reperfusion", "Reversible cerebral vasoconstriction syndrome", "Spontaneous coronary artery dissection", "Ischemic dilated cardiomyopathy", "Hypertrophic cardiomyopathy", "Coronary heart disease", "Cardiac allograft vasculopathy", "Ischemic heart disease", "Stable angina", "Unstable angina", "Cardiomyopathy", "Lung cancer", "Ameloblastoma", "B cell precursor acute lymphoblastic leukemia", "Cholangiocarcinoma", "Colorectal cancer", "Prostate cancer", "Gastric cancer", "Urothelial carcinomas", "Hepatocellular carcinoma", "Non-small cell lung cancer", "Glioblastoma", "Bladder cancer", "Triple-negative breast cancer", "Retinoblastoma", "Renal cell carcinoma", "Clear cell renal cell carcinoma", "Small cell lung cancer", "Ewing sarcoma", "Osteosarcoma", "Ovarian cancer", "Malignant melanoma", "Thyroid cancer", "Pancreatic cancer" ] }
220
CC BY
no
2024-01-14 23:43:48
J Transl Med. 2024 Jan 12; 22:50
oa_package/c9/6f/PMC10787510.tar.gz
PMC10787518
0
[ "<title>Introduction</title>", "<p>High-risk pulmonary embolism (PE) is an obstruction of the pulmonary arterial tree that exceeds 50% of the cross-sectional area, resulting in severe pulmonary compromise and acute cardiovascular failure due to right ventricular overload.<sup>##REF##16397945##1##</sup> A definitive diagnosis requires imaging studies such as ventilation-perfusion scanning (VQ scan), contrast pulmonary angiography (CTPA), computed tomographic [CT] angiography, or echocardiography.<sup>##REF##23940438##2##</sup> The prognosis of PE depends on the severity of clot burden or obstruction.<sup>##REF##23940438##2##</sup> Low-risk PE typically has a good prognosis with less than 1% mortality, while intermediate high-risk and high-risk PE carry mortality of 5-25% and more than 20%, respectively.<sup>##REF##23940438##2##</sup> Management includes urgent systemic thrombolysis and anticoagulation if there are no contraindications or surgical/catheter-based embolectomy in selected critical cases with good outcomes. However, the choice of intervention depends on the patient’s clinical status and the available hospital resources.<sup>##REF##33087152##3##,##REF##30233892##4##</sup> We present a challenging case of high-risk PE leading to multiple cardiac arrests in a female with severe thrombocytopenia in a public healthcare-based facility.</p>", "<p>“The authors took consent from the patient and approval from the institution for the publication of this case report (MRC-04-22-143).”</p>" ]
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[ "<title>Discussion</title>", "<p>High-risk pulmonary embolism (PE) is characterized by a drop in systolic pressure to less than 90 mmHg or a decline in systolic arterial pressure of at least 40 mmHg for at least 15 minutes, which is not caused by new-onset arrhythmias or shock.<sup>##REF##23940438##2##,##REF##23319967##5##</sup> It carries high mortality; thus, early diagnosis is the key to managing PE. In patients with high-risk PE, 50% die within 30 minutes, 70% die within an hour, and more than 85% die within 6 hours of the onset of symptoms,<sup>##REF##23319967##5##</sup> emphasizing the need for rapid evaluation and prompt intervention. According to the British Thoracic Society (BTS), major risk factors for PE include major abdominal or pelvic surgery, hip/knee joint replacement, a history of previous venous thromboembolism (VTE), limited immobility as seen in the prolonged bed-bound state, and malignancies.<sup>##REF##23940438##2##</sup></p>", "<p>Definitive diagnosis is made through imaging studies such as bedside TTE, VQ scan, CT angiography, and CTPA.<sup>##REF##23940438##2##</sup> Management guidelines for high-risk PE include the prompt administration of systemic fibrinolysis/thrombolysis in stable patients without having a risk of bleeding or contraindication or systemic embolectomy/catheter-based thrombectomy in severely hemodynamically compromised patients or those with contraindication to fibrinolysis, which reduces both morbidity and mortality.<sup>##REF##27373187##6##, ####REF##15867775##7##, ##REF##16009801##8####16009801##8##</sup> Certain malignancies are associated with thrombocytopenia and pose a significant risk factor for PE, even in mobile patients.<sup>##REF##23940438##2##,##REF##32286024##9##,##REF##18287133##10##</sup> The patient, in this case, had lymphoma and severe thrombocytopenia9,10 with a platelet count of less than 10,000/μL.</p>", "<p>Furthermore, our patient had high-risk PE, suggested by her underlying lymphoma, elevated d-dimers, and TTE showing RA and RV dilation with McConnell’s sign. She was critically ill, requiring high inotropic support, and experienced multiple consecutive cardiac arrests. In this clinical scenario, mortality was inevitable, and thrombolysis carried an increased risk of major bleeding, such as intracranial hemorrhage. However, after the third arrest, the MICU team decided to give half a bolus dose of the recommended dose of IV Alteplase, i.e., 50mg, as a last resort to saving her life. Her clinical and laboratory parameters improved drastically, leading to successful extubation after two days.</p>", "<p>Severe thrombocytopenia of &lt;100,000/mm3 is a contraindication to thrombolysis therapy in stroke patients.<sup>##REF##26288669##11##</sup> Unfortunately, the literature is deficient, and there is scarce data regarding the management and outcome of high-risk PE associated with thrombocytopenia, commonly associated with autoimmune conditions and immunosuppressive states like malignancies.<sup>##REF##30805221##12##</sup> A half dose of IV thrombolysis has been reported to show similar beneficial effects and bleeding risk in intermediate high-risk or sub-massive PE; however, implementing this approach in high-risk PE remains questionable.<sup>##REF##29979222##13##,##UREF##0##14##</sup></p>" ]
[ "<title>Conclusion</title>", "<p>Malignancy carries a high risk of PE, and certain malignancies, particularly hematological ones, are associated with thrombocytopenia. Thus, in such clinical scenarios, where the management of PE intercepts with thrombocytopenia, decisions become individualized, requiring a delicate balance between benefits and risks. In the presented case, the paper aims to highlight a contemporary approach to managing a life-threatening condition of high-risk PE and thrombocytopenia with a good clinical outcome. Such rare reportable cases bridge the gap in literature deficiency where no standards of care have been established. However, it is imperative to acknowledge the need for further studies to formulate a standardized management approach.</p>" ]
[ "<p>Background: Managing a high-risk pulmonary embolism (PE) in a critically ill patient with severe thrombocytopenia can present a challenging dilemma. There is a high risk of fatal bleeding due to anticoagulation in high-risk PE with thrombocytopenia; therefore, risks and benefits are balanced while dealing with such a critical scenario.</p>", "<p>Case Report: We present a case of a female patient with thrombocytopenia who was admitted for management of lymphoma. Her hospital course was complicated by high-risk PE, leading to acute respiratory failure and hypotension, necessitating urgent transfer to the medical intensive care unit. She was intubated and placed on mechanical ventilation. Multiple cardiac arrests occurred due to compromised cardiac output from a severely dilated right ventricle on bedside transthoracic echocardiography. As a last resort to save her life in this critical state and severe thrombocytopenia, she was given a half bolus dose of the recommended drug, i.e., 50mg IV of Alteplase. Subsequently, she stabilized and was extubated without any further complications.</p>", "<p>Discussion: High-risk PE needs prompt management with anticoagulation to avoid fatal outcomes. However, on the other hand, anticoagulation carries a high risk of bleeding, especially in patients with thrombocytopenia. These challenges prompt a modern perspective in situations where clear guidelines are absent.</p>", "<p>Conclusion: We aim to discuss our contemporary clinical practice in managing such a complex case and highlight the need for further studies.</p>", "<title>Keywords:</title>" ]
[ "<title>Case Presentation</title>", "<p>A 42-year-old Filipino woman with a recent diagnosis of lymphoma made two months before her presentation to the emergency department (ED) arrived with two episodes of fainting within a day. She reported symptoms of palpitations, lightheadedness, and exertional shortness of breath. Despite her lymphoma diagnosis, she had not attended her follow-up visits. Upon presentation, her initial vital signs indicated a low-grade fever of 37.8°C, a heart rate of 110 beats per minute, a respiratory rate of 24 breaths per minute, and normal room air oxygen saturation of 97%. Physical examination revealed a right cervical mass, multiple enlarged cervical, supraclavicular, and inguinal lymph nodes, and splenomegaly.</p>", "<p>An urgent consultation with a hematologist/oncologist was sought for the management of lymphoma. She was subsequently transferred to a local cancer hospital for appropriate care. During her stay, she underwent staging through a positron emission tomography scan (PET CT), and a bone marrow biopsy was performed before initiating induction chemotherapy. Due to thrombocytopenia (platelet count less than 10,000/μL), intravenous immunoglobulins (IVIG) were initiated.</p>", "<p>Within 24 hours of starting the IVIG infusion, she developed hypotension, with a mean arterial pressure (MAP) of 55 mmHg (millimeter of mercury), tachypnea (respiratory rate of 35-38/min), and tachycardia (120 beats per minute). She was afebrile and required oxygen supplementation through a 15-liter non-rebreather mask to maintain an oxygen saturation of around 90%. Due to the severity of her condition, she was urgently transferred to the medical intensive care unit (MICU) within 1-2 hours of clinical deterioration.</p>", "<p>Fluid resuscitation was started, and her initial complete blood count revealed hemoglobin of 7.2 g/dL, thrombocytopenia of 6000/μL, and elevated d-dimers. An urgent bedside echocardiogram revealed a severely dilated right ventricle with a strain pattern (as illustrated in ##FIG##0##Figure 1a##), followed by the official echocardiogram that indicated a highly suspected fresh, early-forming thrombus in the inferior vena cava (IVC), approximately 2.0 cm proximal to the inferior vena cava orifice.</p>", "<p>Concurrently, the cardiothoracic team was consulted for a potential thrombectomy, but the patient’s deteriorating condition rendered her unfit for surgery. Therefore, an interventional radiologist was consulted for possible interventional radiology-guided thrombectomy or the inferior vena cava filter insertion. Still, she was hemodynamically unstable to be transferred out of the MICU ward. Over the next 3-4 hours, her clinical condition worsened, prompting a drop in her Glasgow Coma Scale (GCS) below 8, requiring endotracheal intubation and mechanical ventilation.</p>", "<p>Fifteen minutes after intubation, she developed pulseless ventricular tachycardia. Urgent cardiopulmonary resuscitation (CPR) was started per Advanced Cardiac Life Support (ACLS) protocol, achieving return of spontaneous circulation (ROSC) after one CPR cycle. Blood gases revealed an acidemia with a pH of 6.9, hCO3 of 10 meq/L, CO2 of 80 mmHg, and lactate of 18. Her mean arterial pressure (MAP) barely reached 50 mmHg, necessitating high doses of three different inotropes: noradrenaline, dopamine, and vasopressin.</p>", "<p>A repeat bedside transthoracic echocardiography (TTE) showed severe right atrium (RA) and right ventricle (RV) dilation with McConnell’s sign, as depicted in ##FIG##0##Figure 1b##. Subsequently, she experienced two more cardiac arrests with an initial rhythm of pulseless electrical activity (PEA), requiring one cycle of CPR each time to achieve ROSC. The most likely cause of her recurrent cardiac arrest was PE. However, due to severe thrombocytopenia (6000/μL) with a high risk of life-threatening intracranial bleeding, she was deemed unfit for IV thrombolysis.</p>", "<p>Transferring her out of the MICU to the interventional radiology department was challenging, and bedside thrombectomy was not an available option. Whether administering or withholding IV thrombolytics in both instances, her prognosis remained uncertain. Consequently, systemic thrombolysis therapy with 50mg of IV Alteplase over 10 minutes was administered as a last measure to save her life. Her blood pressure dramatically improved after 30 minutes of thrombolysis on the same day. Inotropic support was gradually reduced and discontinued within the next 2 hours. Her clinical and laboratory parameters also improved, as detailed in ##TAB##0##Table 1## and ##FIG##1##Figure 2##.</p>", "<p>She experienced mild to moderate fresh bleeding from her nose and mouth and received a transfusion with twelve units of platelets. After the event, her condition improved significantly, leading to successful extubation after two days. She then underwent IVC filter insertion and was transferred to the oncology hospital for lymphoma management. She remained stable during the follow-up period of 4-6 weeks.</p>", "<title>Statement of Ethics</title>", "<p>The patient has consented to publish this case. The study is conducted ethically per the World Medical Association Declaration of Helsinki.</p>", "<title>Approval from the Institutional Research Body</title>", "<p>The manuscript completed the review process by the medical research center (MRC) of Hamad Medical Corporation, and the MRC approval number is MRC-04-22-143.</p>", "<title>Conflict of Interest/Disclosure Statement</title>", "<p>The authors certify that they have no conflict of interest or affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.</p>", "<title>Patient Consent</title>", "<p>The subject has given verbal and written informed consent to publish the case.</p>", "<title>Data Availability</title>", "<p>Authors confirm that all relevant data or information are included in the article and are available via open access platform of this journal.</p>" ]
[]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1.</label><caption><p>Transthoracic echocardiography of dilated atrium showing early forming Thrombus (a) and dilated right ventricle with McConnell’s sign (b).</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2.</label><caption><p>Trend of the physiological parameters with black arrows highlighting unstable vitals during cardiac arrest and subsequent improvement post-Tpa in MICU (~time of Tpa: 9:30 am). (Units: Peripheral pulse rate and respiratory rate units in beats per minute, Blood pressure unit in mmHg).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tbl1\"><label>Table 1.</label><caption><p>Blood gas pH during cardiac arrest, pre-Tpa, and Post-Tpa response.</p></caption><table frame=\"box\" rules=\"groups\"><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Blood gas pH</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Normal range: 7.350-7.450</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Interpretation</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">During cardiac arrest</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.807</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Crit low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pre-Tpa</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.133</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Crit low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Post-Tpa after ~ 2 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.252</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Post-Tpa after ~ 4 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.400</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">normal</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Post-Tpa after ~ 6 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.441</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">normal</td></tr></tbody></table></table-wrap>" ]
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[{"element-citation": ["\n"], "surname": ["Yilmaz", "Uzun"], "given-names": ["ES", "O"], "article-title": ["Low-dose thrombolysis for submassive pulmonary embolism"], "source": ["J Investig Med"], "year": ["2021"], "month": ["Dec;"], "volume": ["69"], "issue": ["(8):"], "fpage": ["1439-1446"], "comment": ["doi: 10.1136/jim-2021-001816"]}]
{ "acronym": [], "definition": [] }
14
CC BY
no
2024-01-14 23:43:48
Qatar Med J. 2024 Jan 13; 2023(4):39
oa_package/ce/e2/PMC10787518.tar.gz
PMC10787534
0
[ "<title>Introduction</title>", "<p>The global population is ageing, making it imperative to prioritise public health initiatives that promote cognitive functioning and functional capacity.<sup>\n##UREF##0##1##\n</sup> Digital health refers to the use of digital, mobile, and wireless technologies to achieve health-related outcomes, and it has become a mainstream way to deliver healthcare services to a broad sector of the population, including older adults.<sup>##UREF##1##2##,##REF##37258624##3##</sup></p>", "<p>To ensure the development of effective digital healthcare interventions, evaluating their applicability and feasibility is essential, taking into account the specific needs and characteristics of the clinical populations.<sup>##REF##23925575##4##–##REF##19362699##6##</sup></p>", "<p>From a technological perspective, the applicability and adequacy of digital healthcare interventions are usually determined based on participants’ interaction experience.<sup>\n##REF##32450782##7##\n</sup> The concept of interaction experience (IX) includes usability aspects (i.e. the extent to which a system can be used to achieve specific goals with effectiveness and efficiency); user experience (i.e. hedonic experience and user's perceptions and responses that result from the use and/or anticipated use of a system); and accessibility (i.e. the extent to which a system addresses the needs, characteristics, and abilities of a large spectrum of users with or without impairments).<sup>\n##UREF##2##8##\n</sup> Although these three components are considered the ‘trinity’ of the technological strand of feasibility studies,<sup>\n##REF##32450782##7##\n</sup> issues related to these components when developing digital cognitive platforms are rarely mentioned or just superficially addressed.<sup>\n##UREF##3##9##\n</sup></p>", "<p>Additionally, the impact of non-clinical variables is also overlooked in the development and evaluation process of digital cognitive platforms, with the primary focus being on addressing the barriers and requirements resulting from users’ cognitive impairments with disregard for other crucial user characteristics.<sup>##UREF##0##1##,##UREF##3##9##</sup></p>", "<p>Scientific literature has shown that low digital/computer literacy, negative emotions associated with previous unsuccessful experiences with technology, computer anxiety, and low perceived computer self-efficacy can impact the adoption and use of digital healthcare interventions in older adults.<sup>##REF##24107443##10##–##UREF##5##12##</sup> For example, according to the eHealth Literacy model, using and adapting to computers and new technologies to solve problems (i.e. digital/computer literacy) is crucial to engage and use digital healthcare services.<sup>\n##UREF##6##13##\n</sup> Moreover, negative emotions associated with previous unsuccessful experiences also determine users’ perceived ease of use and technology usefulness. For instance, Portz et al. showed that past negative experiences with technology resulted in computer anxiety (i.e. apprehension or even fear of using technology) and low perceived computer self-efficacy (i.e. confidence in the ability to ‘figure things out’ while using technology), which, in turn, negatively impacted the search for and adoption of digital healthcare services by older adults.<sup>\n##UREF##7##14##\n</sup> These findings align with psychological literature, which shows that an individual's perception of their competency on a specific matter directly influences their resilience when encountering challenges.<sup>\n##REF##847061##15##\n</sup></p>", "<p>The lack of analysis of the impact of non-clinical variables during feasibility testing of digital cognitive platforms can hinder users’ ability and willingness to engage with these services and use them effectively. Therefore, it is essential to adopt a more holistic approach that considers a broader range of user characteristics to create inclusive interventions tailored to individual needs, including the necessities raised by the lack of low familiarity/proficiency in technology use that some users might present.</p>", "<p>Grounded on this rationale, in the present study, we aimed to analyse the potential barriers created by non-clinical factors to adopting and using a digital neuropsychological platform called NeuroVRehab.PT. This platform was designed to promote cognitive functioning and functional capacity to perform an instrumental activity of daily living, namely grocery shopping.<sup>\n##REF##33255869##16##\n</sup> Moreover, by gaining insight into the nature of these barriers and their potential impact on IX,<sup>\n##REF##32450782##7##\n</sup> we aimed to contribute to developing more inclusive and effective digital neuropsychological interventions.</p>" ]
[ "<title>Materials and methods</title>", "<p>In this study, we conducted a phenomenological qualitative study<sup>\n##REF##28597869##17##\n</sup> with the objective of gaining a deeper understanding of the experiences and personal perspectives concerning the strengths and challenges associated with interacting with our platform in a group of older adults with varying levels of computer confidence and computer self-efficacy. Moreover, we adhered to the Consolidated Criteria for Reporting Qualitative Research guidelines<sup>\n##UREF##8##18##\n</sup> to ensure the quality of the study's execution and reporting (see <ext-link xlink:href=\"https://journals.sagepub.com/doi/suppl/10.1177/20552076231223805\" ext-link-type=\"uri\">Table S1 in Supplementary Material</ext-link>).</p>", "<title>Recruitment and participants</title>", "<p>To recruit participants, we visited two non-profit institutions that offer educational and recreational activities specifically designed for older adults (i.e. senior universities). We presented the details of our project to attendees of two Information and Communication Technologies (ICT) classes. Eight ICT attendees (##TAB##0##Table 1##) expressed willingness to participate and were screened against the eligibility criteria shown in ##TAB##1##Table 2##.</p>", "<title>Procedure</title>", "<p>Participants who met the eligibility criteria were assigned to an individual session to use and evaluate NeuroVRehab.PT. Sessions took place in a private room at the senior universities’ facilities and were conducted by a neuropsychologist (FFB, PhD student) with previous experience in qualitative research. The second author (SA), a computer scientist and PhD student, was also present at the first two interviews to ensure the sessions went smoothly without technical issues.</p>", "<p>A Huawei MediaPad T5 tablet (Android 8) (Huawei Technologies Co. Ltd, Shenzhen, China) was placed horizontally at a 25° using a tablet stand on a table in front of participants.</p>", "<p>Before data collection, the objectives, study procedures, and participants’ rights were reviewed once again. Additionally, all participants received an informative sheet detailing the study's objectives, contact information, and the written informed consent form. Participants were encouraged to carefully read both documents and ask any questions they may have had. Demographic data, including age, sex, education level, marital status, digital literacy (self-reported confidence using technology and new technological devices, weekly time browsing the internet), and mhealth use (use of cognitive training mobile apps) were collected. All participants provided written informed consent before the start of the study.</p>", "<title>Instruments</title>", "<p>The study employed individual audio-recorded interviews lasting approximately 60 min and comprosing two parts. During the first part, the Think-Aloud method was used to collect participants’ thoughts and feelings regarding their IX<sup>##REF##32450782##7##,##UREF##2##8##</sup> with the platform. This method was selected for its capacity to bring to the surface complicated thinking processes and problem-solving strategies that participants experience while performing a task.<sup>\n##UREF##9##19##\n</sup> These thoughts are verbalised and collected as data, which researchers can analyse.<sup>\n##UREF##9##19##\n</sup> In parallel with the Think-Aloud exercise, researchers documented participants’ behaviour while interacting with the platform, using field notes from direct observation. Before participants started exploring the platform, the first author (FFB) exemplified how to perform a Think-Aloud exercise using the Gmail website as an example.</p>", "<p>The second part of the session consisted of a semi-structured interview conducted to ensure that relevant aspects of platform IX that might be neglected or omitted during the Think-Aloud exercise were collected. An interview script was collaboratively developed and discussed with the research team (see <ext-link xlink:href=\"https://journals.sagepub.com/doi/suppl/10.1177/20552076231223805\" ext-link-type=\"uri\">Doc S1 in Supplementary Material</ext-link>). This script was used to guide the interviews and encompasses topics and themes that the research team deemed pertinent for exploring with the participants. The script comprised open-ended questions on the platform's usability, inputs and feedback quality, user experience, intention to use, and the most and least appreciated/enjoyable platform features. Examples of the questions included: ‘What difficulties did you experience while using the platform?’, ‘How would you describe your experience with the platform?’, ‘How precise did you find the instructions provided by the platform?’, ‘If available to the public, will you consider using the platform on a daily/weekly basis? Why?’ and ‘What platform features did you enjoy/value most?’.</p>", "<title>Digital neuropsychological rehabilitation platform</title>", "<p>NeuroVRehab.PT is a photo-realistic virtual supermarket designed to promote cognitive functions and functional/behavioural skills involved in shopping activities. The platform was developed using a participatory design approach by a multidisciplinary team of psychologists, neurologists, and computer engineers in collaboration with health professionals and older adults.<sup>\n##REF##33255869##16##\n</sup> The activities incorporated gamification elements such as numeric and non-numeric feedback systems and narrative context to increase users’ engagement and motivation. These activities were grouped into three game modes: supermarket, recipes, and shopping list (see ##FIG##0##Figures 1(a)##, ##FIG##1##2(a)##, ##FIG##2##3(a)##, ##FIG##3##4(a)## and ##FIG##4##5(a)##).</p>", "<p>In NeuroVRehab.PT, users are rewarded with points and positive feedback sounds when they perform correct actions, such as putting a product on the shopping list into the basket. On the other hand, if users perform an incorrect action, such as selecting an item that is not on the shopping list, they receive a negative feedback sound, and no points are awarded. At the end of each level, users are presented with a three-star classification system based on their performance (see ##FIG##0##Figure 1(a)##). For more details about the platform and game modes, see.<sup>\n##REF##33255869##16##\n</sup></p>", "<title>Data analysis</title>", "<p>The audio recordings of the interviews were transcribed (see <ext-link xlink:href=\"https://journals.sagepub.com/doi/suppl/10.1177/20552076231223805\" ext-link-type=\"uri\">Doc S2 in Supplementary Material</ext-link>) and then cross-checked by the first author (FFB) to ensure the accuracy and removal of any identifiable information (such as participant names, locations, and brands). An inductive-deductive informed Thematic Analysis (TA) was conducted following the six-step approach proposed by Braun and Clarke.<sup>##UREF##10##20##–##UREF##12##22##</sup></p>", "<p>Following Braun and Clarke's model, the initial step involves two co-authors (FFB and SA) immersing themselves in the data by carefully reading and re-reading the transcriptions of two randomly selected interviews. An initial set of codes was deductively informed by the research question and interview script. In addition, relevant phrases and recurring ideas were annotated and added to the code list through an inductive process.</p>", "<p>For the purpose of analysis, the unit of analysis was defined as blocks of 20 lines within the transcriptions. Codes were modified or added (for all interview transcripts by revisiting the complete set of interviews) as new codes were identified in the remaining transcriptions. Data saturation was achieved during the content analysis, with no new codes identified in the last three interviews. Then, the research team collated text extracts under each potential theme, and codes were organised to fit into the themes’ organising concept. Finally, the research team discussed and refined the final list of themes (and subthemes) and their headings.</p>" ]
[ "<title>Results</title>", "<p>Based on the analysis of the <italic toggle=\"yes\">corpus</italic> of the interviews conducted with eight older adults, three main themes and eight subthemes (##TAB##2##Table 3##) were identified.</p>", "<title>Theme 1: Interaction experience</title>", "<p><italic toggle=\"yes\">Usability and learnability:</italic> the platform's usability and learnability were determined based on participants’ statements and observed behaviour on their ability to identify the different game modes, follow instructions, and use the game controls. All participants demonstrated the capacity to use the platform independently (to some extent) and could identify and switch between the three game modes. However, some participants initially struggled to navigate inside the virtual supermarket, particularly in understanding the purpose of the arrows as game controls (see ##FIG##1##Figure 2(a)##). For instance, some participants interpreted the arrows as mandatory actions or tips for finding a specific product. After becoming more familiar with the game controls, one participant stated: ‘<italic toggle=\"yes\">… I will not say confusing. But I must train more</italic>” (Participant 5, line 352). To other participants, game controls were ‘<italic toggle=\"yes\">… [not difficult] … as I had the arrows, I would press the arrows, and they would take me where I wanted to go’</italic> (Participant 2, line 412).</p>", "<p>Regarding the instructions provided at the beginning of each game mode and throughout the activities, they were considered by participants necessary, important, and easy to follow ‘<italic toggle=\"yes\">… to those who do not know [the platform], it makes sense (…) I think so’</italic> (Participant 2, line 418). Moreover, two participants emphasised the importance of the instructions being in their native language <italic toggle=\"yes\">‘… there were no foreign words at all, it was all in Portuguese’</italic> (Participant 1, line 684), and ‘… <italic toggle=\"yes\">do not put signboards in English (…) I do not know English</italic>…’ (Participant 4, line 753).</p>", "<p><italic toggle=\"yes\">Hedonic experience:</italic> except for one (Participant 5), all participants enjoyed using NeuroVRehab.PT. One of the aspects most valued by participants was the possibility of navigating within a highly realistic virtual supermarket, ‘<italic toggle=\"yes\">… this is interesting, it shows you the supermarket products, the photos of the objects are great…’</italic> (Participant 8, line 419). Another participant echoed this perspective stating,</p>", "<p>On the other hand, Participant 5 claimed, ‘<italic toggle=\"yes\">… to be honest, I did not like it…’</italic> (line 386). To this participant, the absence of ‘people’ in the aisles and checkout counters made the environment look very artificial compared to what happens in the real world.</p>", "<p><italic toggle=\"yes\">Confidence, self-efficacy, and computer anxiety:</italic> despite using NeuroVRehab.PT and completing all proposed activities, our results showed that some participants were tense and anxious while using the platform. Some participants had to be constantly encouraged and ensured that everything was all right and that they were not looking bad or doing something wrong: <italic toggle=\"yes\">‘… do not be afraid, you are doing great</italic>.’ (Interviewer speech, Participant 3, line 99) and</p>", "<p>In some cases, the lack of confidence was further compounded by participants’ subjective perception of age-related cognitive decline, fear of memory loss and dementia. As one participant expressed:</p>", "<p>Moreover, using someone else's device contributed to uneasiness and nervousness for some participants. One participant explained: ‘<italic toggle=\"yes\">[referring to her mobile device] I am not afraid of making mistakes. Okay, that is out, it is not right, let it be (…) I do not cause prejudice to anyone’</italic> (Participant 3, line 432).</p>", "<p><italic toggle=\"yes\">Reproduction of daily routines:</italic> once inside the virtual supermarket, participants’ choices and behaviours seemed to be based on their routines (i.e. real-life behaviours and strategies). For instance, one participant said, ‘<italic toggle=\"yes\">What will I need? Fish, let's follow what I had planned for today [in real life], to buy fish, and then I buy bread…’</italic> (Participant 1, line 36). Other situations where it was possible to observe that participants rely on their usual behaviour patterns to progress in the game were, for instance, when Participant 2 (line 11) immediately tried to pick up a shopping basket upon entering the virtual supermarket without any specific instructions. Another example was when Participant 3 (line 289) was asked how to check the price of a product, and she instinctively reached out to touch the price tag near the item. The similarity between the virtual supermarket and real-world shopping experiences also helped participants monitor the task's progression and determine the following steps to conclude the activity: ‘<italic toggle=\"yes\">I finished the shopping list, right? Now I am going to the checkout counter. I assume this is the same [as in a real-world supermarket]’</italic> (Participant 5, line 235).</p>", "<p>However, participants looked puzzled and discontent when the system did not allow them the same freedom of actions and choices (i.e. an accurate reproduction of their shopping-related behaviours). For instance, when specific items were included in a shopping list assigned in one of the Supermarket game levels, one participant exclaimed, ‘<italic toggle=\"yes\">But I do not want bananas…. Ah! Do I have to buy bananas? Oh, okay. Oh, I just wanted the mangos</italic>’ (Participant 4, line 167). A similar situation occurred when participants realised that the system did not allow them to choose the quantity of a product to purchase, ‘<italic toggle=\"yes\">Curry, curry in powder. 50 grams. What if I do not want 50 grams of curry? What if I wanted more or less?’</italic> (Participant 6, line 88) and ‘<italic toggle=\"yes\">bananas … 450 grams, why? What if I want more…?</italic>’ (Participant 6, line 276).</p>", "<title>Theme 2: Digital literacy</title>", "<p><italic toggle=\"yes\">Computer/tablet skills:</italic> despite all participants attending ICT classes, for some of them, this was ‘<italic toggle=\"yes\">… the first time I interacted with a tablet’</italic> (Participant 1, line 625), which presented additional challenges during the evaluation session. The unfamiliarity with tablet devices led to difficulties when specific patterns of interaction were required. For instance, using the keyboard posed a challenge for some participants, as one expressed<italic toggle=\"yes\">,</italic> ‘<italic toggle=\"yes\">… I am not used to this [tablet], if it were on a computer, maybe I would be quicker’</italic> (Participant 4, line 606).</p>", "<p>However, the lack of technological proficiency was not limited to tablet use. Participants encountered difficulties with other aspects, such as logging in, closing instruction windows to proceed in the game, and exploring other elements outside the virtual supermarket environment area.</p>", "<p><italic toggle=\"yes\">Gamification and game elements:</italic> our results showed that some participants were not familiar with certain elements commonly found in video games (i.e. game elements; GEs). For instance, when asked about the meaning of the three-star score system (see ##FIG##0##Figure 1(a)##), one participant expressed uncertainty<italic toggle=\"yes\">: ‘That is not décor, for sure! Now, it can be interpreted in two ways … what if there are five stars and I only got three, [and] this orange colour [of the stars] is not good, for sure</italic>…’ (Participant 1, line 348). Other participants had varying interpretations, such as: <italic toggle=\"yes\">‘Three stars is not a lot’</italic> (Participant 5, line 149), ‘<italic toggle=\"yes\">It is sufficient’</italic> (Participant 3, line 399), and ‘<italic toggle=\"yes\">For me, it is [good], I have never done this before, so it is not bad’</italic> (Participant 2, line 372)<italic toggle=\"yes\">.</italic> The lack of familiarity with GEs was also evident when considering the progress bar element (see ##FIG##1##Figure 2(a)##), which was rarely or incorrectly identified: ‘<italic toggle=\"yes\">… this should mean that I already spent 50% [of the budget]’</italic> (Participant 7, line 82).</p>", "<p>On the other hand, all participants understood the meaning of the colour-based feedback system used to indicate correct and wrong actions. Participants’ interpretations were consistent, with comments such as, ‘<italic toggle=\"yes\">It turned red (…) It is not right. Something is not right…’</italic> (Participant 2, line 305), and ‘<italic toggle=\"yes\">[when asked if the last action was correct] Yes. (….) It turned green…’</italic> (Participant 1, line 527).</p>", "<p>Regarding the auditory feedback system, participants had mixed responses. Some participants correctly identified its purpose, stating, ‘<italic toggle=\"yes\">It tells you [that the product] is already in the basket</italic>’ (Participant 4, line 130), and ‘[it tells you] <italic toggle=\"yes\">It is done!’</italic> (Participant 6, line 284). However, one participant completely missed it: ‘<italic toggle=\"yes\">It went unnoticed … I will tell you why … because this is the computer's sound when (…) an email appears, [when you] do something on the computer</italic>’ (Participant 7, line 395).</p>", "<title>Theme 3: Attitudes toward NeuroVRehab.PT</title>", "<p><italic toggle=\"yes\">Intention to use:</italic> overall, participants present an open and positive attitude towards technology and NeuroVRehab.PT. Regarding NeuroVRehab.PT, participants expressed they were ‘<italic toggle=\"yes\">…curious and want to become more familiar with it’</italic> (Participant 3, line 493). Another participant stated,</p>", "<p>Moreover, participating in the evaluation session was described as a positive experience, and it motivated them to seek additional opportunities to engage with technology. Another participant expressed ‘…<italic toggle=\"yes\">willingness to buy one of these things [referring to the tablet] …</italic>’ (Participant 3, line 602).</p>", "<p><italic toggle=\"yes\">Utility, usefulness, and connectedness:</italic> It was apparent that some participants were exploring how the use of NeuroVRehab.PT could help them to shop more efficiently. Among the potential practical (utilitarian) benefits identified by participants, one significant advantage was the possibility of completing a pre-existent shopping list. One participant highlighted this aspect by stating, ‘<italic toggle=\"yes\">It could be useful to visit the [virtual] supermarket, to gather ideas (…) maybe I have not even realised that I needed that product. In this aspect, [the platform] is perfect.</italic>’ (Participant 8, line 234). Another participant expressed:</p>", "<p>Additionally, one participant further developed this idea by suggesting that the shopping list created in the platform could be e-mailed or printed and then used in a real-life shopping activity: ‘<italic toggle=\"yes\">… this shopping list goes somewhere out? Can I bring it [with me] to the supermarket?’</italic> (Participant 6, line 118).</p>", "<p>Another practical aspect identified by participants was the possibility of using the platform to strengthen family bonds, especially with the younger generations. As one participant shared: ‘<italic toggle=\"yes\">Yes. I have grandsons that play (…) they enjoy playing these things. Maybe they would like to play this one too, especially the older one who is 11 years old</italic>’ (Participant 2, line 440).</p>", "<title>Platform improvements and design recommendations</title>", "<p>In the previous section, we presented the insights and suggestions provided by participants, which in turn were used to improve NeuroVRehab.PT (see ##FIG##0##Figures 1(b)##, ##FIG##1##2(b)##, ##FIG##2##3(b)##, ##FIG##3##4(b)## and ##FIG##4##5(b)##). The implemented modifications were thoughtfully selected based on their potential to augment the overall IX with the platform. The changes implemented were categorised into two groups: those directly proposed by participants (i.e. verbally expressed) and those derived from direct observations (e.g. difficulties identified from users’ observed behaviour) (see <ext-link xlink:href=\"https://journals.sagepub.com/doi/suppl/10.1177/20552076231223805\" ext-link-type=\"uri\">Table S2 in the Supplementary Material</ext-link> for details).</p>", "<p>Based on the analysis of participants’ suggestions and changes implemented, we identified seven design recommendations (##TAB##3##Table 4##) for developing digital neuropsychological applications targeting low technological proficient populations.</p>" ]
[ "<title>Discussion</title>", "<p>Perceived ease of use and usefulness of technology may be negatively impacted by users’ lack of familiarity with technology and computer anxiety, which consequently hinder the intention to use digital healthcare services.<sup>##UREF##7##14##,##REF##31230937##23##,##REF##31171391##24##</sup> In this qualitative study, we sought to explore the feasibility of a digital neuropsychological rehabilitation platform called NeuroVRehab.PT, and the influence of non-clinical factors on IX among a group of older adults with varying levels of technology familiarity and computer confidence.</p>", "<p>Our study showed that NeuroVRehab.PT is feasible among older adults with varying computer confidence and computer self-efficacy levels. However, our findings suggest that the continued use of NeuroVRehab.PT may be negatively influenced by participants’ perceived low computer confidence and computer self-efficacy. According to Bandura's Theory of Behavioural Change,<sup>\n##REF##847061##15##\n</sup> individuals who perceive themselves as less competent in a specific domain are less engaged and more prone to disengage when encountering difficulties. In our study, we observed that participants’ perceptions of their low digital skills and the fear of making mistakes negatively impacted their ability to initiate or complete actions without seeking validation from the researcher. As a result, some participants required continuous encouragement to explore and interact with the platform despite demonstrating the skills and knowledge to do it independently.</p>", "<p>After the intervention's initial contact/onboarding phase, continued contact with healthcare professionals has been identified as a promoting factor of adherence to digital interventions.<sup>\n##UREF##13##25##\n</sup> Together with ours, these findings indicate that human support and guidance are essential for fostering a positive IX and cultivating a sense of patient empowerment when utilising digital healthcare platforms. This notion gains further strength when applied to demographic groups with reduced technological proficiency and low familiarity with digital interfaces.</p>", "<p>Our study highlights three other important aspects of designing, implementing, and assessing the feasibility of digital neuropsychological interventions in older adults. First, some of the GEs used in NeuroVRehab.PT were not easily understood by all participants despite being previously identified in other digital neuropsychological interventions.<sup>\n##REF##31518700##26##\n</sup> Video game culture familiarity is gradually increasing among older generations<sup>##UREF##14##27##,##UREF##15##28##</sup> and scientific evidence supports using gamified applications to improve cognitive and other health-related outcomes in older adults.<sup>\n##UREF##16##29##\n</sup> However, our study calls attention to a different aspect of gamification when applied to the health context, namely the importance of conducting comprehensive analyses to assess gamification elements’ adequacy and differential impact, especially in digital interventions targeting older adults who might be less technologically savvy.</p>", "<p>Second, we observed that participants relied on virtual environment (VE) realism to guide their behaviour and progress in the game. The realism of a VE is determined by its ability to simulate both the physical characteristics (i.e. engineering fidelity) and the critical elements that elicit specific behaviours to complete the task (i.e. psychological fidelity).<sup>\n##UREF##17##30##\n</sup> Based on the reports gathered, we can conclude that NeuroVRehab.PT has a high engineering fidelity derived from its photo-realistic rendered VE. For instance, some participants were naturally compelled to execute specific actions simply by being exposed to virtual stimuli that were sufficiently natural to trigger the usual behaviour (e.g. picking up a basket or checking product prices by clicking on the price tags).</p>", "<p>On the other hand, the psychological fidelity of our platform could be enhanced by implementing features that increase the range of routines/behaviours participants usually carry out while shopping. For instance, determining the number and quantity of products included in the shopping list and adding new items to the shopping basket while inside the virtual supermarket were identified as features that would further enrich the IX of our platform.</p>", "<p>Finally, we observed a utilitarian use of NeuroVRehab.PT among participants. More specifically, participants demonstrated a strong focus on identifying potential advantages and real-world benefits of using our platform. Notably, participants associated the use of NeuroVRehab.PT with the opportunity to optimise shopping activities by creating or completing their shopping lists before visiting a real-world supermarket. Previous literature has shown that older adults value (and more easily adhere to) technology when it improves and supports daily-life activities.<sup>\n##UREF##18##31##\n</sup> Although further research is required, these results are particularly encouraging since they suggest NeuroVRehab.PT might contribute to bridging the gap between clinical/experimental context and patients’ daily life routines.</p>", "<title>Limitations and future work</title>", "<p>The results of this study provide valuable insights into the impact of nonclinical factors on the IX and feasibility of NeuroVRehab.PT among older adults with varying levels of computer confidence and computer self-efficacy. However, some limitations should be considered when interpreting the findings.</p>", "<p>First, it is important to note that the study exclusively enrolled participants attending ICT classes. This may have resulted in a group of individuals more motivated and capable of exploring and interacting with the platform. However, excluding participants lacking basic digital skills allowed us to unmask the influence of computer confidence and self-efficacy on our platform's interaction. This, in turn, contributed to our understanding and awareness of how these factors impact the adoption and adherence to digital healthcare platforms.</p>", "<p>On the other hand, for most participants, this study marked their first experience using a tablet device. Consequently, it is challenging to discern whether their motivation stemmed from the platform's ability to engage users or the novelty of interacting with a new mobile device. Future research should explore this aspect by including participants with prior tablet experience or by providing a familiarisation period with the intervention device.</p>", "<p>Additionally, the presence of two researchers during the sessions may have contributed to participants’ feelings of nervousness and anxiety. While controlling for this variable is challenging, we attempted to mitigate its potential impact by (1) explaining the role of each of the researchers present, (2) emphasising that it was the platform that was under evaluation rather than the participant, and (3) reinforcing the value of participant's insights regarding their difficulties and doubts in improving the platform.</p>", "<p>Lastly, it is important to acknowledge that this study only involved a single session of platform use with a small sample. This limitation hinders our ability to draw conclusions regarding long-term adherence and satisfaction with NeuroVRehab.PT, as well as the influence of individual characteristics on platform use. Future studies should involve recruiting a representative sample and extending the intervention exposure period to assess the feasibility of NeuroVRehab.PT among less technologically-savvy populations.</p>" ]
[ "<title>Conclusion</title>", "<p>The study shows that NeuroVRehab.PT is feasible among older adults with different computer confidence and computer self-efficacy levels. The importance of considering non-clinical factors, such as computer confidence and self-efficacy, when designing and implementing digital healthcare services became evident in our study since these variables seemed to influence participants’ IX negatively. Contact with the healthcare professionals throughout the intervention period, the physical and psychological realism of the VE, and the adequacy of the game elements to the targeted populations seemed to be factors determining the adoption and long-term use of digital healthcare services.</p>" ]
[ "<title>Introduction</title>", "<p>Computer confidence and computer self-efficacy can impact an individual's perceived ease of use and usefulness of technology, ultimately determining adherence to digital healthcare services. However, few studies focus on assessing the impact of non-clinical factors on the efficacy and adherence to digital healthcare platforms.</p>", "<title>Objective</title>", "<p>We aimed to analyse the role of non-clinical factors (i.e. computer confidence and computer self-efficacy) in the interaction experience (IX) and the feasibility of a digital neuropsychological platform called NeuroVRehab.PT in a group of older adults with varying levels of computer confidence.</p>", "<title>Methods</title>", "<p>Eight older adults (70.63 ± 6.1 years) evaluated the platform, and data was collected using the Think-Aloud method and a semi-structured interview. Sessions were audio-recorded and analysed through an inductive-deductive informed Thematic Analysis protocol. This study was conducted according to the Consolidated Criteria for Reporting Qualitative Research guidelines.</p>", "<title>Results</title>", "<p>Three main themes were identified (Interaction Experience, Digital Literacy, and Attitudes toward NeuroVRehab.PT). Computer anxiety and fear of making errors were not uncommon, even among older adults who perceive themselves as confident in technology use, and negatively impacted IX. Moreover, some game elements (e.g. three-star system, progression bar) were not intuitive to all participants, leading to misleading interpretations. On the other hand, human support and the platform's realism seemed to impact participants’ IX positively.</p>", "<title>Conclusions</title>", "<p>This study shed light on the barriers raised by non-clinical factors in adopting and using digital healthcare services by older adults. Furthermore, a critical analysis of the platform's features that promote user adoption is done, and suggestions for overcoming limitations are presented.</p>" ]
[ "<title>Supplemental Material</title>" ]
[ "<title>Acknowledgements:</title>", "<p>The authors wish to thank all participants in this study.</p>" ]
[ "<fig position=\"float\" id=\"fig1-20552076231223805\"><label>Figure 1.</label><caption><p>(a) Former supermarket game mode interface. The default game mode was the virtual supermarket with different game levels; (b) challenges interface with the new features. In the latest version (after this qualitative study) default game mode is the shopping list game.</p></caption></fig>", "<fig position=\"float\" id=\"fig2-20552076231223805\"><label>Figure 2.</label><caption><p>(a) Former supermarket virtual environment and interface; (b) supermarket virtual environment and interface with the (new) features identified through this qualitative study.</p></caption></fig>", "<fig position=\"float\" id=\"fig3-20552076231223805\"><label>Figure 3.</label><caption><p>(a) Former recipe game mode interface; (b) the new recipe game mode interface where users can skip subtasks (e.g. task segmentation – organization of the ingredients according to product's category).</p></caption></fig>", "<fig position=\"float\" id=\"fig4-20552076231223805\"><label>Figure 4.</label><caption><p>(a) Former shopping list interface with a two-column shopping list; (b) new shopping list interface (after this qualitative study) with 1-column shopping list organised by product's category.</p></caption></fig>", "<fig position=\"float\" id=\"fig5-20552076231223805\"><label>Figure 5.</label><caption><p>(a) Former shopping list game mode; (b) shopping list game mode with the new features (identified through this qualitative study).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"table1-20552076231223805\"><label>Table 1.</label><caption><p>Demographic information and self-reported confidence using technology (n = 8).</p></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">ID</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">GN</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Age</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Education</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Marital status</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Confidence using technology</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Confidence using new devices</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Time browsing the internet/week</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Play video games/other apps</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">P1</td><td rowspan=\"1\" colspan=\"1\">M</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">68</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">High school</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Married</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">14 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">No</td></tr><tr><td rowspan=\"1\" colspan=\"1\">P2</td><td rowspan=\"1\" colspan=\"1\">F</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">78</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">High school</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Widow</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Little confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Little confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">11 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">Sudoku game and Solitaire Spider Microsoft<sup>®</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">P3</td><td rowspan=\"1\" colspan=\"1\">F</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">77</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">High school</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Married</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Little confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Little confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">4 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">No</td></tr><tr><td rowspan=\"1\" colspan=\"1\">P4</td><td rowspan=\"1\" colspan=\"1\">F</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">67</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Primary education</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Married</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">5 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">No</td></tr><tr><td rowspan=\"1\" colspan=\"1\">P5</td><td rowspan=\"1\" colspan=\"1\">F</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">68</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">University</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Single</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">5 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">Facebook<sup>®</sup> math exercises</td></tr><tr><td rowspan=\"1\" colspan=\"1\">P6</td><td rowspan=\"1\" colspan=\"1\">F</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">61</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">High school</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Divorced</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Little confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Little confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">8 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">No</td></tr><tr><td rowspan=\"1\" colspan=\"1\">P7</td><td rowspan=\"1\" colspan=\"1\">M</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">69</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">High school</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Single</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Very confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Very confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">10 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">No</td></tr><tr><td rowspan=\"1\" colspan=\"1\">P8</td><td rowspan=\"1\" colspan=\"1\">M</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">77</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">University</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\">Widower</td><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\">Confident</td><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">10 h</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\">No</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n</td><td rowspan=\"1\" colspan=\"1\">\n</td><td style=\"width: 133px;\" rowspan=\"1\" colspan=\"1\">70.6 ± 6.1 mean age</td><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\"/><td style=\"width: 102px;\" rowspan=\"1\" colspan=\"1\"/><td style=\"width: 210px;\" rowspan=\"1\" colspan=\"1\"/><td style=\"width: 208px;\" rowspan=\"1\" colspan=\"1\"/><td style=\"width: 220px;\" rowspan=\"1\" colspan=\"1\">8.38 ± 3.50 mean time</td><td style=\"width: 198px;\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table2-20552076231223805\"><label>Table 2.</label><caption><p>Study's eligibility criteria.</p></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Eligibility criteria</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">(a) 60 years or older</td></tr><tr><td rowspan=\"1\" colspan=\"1\">(b) Be fluent in Portuguese (spoken and written)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">(c) Live autonomously in the community</td></tr><tr><td rowspan=\"1\" colspan=\"1\">(d) Have basic skills or willingness to learn to use a tablet.</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table3-20552076231223805\"><label>Table 3.</label><caption><p>Themes and subthemes identified based on eight interviews analyzed using a TA protocol.</p></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/></colgroup><tbody><tr><td rowspan=\"1\" colspan=\"1\">Interaction experience</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Usability and learnability</p></list-item><list-item><p>Hedonic experience</p></list-item><list-item><p>Confidence, self-efficacy and computer anxiety</p></list-item><list-item><p>Reproduction of daily routines</p></list-item></list>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Digital literacy</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Computer/tablet skills</p></list-item><list-item><p>Gamification and game elements</p></list-item></list>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Attitudes toward NeuroVRehab.PT</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Intention to use</p></list-item><list-item><p>Utility, usefulness and connectedness</p></list-item></list>\n</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table4-20552076231223805\"><label>Table 4.</label><caption><p>Design recommendation for low technological proficient populations.</p></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Design recommendations</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">1. Avoid using technical terms or providing information written in a different language from users’ native language.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">2. Ensure users understand that the actions carried out on the platform do not have real-world implications or consequences.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3. The platform's capacity to reproduce the physical, psychological, and cognitive demands of a task when executed in the real world should be considered a crucial aspect during the design and development process.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">4. Divide tasks into segments and give users a transparent sense of the task's status, indicating its current phase and whether it has been successfully completed.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5. Introduce new material, tasks, and information periodically to ensure that users continue to feel challenged and engaged.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">6. Provide sufficient training on platform/device usage, ideally supported by human guidance before the intervention starts.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">7. Whenever feasible, users should have access to the platform on their own devices, rather than on a shared device or a device owned by someone else.</td></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<disp-quote><p><italic toggle=\"yes\">… it is interesting the possibility of navigating [within the supermarket] because it motivates you to plan your shopping list in your head. It is interesting. And then you see, I need this [product] while walking through the aisles…. I think it is interesting, yes</italic>!’ (Participant 6, line 650).</p></disp-quote>", "<disp-quote><p><italic toggle=\"yes\">… there is no one [in the aisles], the checkout counters are empty (…) maybe [including non-playable characters] would be confusing for the people who will use the platform. But I considered it too artificial; it looked more like a huge freezer than a supermarket</italic>. (Participant 5, line 341), and then added, ‘<italic toggle=\"yes\">… usually, when I go to the supermarket, I think about the time of the day [to go during low affluence periods]. But not having anyone at the checkout counter! … [seems implausible]’</italic> (Participant 5, line 421).</p></disp-quote>", "<disp-quote><p><italic toggle=\"yes\">You are doing well, do not worry. You are using something for the first time, and we are all a little like that (…) we are a little unsure, and we have to explore [the platform], we do not know what we are working with, so do not worry.</italic> (Interviewer speech, Participant 1, line 169)</p></disp-quote>", "<disp-quote><p><italic toggle=\"yes\">… the problem is me, my memory. If I had a better memory like I used to have in the old days, I would not have forgotten all the details and would have assimilated it all and go straight [to the point].</italic> (Participant 1, line 626)</p></disp-quote>", "<disp-quote><p><italic toggle=\"yes\">This is interesting … because … how can I explain, this makes us want to explore with the finger, try to find [a product], to rummage [the supermarket], for me, it would be great to be here one or two hours exploring the whole supermarket…’</italic> (Participant 4, line 619).</p></disp-quote>", "<disp-quote><p><italic toggle=\"yes\">Being able to create a shopping list with this [while visiting the virtual supermarket], it seems like a good idea. Sometimes, I am at home, [searching for] what I need [to buy], and maybe …, it would come to me [to my mind] easier …. the products that I need (…) The fact that we can visit the aisles… it helps us to identify what we need.</italic> (Participant 6, line 655)</p></disp-quote>" ]
[]
[ "<supplementary-material id=\"supp1-20552076231223805\" position=\"float\" content-type=\"local-data\"><caption><title>sj-docx-1-dhj-10.1177_20552076231223805 - Supplemental material for Digital health and patient adherence: A qualitative study in older adults</title></caption><p>Supplemental material, sj-docx-1-dhj-10.1177_20552076231223805 for Digital health and patient adherence: A qualitative study in older adults by Filipa Ferreira-Brito, Sérgio Alves, Tiago Guerreiro, Osvaldo Santos, Cátia Caneiras, Luís Carriço and Ana Verdelho in DIGITAL HEALTH</p></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"table-fn1-20552076231223805\"><p>GN: gender.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"other\"><p><bold>Contributorship:</bold> Conceptualisation: FFB, TG, OS, CC, LC and AV; methodology: FFB, SA, TG, OS, CC, LC and AV; validation: FFB, SA, TG and OS; formal analysis: FFB, SA, TG and OS; investigation: FFB and SA; software: SA; resources: TG, CC and LC; data curation: FFB and SA; writing—original draft preparation: FFB; writing—review and editing: FFB, SA, TG, OS, CC, LC and AV; visualisation: FFB; supervision: FFB, TG, OS, CC, LC and AV; project administration: FFB, CC, LC and AV. All authors have read and agreed to the published version of the manuscript.</p></fn><fn fn-type=\"other\"><p><bold>Declaration of conflicting interests:</bold> The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.</p></fn><fn fn-type=\"other\"><p><bold>Ethical approval:</bold> This study was conducted in compliance with the Declaration of Helsinki, and ethical approval was obtained from the Ethics Committee of the Centro Hospitalar de Lisboa Norte e Centro Académico de Medicina de Lisboa (ref. 546/19) and Comissão de Ética para Recolha e Proteção de Dados de Ciências (ref. CERPDC/16/2019).</p></fn><fn fn-type=\"other\"><p><bold>Funding:</bold> The authors received the following financial support for research, authorship, and publication of this article: FFB received a scholarship from the following entities: Nippon Gases Portugal and Fundação para a Ciência e a Tecnologia through European Social Fund and Human Capital Operational Programme, co-financed by Portugal 2020 and European Union [Ref: PDE/BDE/127784/2016]. This work was also supported by Fundação para a Ciência e Tecnologia through the LASIGE Research Unit [Ref. UIDB/00408/2020, UIDP/00408/2020], and Project 41, HfPT: Health from Portugal, funded by the Portuguese Plano de Recuperação e Resiliência.</p></fn><fn fn-type=\"other\"><p><bold>Guarantor:</bold> FFB.</p></fn><fn fn-type=\"other\"><p><bold>Disclosure:</bold> The writing AI assistant ChatGPT was used for linguist improvements. All the content in the manuscript is solely authored by the authors of the article. None of the scientific content (i.e. 0%) presented in the manuscript was generated by this or any other AI system.</p></fn><fn fn-type=\"other\"><p><bold>Supplemental material:</bold> Supplemental material for this article is available online.</p></fn><fn fn-type=\"other\"><p><bold>ORCID iDs:</bold> Filipa Ferreira-Brito <ext-link xlink:href=\"https://orcid.org/0000-0001-5203-5174\" ext-link-type=\"uri\">https://orcid.org/0000-0001-5203-5174</ext-link></p><p>Cátia Caneiras <ext-link xlink:href=\"https://orcid.org/0000-0002-3735-8554\" ext-link-type=\"uri\">https://orcid.org/0000-0002-3735-8554</ext-link></p></fn></fn-group>" ]
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WHO"], "ext-link": ["https://www.who.int/reproductivehealth/publications/mhealth/digital-health-interventions/en/%0Ahttp://www.who.int/reproductivehealth/publications/mhealth/digital-health-interventions/en/"], "year": ["2016"]}, {"label": ["8"], "collab": ["International Standard Organisation"], "comment": ["ISO 9241-210:2019, ergonomics of human-system interaction \u2014 part 210: human-centred design for interactive systems. [cited 2021 Sep 30]"], "ext-link": ["https://www.iso.org/obp/ui/#iso:std:iso:9241:-210:ed-2:v1:en"], "year": ["2019"]}, {"label": ["9"], "person-group": ["\n"], "surname": ["Meiland", "Innes", "Mountain"], "given-names": ["F", "A", "G"], "etal": ["et al."], "article-title": ["Technologies to support community-dwelling persons with dementia: a position paper on issues regarding development, usability, effectiveness and cost-effectiveness, deployment, and ethics"], "source": ["JMIR Rehabil Assist Technol"], "year": ["2017"], "volume": ["4"], "comment": ["e1"]}, {"label": ["11"], "person-group": ["\n"], "surname": ["Davis"], "given-names": ["FD"], "article-title": ["Perceived usefulness, perceived ease of use, and user acceptance of information technology"], "source": ["MIS Q Manag Inf Syst"], "year": ["1989"], "volume": ["13"], "fpage": ["319"], "lpage": ["340"]}, {"label": ["12"], "person-group": ["\n", "\n"], "surname": ["Winn", "Ferdig"], "given-names": ["BM", "R"], "part-title": ["The design, play, and experience framework"], "source": ["Handb Res Eff Electron Gaming Educ"], "comment": [" 2009"], "fpage": ["1010"], "lpage": ["1024"]}, {"label": ["13"], "person-group": ["\n"], "surname": ["Norman", "Skinner"], "given-names": ["CD", "HA"], "article-title": ["Ehealth literacy: essential skills for consumer health in a networked world"], "source": ["J Med Internet Res"], "year": ["2006"], "volume": ["8"], "comment": ["e9"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Portz", "Bayliss", "Bull"], "given-names": ["JD", "EA", "S"], "etal": ["et al."], "article-title": ["Using the technology acceptance model to explore user experience, intent to use, and use behavior of a patient portal among older adults with multiple chronic conditions: descriptive qualitative study"], "source": ["J Med Internet Res"], "year": ["2019"], "volume": ["21"], "fpage": ["1"], "lpage": ["18"]}, {"label": ["18"], "person-group": ["\n"], "surname": ["Tong", "Sainsbury", "Craig"], "given-names": ["A", "P", "J"], "article-title": ["Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups"], "source": ["Int J Qual Heal Care"], "year": ["2007"], "volume": ["19"], "fpage": ["349"], "lpage": ["357"]}, {"label": ["19"], "person-group": ["\n"], "surname": ["Someren", "Barnard", "Sandberg"], "given-names": ["M", "YF", "J"], "source": ["The think aloud method: a practical guide to modelling cognitive processes"], "comment": ["Information Processing & Management"], "publisher-loc": ["London"], "publisher-name": ["Academic Press"], "year": ["1994"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Braun", "Clarke"], "given-names": ["V", "V"], "article-title": ["Using thematic analysis in psychology"], "source": ["Qual Res Psychol"], "year": ["2006"], "volume": ["3"], "fpage": ["77"], "lpage": ["101"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Braun", "Clarke"], "given-names": ["V", "V"], "article-title": ["Qualitative research in psychology using thematic analysis in psychology using thematic analysis in psychology"], "source": ["Qual Res Psychol"], "year": ["2006"], "volume": ["3"], "fpage": ["77"], "lpage": ["101"], "ext-link": ["http://www.tandfonline.com/action/journalInformation?journalCode=uqrp20%5Cnhttp://www.tandfonline.com/action/journalInformation?journalCode=uqrp20"]}, {"label": ["22"], "person-group": ["\n", "\n"], "surname": ["Braun", "Clarke", "Cooper", "Camic", "Long"], "given-names": ["V", "V", "H", "PM", "DL"], "part-title": ["Thematic analysis"], "etal": ["et al."], "source": ["APA handbook of research methods in psychology, vol 2: research designs: quantitative, qualitative, neuropsychological, and biological"], "publisher-loc": ["Washington, DC"], "publisher-name": ["American Psychological Association"], "year": ["2012"], "fpage": ["57"], "lpage": ["71"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Jakob", "Harperink", "Rudolf"], "given-names": ["R", "S", "AM"], "etal": ["et al."], "article-title": ["Factors influencing adherence to mHealth apps for prevention or management of noncommunicable diseases: systematic review"], "source": ["J Med Internet Res"], "year": ["2022"], "volume": ["24"], "comment": ["e35371"]}, {"label": ["27"], "collab": ["ESA"], "comment": ["2020 essential facts about the video game industry. Cision"], "fpage": ["1"], "lpage": ["2"], "ext-link": ["https://www.prnewswire.com/news-releases/new-survey-2020-essential-facts-about-the-video-game-industry-301093972.html"], "year": ["2020"]}, {"label": ["28"], "collab": ["ESA"], "comment": ["2019 essential facts about the computer and video game industry. Entertain Software Association"], "fpage": ["3"], "ext-link": ["https://www.theesa.com/wp-content/uploads/2019/05/ESA_Essential_facts_2019_final.pdf", "https://www.theesa.com/esa-research/2019-essential-facts-about-the-computer-and-video-game-industry/"], "year": ["2019"]}, {"label": ["29"], "person-group": ["\n"], "surname": ["Koivisto", "Malik"], "given-names": ["J", "A"], "article-title": ["Gamification for older adults: a systematic literature review"], "source": ["Gerontologist"], "year": ["2021"], "volume": ["61"], "comment": ["e360"], "lpage": ["e372"]}, {"label": ["30"], "person-group": ["\n"], "surname": ["Maran", "Glavin"], "given-names": ["NJ", "R"], "article-title": ["Low- to high-fidelity simulation \u2013 a continuum of medical education?"], "source": ["Med Educ"], "year": ["2003"], "volume": ["37"], "fpage": ["22"], "lpage": ["28"]}, {"label": ["31"], "person-group": ["\n"], "surname": ["Mitzner", "Boron", "Fausset"], "given-names": ["TL", "JB", "CB"], "etal": ["et al."], "article-title": ["Older adults talk technology: technology usage and attitudes"], "source": ["Comput Hum Behav"], "year": ["2010"], "volume": ["26"], "fpage": ["1710"], "lpage": ["1721"]}]
{ "acronym": [], "definition": [] }
31
CC BY
no
2024-01-14 23:43:48
Digit Health. 2024 Jan 12; 10:20552076231223805
oa_package/a8/71/PMC10787534.tar.gz
PMC10787539
0
[ "<title>INTRODUCTION</title>", "<p>Gastro-oesophageal reflux disease (GORD) is a common, chronic gastrointestinal condition in which gastric contents flow back into the oesophagus, resulting in symptoms of heartburn, regurgitation, bloating, excessive salivation, and impaired sleep.##UREF##0##1## GORD also increases the risk of developing the precancerous condition Barrett’s oesophagus and oesophageal cancer.##REF##32178772##2## Nearly 10% of the UK population are estimated to be affected by GORD.##REF##32178772##2##</p>", "<p>Currently, the National Institute for Health and Care Excellence (NICE) recommends that patients treated within the National Health Service (NHS) receive proton pump inhibitors (PPI) as initial treatment for GORD. Surgical management (ie, a laparoscopic Nissen fundoplication or magnetic sphincter augmentation [MSA]) using the LINX<sup>®</sup> system may be offered to patients who are intolerant to or unwilling to accept prolonged treatment with PPIs.##UREF##1##3,4## During a laparoscopic Nissen fundoplication procedure, the top of the stomach is folded and stitched to reduce the size of the opening between the stomach and oesophagus.##UREF##1##3## Similarly, MSA with the LINX<sup>®</sup> system also aims to reduce the size of this opening by inserting a ring of magnetic beads around the oesophagus, just above the stomach.##UREF##2##4##</p>", "<p>In light of the limited treatment options comprising the standard of care within the NHS in England and Wales, recent years have brought potential advances in the treatment of GORD. MSA using the LINX<sup>®</sup> system has been used in patients who do not respond well to treatment with PPIs##REF##32676727##5,6## and in January 2023 was recommended by NICE as part of routine care.##UREF##2##4## Recently, a novel implantable, nonactive, single-use device (RefluxStop™, Implantica, Zug, Switzerland), which restores normal oesophageal anatomy and function without affecting the passage of food, has been developed as a treatment for patients with GORD who are eligible for laparoscopic surgery. The RefluxStop™ procedure involves laparoscopic surgery where the device is implanted on the outer stomach wall.##REF##32689979##7## RefluxStop™ received a CE mark across the European Union (then including the United Kingdom) in August 2018, allowing for RefluxStop™ to be marketed and sold in these countries for the treatment of acid reflux.##UREF##3##8## CE mark approval was based on the positive results of a prospective, single-arm, multicenter study enrolling 50 patients with chronic GORD who required daily PPI treatment.##REF##32689979##7## This study demonstrated that RefluxStop™ was a safe, well-tolerated treatment for GORD, with efficacy that may improve upon the current standard of care.##REF##32689979##7## The study met its primary efficacy endpoint; at 1 year post-surgery, patients implanted with RefluxStop™ experienced on average 86% improvement in GORD-related symptoms measured using a well-established, disease-specific questionnaire (GORD–Health-Related Quality of Life [GORD-HRQL]##REF##8784314##9##).##REF##32689979##7## Laparoscopic implantation of RefluxStop™ also resulted in a decline in objectively measured GORD severity; at 6 months post-surgery, 98% of the patients displayed normal oesophageal pH on 24-hour monitoring, meeting a secondary outcome of the study.##REF##32689979##7## The safety profile of RefluxStop™ in the CE mark trial was consistent with laparoscopic surgery, as no serious adverse events relating to the RefluxStop™ device were reported and any complications were related to laparoscopic surgery rather than the RefluxStop™ device.##REF##32689979##7##</p>", "<p>A recent health-economic evaluation demonstrated the cost-effectiveness of RefluxStop™ when assessed over a patients’ lifetime horizon from the perspective of the NHS in England and Wales.##UREF##4##10## The objective of the current study was to estimate the more immediate, short-term clinical and economic effects of introducing RefluxStop™ as a therapeutic option for patients with GORD treated within NHS England and Wales.</p>" ]
[ "<title>METHODS</title>", "<title>Model Overview</title>", "<p>A budget impact model adherent to the recommendations of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR)##UREF##5##11## was developed. The model utilized a 1-year cycle length and estimated the budget impact of introducing RefluxStop™ over a 5-year time horizon from the perspective of the NHS in England and Wales. Two hypothetical scenarios were considered: one with existing interventions (PPI-based medical management, laparoscopic Nissen fundoplication, and MSA using the LINX<sup>®</sup> system) but without RefluxStop™, and another in which RefluxStop™ was introduced in addition to the currently existing interventions.</p>", "<p><bold>Supplementary Figure 1</bold> presents the model schematic. First, the population potentially eligible for treatment with RefluxStop™ (ie, patients diagnosed with GORD who currently take PPI medication and/or who have not had previous surgery and are eligible for/willing to undergo surgery) and the change in the size of this population over the 5-year model horizon were identified. The population was consistent with those who participated in the RefluxStop™ CE mark trial. Market shares of the available treatment options were then applied to the eligible population, and costs specific to each intervention (including the cost savings arising from the benefits of each technology, ie, cost offsets) were evaluated to obtain the total costs associated with the scenario with RefluxStop™ and those without RefluxStop™. The difference between these two scenarios represents the net budget impact of introducing RefluxStop™.</p>", "<title>Modeled Population</title>", "<p>The modeled population included all those who may potentially benefit from the introduction of RefluxStop™, namely, patients with GORD who receive PPI medication or who have not had previous surgical treatment for GORD and are eligible and willing to undergo surgery. The model considered both prevalent patients with GORD and incident cases (ie, patients newly developing GORD during the model time horizon).</p>", "<p>Estimates of population size and its projected growth in England and Wales were obtained from the Office for National Statistics (ONS) database.##UREF##6##12,13## GORD prevalence and incidence rates in the UK general population, and the annual incidence of laparoscopic antireflux surgery were obtained from the literature. Prevalence of GORD (14.5%) and annual incidence of new GORD cases (0.5%) were based on systematic reviews of GORD epidemiology.##REF##32242117##14,15## The proportion of GORD patients treated within the NHS (6.6%) was derived from a pragmatic analysis of healthcare cost of managing GORD in the Dorset Clinical Commissioning Group, extrapolated to the entire NHS England and Wales.##UREF##8##16## The number of antireflux surgeries among patients with GORD was 4.9 per 100 000 persons per year, based on a review of laparoscopic antireflux surgery in England.##REF##29368285##17##</p>", "<title>Market Shares</title>", "<p>Market shares of PPI-based medical management, laparoscopic Nissen fundoplication, and MSA, as well as the projected market share of RefluxStop™, were obtained from market research conducted by RefluxStop’s manufacturer, Implantica, and from expert opinion. The market shares are presented in <bold>##TAB##0##Table 1##</bold> for both the scenario without RefluxStop™ and the scenario with RefluxStop™. To accommodate for the uncertainty associated with real-world uptake of RefluxStop™, 2 additional scenarios were also considered. In one scenario, the rate of RefluxStop™ uptake was halved, while in the other scenario, the rate was doubled. The corresponding market shares in each model year are presented in <bold>##TAB##0##Table 1##</bold>.</p>", "<p>For the model calculations, the market share values were multiplied by the population size to estimate the number of people receiving each of the treatment options over the 5 years. Due to the surgical nature of most of the treatments considered in the model, it was assumed that those people who had the procedure previously would not switch to another treatment. Treatment switching in patients who underwent surgery after initially receiving PPI-based medical management was included in the per-patient cost estimates. Therefore, the population entering the model included all prevalent GORD patients in Year 1, and only incident cases in Years 2-5.</p>", "<title>Costs</title>", "<p>The cost per patient per year for each treatment reflected all costs associated with each treatment option and was obtained from the previously published cost-effectiveness model of RefluxStop™, which also applied the perspective of NHS England and Wales.##UREF##4##10## The methodology of the cost-effectiveness model has been described previously.##UREF##4##10## Briefly, the major cost categories captured were (1) treatment costs, comprising the costs of PPI medications and surgical treatments, with the latter including procedure costs and, for MSA and RefluxStop™ only, device and training costs; (2) the costs of diagnosing and treating Barrett’s oesophagus and oesophageal cancer in patients who developed the conditions; (3) the costs of managing adverse events associated with PPIs (chronic kidney disease,##UREF##9##18## cardiovascular events,##REF##30178881##19## fractures,##REF##30237993##20## pneumonia,##UREF##10##21##\n<italic toggle=\"yes\">Clostridium difficile</italic> infections,##REF##29085200##22## and stomach cancer##REF##35804824##23##) and the costs of managing adverse events associated with surgical treatment (conversion from laparoscopic to open surgery, oesophageal dilation, additional surgery for major complications and, for RefluxStop™ and MSA only, device removal). To ensure the costs used in the model were as relevant to NHS England as possible, unit costs were sourced from NHS schedule costs 2019/20##UREF##11##24## whenever possible. Where data are used from older sources in the literature, the values were inflated using the Personal Social Services Research Unit (PSSRU) inflation indices.##UREF##12##25##</p>", "<p>The total annual per-patient costs associated with each modeled treatment are presented in <bold>##TAB##1##Table 2##</bold>. The costs in year 1 were higher for surgical treatment options than for medical management, as they included the surgery and device costs. In contrast, the costs incurred during years 2-5 with the surgical treatment options were mainly related to follow-up, and therefore lower than for medical management.</p>", "<title>Analysis</title>", "<p>The total budget required for each treatment option was determined by multiplying the number of people receiving each treatment by the cost per specific year associated with that treatment option, so that patients in year 2 of their treatment would receive the year 2 cost, those in year 3 of their treatment would receive year 3 costs, etc. The total costs for each year were then summarized over the 5-year time horizon to generate the final healthcare system budget required for the scenarios with and without RefluxStop™. The net budget impact of introducing RefluxStop™ was calculated as the difference between the budget required to fund the scenario with RefluxStop™ and that without RefluxStop™. Scenario analysis were used to test the robustness of the model to changes in market uptake rates (the key driver of budget impact).</p>" ]
[ "<title>RESULTS</title>", "<p>The number of patients who receive RefluxStop™ was estimated at 150 in the first year of its availability and increased to 650 in the fifth year, with a corresponding decline in the annual number of patients who undergo Nissen fundoplication, from 2626 in year 1 to 1685 in year 5 (##TAB##2##Table 3##).</p>", "<p>Over the model’s 5-year time horizon, introduction of RefluxStop™ was associated with improved clinical outcomes, with 347 surgical failures, 39 reoperations, and 239 endoscopic oesophageal dilations avoided (representing 6.8%, 7.2%, and 15.7% reduction, respectively) relative to the scenario without RefluxStop™ (<bold>##FIG##0##Figure 1##</bold>).</p>", "<p>The total costs over the 5-year model time horizon associated with the scenarios including and excluding RefluxStop™ are presented in <bold>##FIG##1##Figure 2##</bold>. The net 1-year, 3-year, and 5-year budget impact of introducing RefluxStop™ was £711 014, £2 115 929, and £3 029 702 per year, respectively, which corresponded to 0.39%, 1.14%, and 1.68% increases per year in overall NHS expenditure for GORD treatment in England and Wales (<bold>##TAB##3##Table 4##</bold>).</p>", "<p>In the scenario in which the rate of uptake of RefluxStop™ was halved relative to the base case, the 5-year reductions in the number of surgical failures, reoperations, and endoscopic dilations were 174, 20, and 120, respectively. The 1-year budget impact of introducing RefluxStop™ in this scenario was £335 507, corresponding to an 0.20% increase in overall NHS expenditure for GORD treatment. The 5-year budget impact was £1 514 851, amounting to an 0.84% increase in NHS expenditure.</p>", "<p>In the scenario exploring a doubled rate of RefluxStop™ uptake, the 5-year reductions in the number of surgical failures, reoperations and endoscopic dilations were 695, 79, and 479, respectively. The budget impact associated with increased uptake of RefluxStop™ in the UK was £1 422 029 over 1 year, which translated to an 0.79% increase in NHS spending on GORD treatment, and £6 059 403 over 5 years, translating to a 3.36% increase in spending.</p>" ]
[ "<title>DISCUSSION</title>", "<p>RefluxStop™ has demonstrated a favorable safety and efficacy profile in its pivotal CE mark study,##REF##32689979##7## and a recent analysis suggests it is highly likely to be a cost-effective treatment for GORD patients in England and Wales at the standard cost-effectiveness threshold of £20 000 per quality-adjusted life-year gained.##UREF##4##10## The current analysis focused on the short-term health and economic impact of introducing RefluxStop™ as a treatment option on the NHS. While the time horizon of 5 years was too short to meaningfully capture some of the adverse events of PPIs and complications of GORD itself, such as the development of Barrett’s oesophagus or oesophageal cancer, the use of RefluxStop™ was associated with a substantial reduction in surgical complications, including surgical failures, reoperations, and endoscopic oesophageal dilations. This favorable clinical profile resulted in cost offsets for the NHS and contributed to the marginal budget impact of RefluxStop™ estimated in the current analysis.</p>", "<p>PPIs and, in selected patients, laparoscopic Nissen fundoplication, are recommended in the NICE guideline for GORD management##UREF##1##3## and form the mainstay of GORD treatment within the NHS. However, long-term PPI use is associated with a number of potential adverse events, including kidney disease, infections, and myocardial infarction, among others.##REF##27825371##26## Furthermore, up to a third of GORD patients do not achieve sufficient symptom relief with PPI treatment.##UREF##13##27## With regard to laparoscopic Nissen fundoplication, although this type of surgery has been reported to provide superior control of GORD symptoms, it is also associated with an up to 5-fold increase in the risk of dysphagia compared with PPI treatment.##REF##26544951##28## Furthermore, by 5 to 10 years post-surgery, some patients experience a recurrence of burdensome GORD symptoms such as heartburn and regurgitation.##REF##24574753##29## Therefore, there remains a group of patients who do not achieve optimal control of GORD symptoms with standard of care treatments. A novel treatment modality, MSA using the LINX<sup>®</sup> system, has recently been recommended by NICE as part of routine care,##UREF##2##4## and its adoption is likely to be gradual. Consequently, there remains a need for additional treatment options for patients with GORD.</p>", "<p>New treatments for GORD are likely to become even more important from the population health perspective over the coming years with the aging population of the UK. The Global Burden of Disease Study 2017 estimated that the number of cases of GORD as well as the absolute number of years lived with disability due to the condition increased in the UK between 1990 and 2017; however, the corresponding age-standardized rates did not increase, consistent with the higher prevalence of GORD in older age groups and the aging of the population.##REF##32178772##2## As the UK population continues to age in the coming years, GORD is likely to become even more prevalent, with more patients seeking access to innovative, effective treatment. Given that GORD patients with frequent and/or severe symptoms, who are perhaps most likely to seek surgical treatment, experience a substantial impairment of both health-related quality of life and work productivity,##REF##22486579##30## the broader societal benefits of a novel effective treatment for the condition are likely to be substantial.</p>", "<p>With regard to uncertainties and limitations of the current analysis, it should be noted that the results of the model are largely driven by the market share inputs. As these are associated with substantial uncertainty, and the real-world uptake of RefluxStop™ is difficult to predict, scenario analyses were conducted to assess the effect of halving or doubling the rates of RefluxStop™ uptake. In both of these scenarios, the budget impact of introducing the treatment remained small and manageable. Other inputs that are likely to substantially impact the model results are the size of the eligible patient population, and therefore the prevalence and incidence of GORD, the proportion of patients on PPIs who have not had prior surgical treatment for GORD, and the proportion of those willing to undergo surgery and eligible for it. An additional source of uncertainty around the results of the current analysis arises from the estimates of the total costs, which were based on a previously published cost-effectiveness model of RefluxStop™.##UREF##4##10## Although there was uncertainty around some of the inputs of that model, it predominantly used well-established, standard cost sources for England and its results proved robust to rigorous deterministic and probabilistic sensitivity analyses,##UREF##4##10## increasing confidence in using the model as a source of cost data for the current analysis.</p>" ]
[ "<title>CONCLUSION</title>", "<p>Introducing RefluxStop™ as a treatment option for patients with GORD treated within NHS England and Wales may be associated with clinical benefits at the expense of a marginal budget impact on the NHS over a 5-year time horizon, assuming the model is reflective of real-world outcomes. Considering the likely increase in both medical costs and the wider societal impact of GORD as the UK population continues to age, an effective and cost-effective treatment option for GORD patients that is also economically acceptable in the short term is likely to provide substantial benefits to patients, the NHS, and the broader society.</p>", "<title>Disclosures</title>", "<p>S.M., S.H., and L.G. work for a consulting company that works for a range of medical devices companies, including Implantica. They have no other interests to declare. E.G., P.G., and A.R.A have no interest to declare.</p>" ]
[ "<p><bold>Corresponding author:</bold> Email: [email protected]</p>", "<p><bold>Background:</bold> Gastro-oesophageal reflux disease (GORD) is a common condition associated with heartburn and regurgitation. Standard of care for GORD patients in the UK involves initial treatment with proton pump inhibitors (PPIs) and laparoscopic antireflux surgery in patients unwilling to continue or intolerant of long-term PPI treatment. Recently, RefluxStop™, a novel, implantable medical device, has proven to be an efficacious and cost-effective treatment for patients with GORD. The current analysis aimed to describe the budget impact of introducing RefluxStop™ within National Health Service (NHS) England and Wales.</p>", "<p><bold>Objectives:</bold> To estimate the more immediate, short-term clinical and economic effects of introducing RefluxStop™ as a therapeutic option for patients with GORD treated within NHS England and Wales.</p>", "<p><bold>Methods:</bold> A model adherent to international best practice guidelines was developed to estimate the budget impact of introducing RefluxStop™ over a 5-year time horizon, from an NHS perspective. Two hypothetical scenarios were considered, one without RefluxStop™ (comprising PPI treatment, laparoscopic Nissen fundoplication, and magnetic sphincter augmentation using the LINX® system) and one with RefluxStop™ (adding RefluxStop™ to the aforementioned treatment options). Clinical benefits and costs associated with each intervention were included in the analysis.</p>", "<p><bold>Results:</bold> Over 5 years, introducing RefluxStop™ allowed the avoidance of 347 surgical failures, 39 reoperations, and 239 endoscopic esophageal dilations. The financial impact of introducing RefluxStop™ was £3 029 702 in year 5, corresponding to a 1.68% increase in annual NHS spending on GORD treatment in England and Wales.</p>", "<p><bold>Discussion:</bold> While the time horizon was too short to capture some of the adverse events of PPIs and complications of GORD, such as the development of Barrett’s esophagus or esophageal cancer, the use of RefluxStop™ was associated with a substantial reduction in surgical complications, including surgical failures, reoperations, and endoscopic esophageal dilations. This favorable clinical profile resulted in cost offsets for the NHS and contributed to the marginal budget impact of RefluxStop™ estimated in the current analysis.</p>", "<p><bold>Conclusions:</bold> Introducing RefluxStop™ as a treatment option for patients with GORD in England and Wales may be associated with clinical benefits at the expense of a marginal budget impact on the NHS.</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>Medical writing and editorial assistance with the preparation of the manuscript was provided by Karolina Badora, PhD.</p>" ]
[ "<fig position=\"float\" id=\"attachment-190191\"><object-id pub-id-type=\"publisher-id\">190191</object-id><label>Figure 1.</label><caption><title>Clinical Outcomes of Base Case Scenarios With and Without RefluxStop™ Over the 5-Year Model Time Horizon</title></caption></fig>", "<fig position=\"float\" id=\"attachment-190192\"><object-id pub-id-type=\"publisher-id\">190192</object-id><label>Figure 2.</label><caption><title>Total Costs of Base Case Scenarios With and Without RefluxStop™</title></caption></fig>", "<fig position=\"float\" id=\"attachment-192126\"><object-id pub-id-type=\"publisher-id\">192126</object-id><caption><title>Supplementary Online Material</title></caption><ext-link xlink:href=\"https://jheor.org/article/90924-budget-impact-of-refluxstop-as-a-treatment-for-patients-with-refractory-gastro-oesophageal-reflux-disease-in-the-united-kingdom/attachment/192126.pdf\" ext-link-type=\"uri\"/></fig>" ]
[ "<table-wrap position=\"float\" id=\"attachment-190189\"><object-id pub-id-type=\"publisher-id\">190189</object-id><label>Table 1.</label><caption><title>Market Share of Interventions Considered in the Scenarios With and Without RefluxStop™</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" colspan=\"1\">\n<bold>Treatment</bold>\n</th><th colspan=\"5\" rowspan=\"1\">\n<bold>Market Share (%)</bold>\n</th></tr><tr><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 1</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 2</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 3</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 4</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 5</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Scenario without RefluxStop™ (all scenarios)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Nissen fundoplication</td><td rowspan=\"1\" colspan=\"1\">89.7</td><td rowspan=\"1\" colspan=\"1\">79.6</td><td rowspan=\"1\" colspan=\"1\">69.6</td><td rowspan=\"1\" colspan=\"1\">63.0</td><td rowspan=\"1\" colspan=\"1\">56.4</td></tr><tr><td rowspan=\"1\" colspan=\"1\">MSA</td><td rowspan=\"1\" colspan=\"1\">10.3</td><td rowspan=\"1\" colspan=\"1\">20.4</td><td rowspan=\"1\" colspan=\"1\">30.4</td><td rowspan=\"1\" colspan=\"1\">37.0</td><td rowspan=\"1\" colspan=\"1\">43.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Base case scenario with RefluxStop™</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">RefluxStop™</td><td rowspan=\"1\" colspan=\"1\">5.1</td><td rowspan=\"1\" colspan=\"1\">10.2</td><td rowspan=\"1\" colspan=\"1\">15.2</td><td rowspan=\"1\" colspan=\"1\">18.5</td><td rowspan=\"1\" colspan=\"1\">21.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Nissen fundoplication</td><td rowspan=\"1\" colspan=\"1\">84.6</td><td rowspan=\"1\" colspan=\"1\">69.4</td><td rowspan=\"1\" colspan=\"1\">54.4</td><td rowspan=\"1\" colspan=\"1\">44.5</td><td rowspan=\"1\" colspan=\"1\">34.7</td></tr><tr><td rowspan=\"1\" colspan=\"1\">MSA</td><td rowspan=\"1\" colspan=\"1\">10.3</td><td rowspan=\"1\" colspan=\"1\">20.4</td><td rowspan=\"1\" colspan=\"1\">30.4</td><td rowspan=\"1\" colspan=\"1\">37.0</td><td rowspan=\"1\" colspan=\"1\">43.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Scenario with RefluxStop™ (reduced rate of RefluxStop™ uptake)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">RefluxStop™</td><td rowspan=\"1\" colspan=\"1\">2.6</td><td rowspan=\"1\" colspan=\"1\">5.1</td><td rowspan=\"1\" colspan=\"1\">7.6</td><td rowspan=\"1\" colspan=\"1\">9.3</td><td rowspan=\"1\" colspan=\"1\">10.9</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Nissen fundoplication</td><td rowspan=\"1\" colspan=\"1\">87.2</td><td rowspan=\"1\" colspan=\"1\">74.5</td><td rowspan=\"1\" colspan=\"1\">62.0</td><td rowspan=\"1\" colspan=\"1\">53.7</td><td rowspan=\"1\" colspan=\"1\">45.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">MSA</td><td rowspan=\"1\" colspan=\"1\">10.3</td><td rowspan=\"1\" colspan=\"1\">20.4</td><td rowspan=\"1\" colspan=\"1\">30.4</td><td rowspan=\"1\" colspan=\"1\">37.0</td><td rowspan=\"1\" colspan=\"1\">43.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Scenario with RefluxStop™ (increased rate of RefluxStop™ uptake)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">RefluxStop™</td><td rowspan=\"1\" colspan=\"1\">10.3</td><td rowspan=\"1\" colspan=\"1\">20.4</td><td rowspan=\"1\" colspan=\"1\">30.4</td><td rowspan=\"1\" colspan=\"1\">37.0</td><td rowspan=\"1\" colspan=\"1\">43.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Nissen fundoplication</td><td rowspan=\"1\" colspan=\"1\">79.5</td><td rowspan=\"1\" colspan=\"1\">59.2</td><td rowspan=\"1\" colspan=\"1\">39.1</td><td rowspan=\"1\" colspan=\"1\">26.0</td><td rowspan=\"1\" colspan=\"1\">12.9</td></tr><tr><td rowspan=\"1\" colspan=\"1\">MSA</td><td rowspan=\"1\" colspan=\"1\">10.3</td><td rowspan=\"1\" colspan=\"1\">20.4</td><td rowspan=\"1\" colspan=\"1\">30.4</td><td rowspan=\"1\" colspan=\"1\">37.0</td><td rowspan=\"1\" colspan=\"1\">43.6</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"attachment-190190\"><object-id pub-id-type=\"publisher-id\">190190</object-id><label>Table 2.</label><caption><title>Cost per Patient per Year for the Different Treatments Assessed</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" colspan=\"1\">\n<bold>Treatment</bold>\n</th><th colspan=\"6\" rowspan=\"1\">\n<bold>Per-patient Cost (£)</bold>\n</th></tr><tr><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 1</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 2</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 3</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 4</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 5</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Total (Years 1-5)</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Medical management</td><td rowspan=\"1\" colspan=\"1\">286</td><td rowspan=\"1\" colspan=\"1\">292</td><td rowspan=\"1\" colspan=\"1\">275</td><td rowspan=\"1\" colspan=\"1\">260</td><td rowspan=\"1\" colspan=\"1\">246</td><td rowspan=\"1\" colspan=\"1\">1359</td></tr><tr><td rowspan=\"1\" colspan=\"1\">RefluxStop™</td><td rowspan=\"1\" colspan=\"1\">10 489</td><td rowspan=\"1\" colspan=\"1\">45</td><td rowspan=\"1\" colspan=\"1\">45</td><td rowspan=\"1\" colspan=\"1\">48</td><td rowspan=\"1\" colspan=\"1\">50</td><td rowspan=\"1\" colspan=\"1\">10 677</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Nissen fundoplication</td><td rowspan=\"1\" colspan=\"1\">5749</td><td rowspan=\"1\" colspan=\"1\">86</td><td rowspan=\"1\" colspan=\"1\">77</td><td rowspan=\"1\" colspan=\"1\">80</td><td rowspan=\"1\" colspan=\"1\">81</td><td rowspan=\"1\" colspan=\"1\">6073</td></tr><tr><td rowspan=\"1\" colspan=\"1\">MSA</td><td rowspan=\"1\" colspan=\"1\">8856</td><td rowspan=\"1\" colspan=\"1\">134</td><td rowspan=\"1\" colspan=\"1\">114</td><td rowspan=\"1\" colspan=\"1\">116</td><td rowspan=\"1\" colspan=\"1\">117</td><td rowspan=\"1\" colspan=\"1\">9337</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"attachment-190369\"><object-id pub-id-type=\"publisher-id\">190369</object-id><label>Table 3.</label><caption><title>Number of Patients Receiving Each Treatment per Model Year in Base Case Scenarios With and Without RefluxStop™</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"3\" colspan=\"1\">\n<bold>Modeled Year</bold>\n</th><th colspan=\"7\" rowspan=\"1\">\n<bold>Patients Receiving Treatment (n)</bold>\n</th></tr><tr><th colspan=\"3\" rowspan=\"1\">\n<bold>Scenario Without RefluxStop™</bold>\n</th><th colspan=\"4\" rowspan=\"1\">\n<bold>Scenario With RefluxStop™</bold>\n</th></tr><tr><th rowspan=\"1\" colspan=\"1\">\n<bold>PPI-Based Medical Management</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Nissen Fundoplication</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>MSA</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>PPI-Based Medical Management</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Nissen Fundoplication</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>MSA</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>RefluxStop™</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">569 777</td><td rowspan=\"1\" colspan=\"1\">2626</td><td rowspan=\"1\" colspan=\"1\">300</td><td rowspan=\"1\" colspan=\"1\">569 777</td><td rowspan=\"1\" colspan=\"1\">2626</td><td rowspan=\"1\" colspan=\"1\">300</td><td rowspan=\"1\" colspan=\"1\">150</td></tr><tr><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">16 873</td><td rowspan=\"1\" colspan=\"1\">2342</td><td rowspan=\"1\" colspan=\"1\">600</td><td rowspan=\"1\" colspan=\"1\">16 873</td><td rowspan=\"1\" colspan=\"1\">2342</td><td rowspan=\"1\" colspan=\"1\">600</td><td rowspan=\"1\" colspan=\"1\">300</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">16 851</td><td rowspan=\"1\" colspan=\"1\">2057</td><td rowspan=\"1\" colspan=\"1\">900</td><td rowspan=\"1\" colspan=\"1\">16 851</td><td rowspan=\"1\" colspan=\"1\">2057</td><td rowspan=\"1\" colspan=\"1\">900</td><td rowspan=\"1\" colspan=\"1\">450</td></tr><tr><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">16 831</td><td rowspan=\"1\" colspan=\"1\">1872</td><td rowspan=\"1\" colspan=\"1\">1100</td><td rowspan=\"1\" colspan=\"1\">16 831</td><td rowspan=\"1\" colspan=\"1\">1872</td><td rowspan=\"1\" colspan=\"1\">1100</td><td rowspan=\"1\" colspan=\"1\">550</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">16 811</td><td rowspan=\"1\" colspan=\"1\">1685</td><td rowspan=\"1\" colspan=\"1\">1300</td><td rowspan=\"1\" colspan=\"1\">16 811</td><td rowspan=\"1\" colspan=\"1\">1685</td><td rowspan=\"1\" colspan=\"1\">1300</td><td rowspan=\"1\" colspan=\"1\">650</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"attachment-190193\"><object-id pub-id-type=\"publisher-id\">190193</object-id><label>Table 4.</label><caption><title>Total Costs Estimated in Base Case Scenarios With and Without RefluxStop™ and Budget Impact</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 1</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 2</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 3</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 4</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Year 5</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Eligible population (n)</td><td rowspan=\"1\" colspan=\"1\">572 703</td><td rowspan=\"1\" colspan=\"1\">592 519</td><td rowspan=\"1\" colspan=\"1\">612 327</td><td rowspan=\"1\" colspan=\"1\">632 130</td><td rowspan=\"1\" colspan=\"1\">651 926</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Population expected to receive RefluxStop™ (n)</td><td rowspan=\"1\" colspan=\"1\">150</td><td rowspan=\"1\" colspan=\"1\">450</td><td rowspan=\"1\" colspan=\"1\">900</td><td rowspan=\"1\" colspan=\"1\">1450</td><td rowspan=\"1\" colspan=\"1\">2100</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Cost of treatment pathway without RefluxStop™ (£)</td><td rowspan=\"1\" colspan=\"1\">180 963 494</td><td rowspan=\"1\" colspan=\"1\">190 134 285</td><td rowspan=\"1\" colspan=\"1\">186 886 832</td><td rowspan=\"1\" colspan=\"1\">183 730 506</td><td rowspan=\"1\" colspan=\"1\">180 906 837</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Cost of treatment pathway with RefluxStop™ (£)</td><td rowspan=\"1\" colspan=\"1\">181 674 508</td><td rowspan=\"1\" colspan=\"1\">191 550 209</td><td rowspan=\"1\" colspan=\"1\">189 002 761</td><td rowspan=\"1\" colspan=\"1\">186 304 637</td><td rowspan=\"1\" colspan=\"1\">183 936 538</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Nominal net budget impact (£)</td><td rowspan=\"1\" colspan=\"1\">711 014</td><td rowspan=\"1\" colspan=\"1\">1 415 924</td><td rowspan=\"1\" colspan=\"1\">2 115 929</td><td rowspan=\"1\" colspan=\"1\">2 574 132</td><td rowspan=\"1\" colspan=\"1\">3 029 702</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Net budget impact as percentage of NHS spending for<break/>GORD treatment (%)</td><td rowspan=\"1\" colspan=\"1\">0.39</td><td rowspan=\"1\" colspan=\"1\">0.75</td><td rowspan=\"1\" colspan=\"1\">1.13</td><td rowspan=\"1\" colspan=\"1\">1.40</td><td rowspan=\"1\" colspan=\"1\">1.68</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>Abbreviation: MSA, magnetic sphincter augmentation.</p></table-wrap-foot>", "<table-wrap-foot><p>Abbreviation: MSA, magnetic sphincter augmentation.</p></table-wrap-foot>", "<table-wrap-foot><p>Abbreviations: MSA, magnetic sphincter augmentation; PPI, proton pump inhibitor.</p></table-wrap-foot>", "<table-wrap-foot><p>Abbreviations: GORD, gastro-oesophageal reflux disease; NHS, National Health Service.</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"jheor_2024_11_1_90924_190191\" position=\"float\"/>", "<graphic xlink:href=\"jheor_2024_11_1_90924_190192\" position=\"float\"/>" ]
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[{"article-title": ["A narrative review of the prevalence of gastroesophageal reflux disease (GERD)"], "source": ["Ann Oesophagus"], "person-group": ["\n"], "surname": ["Boulton", "Dettmar"], "given-names": ["K.H.A.", "P.W."], "year": ["2021"], "volume": ["5"], "fpage": ["7"], "mixed-citation": ["Boulton KHA, Dettmar PW. A narrative review of the prevalence of gastroesophageal reflux disease (GERD). "], "italic": ["Ann Oesophagus"]}, {"source": ["Clinical guideline [CG184]: Gastro-oesophageal reflux disease and dyspepsia in adults: investigation and management"], "person-group": ["\n"], "collab": ["National Institute for Health and Care Excellence"], "ext-link": ["https://www.nice.org.uk/guidance/cg184"], "mixed-citation": ["National Institute for Health and Care Excellence. Clinical guideline [CG184]: Gastro-oesophageal reflux disease and dyspepsia in adults: investigation and management. Accessed June 19, 2023. https://www.nice.org.uk/guidance/cg184"]}, {"source": ["Interventional procedures guidance [IPG749]: Laparoscopic insertion of a magnetic ring for gastrooesophageal reflux disease"], "person-group": ["\n"], "collab": ["National Institute for Health and Care Excellence"], "ext-link": ["https://www.nice.org.uk/guidance/ipg749"], "mixed-citation": ["National Institute for Health and Care Excellence. Interventional procedures guidance [IPG749]: Laparoscopic insertion of a magnetic ring for gastrooesophageal reflux disease. Accessed June 19, 2023. https://www.nice.org.uk/guidance/ipg749"]}, {"source": ["Implantica receives CE mark approval for RefluxStop\u2122, a potential paradigm shift in the treatment for acid reflux"], "person-group": ["\n"], "collab": ["Implantica"], "ext-link": ["https://www.implantica.com/media/press-releases/2018/implantica-receives-ce-mark-approval-for-refluxstop-a-potential-paradigm-shift-in-the-treatment-for-acid-reflux/"], "mixed-citation": ["Implantica. Implantica receives CE mark approval for RefluxStopTM, a potential paradigm shift in the treatment for acid reflux. Accessed June 19, 2023. https://www.implantica.com/media/press-releases/2018/implantica-receives-ce-mark-approval-for-refluxstop-a-potential-paradigm-shift-in-the-treatment-for-acid-reflux/"]}, {"article-title": ["Cost-effectiveness of a novel, non-active implantable device as a treatment for refractory gastro-esophageal reflux disease"], "source": ["Journal of Medical Economics"], "person-group": ["\n"], "surname": ["Harper", "Grodzicki", "Mealing", "Gemmill", "Goldsmith", "Ahmed"], "given-names": ["Sam", "Lukasz", "Stuart", "Liz", "Paul J.", "Ahmed R."], "day": ["2"], "month": ["5"], "year": ["2023"], "volume": ["26"], "issue": ["1"], "fpage": ["603"], "lpage": ["613"], "issn": ["1369-6998"], "pub-id": ["10.1080/13696998.2023.2201063", "10.1080/13696998.2023.2201063"], "mixed-citation": ["Harper S, Grodzicki L, Mealing S, Gemmill L, Goldsmith PJ, Ahmed AR. Cost-effectiveness of a novel, non-active implantable device as a treatment for refractory gastro-esophageal reflux disease. "], "italic": ["J Med Econ"]}, {"article-title": ["Budget impact analysis-principles of good practice: report of the ISPOR 2012 Budget Impact Analysis Good Practice II Task Force"], "source": ["Value in Health"], "person-group": ["\n"], "surname": ["Sullivan", "Mauskopf", "Augustovski", "Jaime Caro", "Lee", "Minchin", "Orlewska", "Penna", "Rodriguez Barrios", "Shau"], "given-names": ["Sean D.", "Josephine A.", "Federico", "J.", "Karen M.", "Mark", "Ewa", "Pete", "Jose-Manuel", "Wen-Yi"], "month": ["1"], "year": ["2014"], "volume": ["17"], "issue": ["1"], "fpage": ["5"], "lpage": ["14"], "issn": ["1098-3015"], "pub-id": ["10.1016/j.jval.2013.08.2291", "10.1016/j.jval.2013.08.2291"], "mixed-citation": ["Sullivan SD, Mauskopf JA, Augustovski F, et al. Budget impact analysis-principles of good practice: report of the ISPOR 2012 Budget Impact Analysis Good Practice II Task Force. "], "italic": ["Value Health"]}, {"source": ["Estimates of the population for the UK, England and Wales, Scotland and Northern Ireland. 2019 mid year estimates"], "person-group": ["\n"], "collab": ["Office for National Statistics (ONS)"], "ext-link": ["https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationestimates/datasets/populationestimatesforukenglandandwalesscotlandandnorthernireland"], "mixed-citation": ["Office for National Statistics (ONS). Estimates of the population for the UK, England and Wales, Scotland and Northern Ireland. 2019 mid year estimates. Accessed June 19, 2023. https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationestimates/datasets/populationestimatesforukenglandandwalesscotlandandnorthernireland"]}, {"source": ["Population projections: national population projections: 2018-based"], "person-group": ["\n"], "collab": ["Office for National Statistics (ONS)"], "ext-link": ["https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationprojections/bulletins/nationalpopulationprojections/2018based"], "mixed-citation": ["Office for National Statistics (ONS). Population projections: national population projections: 2018-based. Accessed June 19, 2023. https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationprojections/bulletins/nationalpopulationprojections/2018based"]}, {"source": ["In Focus: High-Cost PPIs"], "person-group": ["\n"], "collab": ["NHS Dorset Clinical Commissioning Group"], "ext-link": ["https://www.dorsetccg.nhs.uk/Downloads/aboutus/medicines-management/Other%20Guidelines/Gastroenterology%20High%20cost%20PPI%20in%20focus%200519%20%282%29.pdf?UNLID=97481392021984165"], "comment": ["Accessed June 19, 2023."], "mixed-citation": ["NHS Dorset Clinical Commissioning Group. In Focus: High-Cost PPIs. Accessed June 19, 2023. https://www.dorsetccg.nhs.uk/Downloads/aboutus/medicines-management/Other%20Guidelines/Gastroenterology%20High%20cost%20PPI%20in%20focus%200519%20%282%29.pdf?UNLID=97481392021984165"]}, {"article-title": ["The association between proton pump inhibitor use and the risk of adverse kidney outcomes: a systematic review and meta-analysis"], "source": ["Nephrology Dialysis Transplantation"], "person-group": ["\n"], "surname": ["Nochaiwong", "Ruengorn", "Awiphan", "Koyratkoson", "Chaisai", "Noppakun", "Chongruksut", "Thavorn"], "given-names": ["Surapon", "Chidchanok", "Ratanaporn", "Kiatkriangkrai", "Chayutthaphong", "Kajohnsak", "Wilaiwan", "Kednapa"], "year": ["2018"], "volume": ["33"], "issue": ["2"], "fpage": ["331"], "lpage": ["342"], "issn": ["0931-0509"], "pub-id": ["10.1093/ndt/gfw470", "10.1093/ndt/gfw470"], "mixed-citation": ["Nochaiwong S, Ruengorn C, Awiphan R, et al. The association between proton pump inhibitor use and the risk of adverse kidney outcomes: a systematic review and meta-analysis. "], "italic": ["Nephrol Dial Transplant"]}, {"article-title": ["Meta-analysis of proton pump inhibitors induced risk of community-acquired pneumonia"], "source": ["International Journal for Quality in Health Care"], "person-group": ["\n"], "surname": ["Nguyen", "Islam", "Galvin", "Chang", "An", "Yang", "Huang", "Li", "Iqbal"], "given-names": ["Phung Anh", "Mohaimenul", "Cooper J", "Chih-Cheng", "Soo Yeon", "Hsuan-Chia", "Chih-Wei", "Yu-Chuan (Jack)", "Usman"], "day": ["21"], "month": ["5"], "year": ["2020"], "volume": ["32"], "issue": ["5"], "fpage": ["292"], "lpage": ["299"], "issn": ["1353-4505"], "pub-id": ["10.1093/intqhc/mzaa041", "10.1093/intqhc/mzaa041"], "mixed-citation": ["Nguyen PA, Islam M, Galvin CJ, et al. Meta-analysis of proton pump inhibitors induced risk of community-acquired pneumonia. "], "italic": ["Int J Qual Health Care"]}, {"source": ["2019/20 National Cost Collection Data Publication"], "person-group": ["\n"], "collab": ["NHS England"], "ext-link": ["https://www.england.nhs.uk/publication/2019-20-national-cost-collection-data-publication/"], "comment": ["Accessed June 19, 2023."], "mixed-citation": ["NHS England. 2019/20 National Cost Collection Data Publication. Accessed June 19, 2023. https://www.england.nhs.uk/publication/2019-20-national-cost-collection-data-publication/"]}, {"source": ["Unit Costs of Health and Social Care 2021"], "person-group": ["\n"], "surname": ["Jones", "Burns"], "given-names": ["K.", "A."], "year": ["2021"], "ext-link": ["https://www.pssru.ac.uk/project-pages/unit-costs/unit-costs-of-health-and-social-care-2021/"], "mixed-citation": ["Jones K, Burns A. Unit Costs of Health and Social Care 2021. Published 2021. https://www.pssru.ac.uk/project-pages/unit-costs/unit-costs-of-health-and-social-care-2021/"]}, {"article-title": ["Editor\u2019s pick: Refractory gastroesophageal reflux disease: pathophysiology, diagnosis, and management"], "source": ["EMJ Gastroenterology"], "person-group": ["\n"], "surname": ["Nabi", "Karyampudi", "Nageshwar Reddy"], "given-names": ["Zaheer", "Arun", "D."], "day": ["5"], "month": ["12"], "year": ["2019"], "volume": ["2019"], "fpage": ["62"], "lpage": ["71"], "issn": ["2054-6203"], "pub-id": ["10.33590/emjgastroenterol/10314735", "10.33590/emjgastroenterol/10314735"], "mixed-citation": ["Nabi Z, Karyampudi A, Nageshwar Reddy D. Editor\u2019s pick: Refractory gastroesophageal reflux disease: pathophysiology, diagnosis, and management. "], "italic": ["EMJ Gastroenterol"]}]
{ "acronym": [], "definition": [] }
30
CC BY
no
2024-01-14 23:43:48
J Health Econ Outcomes Res.; 11(1):1-7
oa_package/f5/c8/PMC10787539.tar.gz
PMC10787541
0
[ "<title>Introduction</title>", "<p>Thrace, located on the western side of Turkey, is a prominent agricultural region and produces large quantities of wheat, canola, sunflower, rice, and grapes due to its fertile soils, water availability, and ideal climatic conditions. This region alone provided 44.5% of the domestic rice and 41.1% of <italic toggle=\"yes\">Helianthus annuus</italic> L. (sunflower) production in 2021 (##UREF##25##TUIK, 2022##). Furthermore, the agricultural productivity of this region has created a rich agricultural industry processing these crops, including oil and feed factories, mills, and food companies. It should be noted that although this region produces a majority of the sunflower and rice that Turkey consumes, one-third of sunflower and one-fifth of rice consumed in Turkish markets are imported from abroad (##UREF##25##TUIK, 2022##). Moreover, Turkey ranked first among countries that import sunflower in 2021 (##UREF##16##OEC, 2023##). Under pressure to increase productivity, growers from this region increasingly rely on chemical means to control pests such as weeds and diseases and have reduced the numbers of fields in fallow. Due to the reduction in fallow fields, sunflower fields and rice paddies are often adjacent.</p>", "<p>Chemical weed control <italic toggle=\"yes\">via</italic> herbicides has several advantages for many farmers including rice farmers; it is economical, easy, and overall efficient. However, among weed species that are common in rice paddies, a number of biotypes have developed resistance to herbicides due to over-reliance on only a few herbicidal sites of action (SOA) (##UREF##1##Altop et al., 2014##; ##UREF##5##Haghnama &amp; Mennan, 2020##; ##UREF##6##Kacan et al., 2020##). Herbicides are classified based on their SOA which is the specific protein where the herbicide binds and subsequently inhibits weed growth; this classification helps growers manage herbicide resistance. For instance, group 1 and 2 herbicides inhibit acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS), respectively, while group 4 herbicides like quinclorac act as synthetic auxin (##UREF##10##Mallory-Smith &amp; Retzinger, 2003##). Farmers who were living in the Thrace Region of Turkey have increased rates of ALS and ACCase inhibiting herbicides due to the failure of recommended rates (##UREF##22##Serim et al., 2020##). To address the increasing weed resistance to the commonly used herbicide SOA, some pesticide producing companies launched alternatives to ALS and ACCase inhibiting herbicides, including synthetic auxins in 2019 (##UREF##17##PPPD, 2022##).</p>", "<p>Many rice farmers have been making multiple applications by using auxinic rice herbicides, which were registered to control weeds in a single season to achieve effective weed control. Spot spraying in rice fields is often done using a knapsack mist blower. This practice of spot spraying late in the season has increased the off-target movement (OTM) risk of herbicides, such as synthetic auxins from rice fields to sunflower fields of the Thrace Region. Previous research indicates that the severity of the drift caused by herbicides may change depending on the herbicide molecule, rate of herbicide, application parameters, weather conditions, growth stage, and non-target crop species (##REF##28731230##Cederlund, 2017##). Sunflower is considered one of the most sensitive crops to herbicide residues and drift, especially synthetic auxins, ALS and EPSP synthase inhibitors (##UREF##4##Greenshields &amp; Putt, 1958##; ##UREF##8##Lanini &amp; Carrithers, 2000##; ##UREF##23##Serim &amp; Maden, 2014##; ##UREF##21##Serim, 2022##). ##UREF##26##Wall (1996)## estimated that 2.4-D amine can result in a 93–100% yield reduction when applied at 151.2 g ai ha<sup>−1</sup>(24% of the low recommended rate) in sunflower. Additionally, herbicide drift has threatened the sustainable use of herbicides in agricultural fields. For instance, recent studies have shown that sub-lethal rates of herbicides, such as those caused by OTM, may reduce the sensitivity of weeds to herbicides (##REF##32034222##Vieira et al., 2020##).</p>", "<p>Quinclorac and FPB are new herbicide active ingredients registered to control weeds in paddy fields of Turkey as of three years ago (##UREF##17##PPPD, 2022##). Quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) is a Group 4 herbicide that is thought to have two modes of action: (1) it seems to prevent cell wall biosynthesis and increases ethylene and cyanide production in grassy weeds while (2) it acts to mimic native auxin when applied to broadleaves weeds (##UREF##28##Weed Science Society of America, 2014##). OTM of this herbicide can cause severe injury on sensitive crops that grow adjacent to fields where it is applied, especially broadleaf crops, including tomato, pepper, cotton, and tree species (##UREF##24##Snipes, Street &amp; Mueller, 1992##; ##UREF##9##Lovelace et al., 2007##; ##UREF##0##Adams et al., 2017##; ##UREF##7##Kaya et al., 2023##). Florpyrauxifen-benzyl is an auxin-type herbicide (Group 4) belonging to the arylpicolinate chemical family that is used to control weed species in rice fields (##UREF##28##Weed Science Society of America, 2014##). This herbicide molecule is unique because of its wide herbicidal spectrum (##UREF##13##Miller &amp; Norsworthy, 2018a##). Previously developed auxin herbicides like quinclorac are used to control narrow-leaf grass weeds, whereas florpyrauxifen-benzyl also has the ability to control many broadleaf weed species. Furthermore, it is often used to kill weed biotypes resistant to ACCs (Group 1) and ALS (Group 2) inhibiting herbicides in paddy fields such as <italic toggle=\"yes\">Echinocloa</italic> spp.</p>", "<p>Several dicot crop species are sensitive to florpyrauxifen-benzyl. ##UREF##14##Miller &amp; Norsworthy (2018b)## note that soybean was the most sensitive to florpyrauxifen-benzyl in tested crops, and moderate injury was observed on the seedlings at 14 DAT at 1/100 of the recommended rate. ##UREF##20##Schwartz-Lazaro et al. (2017)## stated that florpyrauxifen-benzyl applied to soybean resulted in 71 and 31% injury at 21 DAT when applied with 1/20 and 1/80 drift rates, respectively. Cotton and sunflower also show severe injury at the 1/10 rate of the herbicide at 14 DAT and the herbicidal impact was mitigated by 28 DAT, especially at the 1/100 and 1/500 rates (##UREF##14##Miller &amp; Norsworthy, 2018b##). Grass crops such as grain sorghum and corn seem to be unaffected by the herbicide (##UREF##14##Miller &amp; Norsworthy, 2018b##). Because of the sensitivity of some broadleaf crops, florpyrauxifen-benzyl should be used with mitigation measurements to prevent damage on broadleaf crops. Therefore, recommendations should include a buffer zone and/or the use of drift reduction nozzles to protect non-target plants in some countries (##REF##32626021##Arena et al., 2018##).</p>", "<p>Although some studies have investigated the impact of sub-lethal doses of previously available synthetic auxin herbicides on sunflower, the impact of florpyrauxifen-benzyl has not yet been studied in Thracian sunflower production. The OTM of synthetic auxins is not unique to Turkey and has the potential to be problematic in many countries where rice and sunflower fields are in close proximity such as Italy, Greece, Russia, Argentina, Spain, Ukraine, and Bulgaria. The aim of this study was to determine the impact of sub-lethal rates of florpyrauxifen-benzyl + penoxsulam and quinclorac used in rice fields on the growth and yield of sunflower cultivars.</p>" ]
[ "<title>Material and Methods</title>", "<title>Growth chamber experiments</title>", "<p>A bioassay study was conducted in a growth chamber adjusted to 25 ± 1 °C/20 ± 1 °C with a 12/12 h day/night photoperiod to determine the sensitivity level of two commercial sunflower cultivars, Bosfora (Bosfora®; Syngenta Corporation, İzmir, Turkey) and Tunca (Tunca®; Limagrain Corporation, Bursa, Turkey), to sub-lethal rates of florpyrauxifen-benzyl + penoxsulam (BAXIGA® 32.5 OD; Corteva Agriscience Corporation, Istanbul, Turkey) and quinclorac (Facet®; BASF Corporation, Istanbul, Turkey). The growth chamber was set light to HPI-T Plus Metal Halide lamps (400 W). Three to four sunflower seeds were sown in plastic pots (7 × 7 × 8.5 cm) filled with white peat bedding substrate (TS 1, Klassman-Deilmann GmbH consisting of TS 1 fine + 15% perlite). After emergence seedlings were removed so that only two plants remained per pot. A high-performance air conditioning system was used to acclimatize the growth chamber because of adjusting of high temperature released by metal halide lamps. Therefore, the pots were arranged in a randomized complete block design with four replications according to acclimatization.</p>", "<p>In the experiment, the seedlings were treated with seven herbicide rates (0, 2.93, 5.86, 11.72, 23.44, 46.88, and 93.75 g ai ha<sup>−1</sup> for quinclorac and 0, 0.51, 1.02, 2.03, 4.06, 8.13, and 16.25 g ai ha<sup>−1</sup> for florpyrauxifen-benzyl + penoxsulam) at the 2-4 true leaf stage. The quinclorac and florpyrauxifen-benzyl + penoxsulam label rates were 375 and 65 g ai ha<sup>−1</sup> respectively (##UREF##17##PPPD, 2022##). Herbicides that were dissolved in tap water were applied using a motorized backpack sprayer equipped with two flat-fan nozzles mounted on a hand-held boom (Teejet XR11002) and calibrated to deliver 195 L ha<sup>−1</sup>. Sprayed seedlings were kept in the spraying place for 1 day after treatment. The seedlings were moved to the growth chamber and irrigated using tap water as needed. The seedlings were cut from ground level at 28 DAT and stored in a drying oven at 60 °C for 48 h to determine the above ground dry weight.</p>", "<title>Field experiments</title>", "<p>Two field experiments were conducted at Bilecik Seyh Edebali University Aşağıköy Agricultural Application and Research Centre (AAARC) in 2021 and 2022 to determine the response of sunflower cultivars to sub-lethal rates of florpyrauxifen-benzyl + penoxsulam and quinclorac under non-irrigated conditions. The soil texture at AARC was silty clay with 2.4% organic matter, pH 8.13. Field studies were arranged within a randomized complete block design with four replications. Conventional sunflower cultivars, Bosfora and Tunca, were sown to 70 cm spacing in plots consisting of four parallel rows on April 11, 2021, and May 02, 2022, respectively. Di-ammonium phosphate was applied at the time of sowing at 80 kg ha<sup>−1</sup>. In this study, the temperature and rainfall in 2021 and 2022 were close to long-term averages (12.1 and 12.7 °C and 449.2 and 478.9 mm, respectively).</p>", "<p>Florochloridone was applied prior to emergence at a 700 g ai ha<sup>−1</sup> rate in 2021, while pendimethalin was used two days after sowing at a 1.350 g ai ha<sup>−1</sup> rate to control weeds in 2022. Plots consisted of 4 70-cm-spaced rows 10 m long. Two crop rows between the herbicide-applied plots were left as alleys to avoid contamination between the plots.</p>", "<p>Quinclorac and florpyrauxifen-benzyl + penoxsulam rates were 11.72, 23.44, and 46.88 g ai ha<sup>−1</sup> and 2.03, 4.06, and 8.13 g ai ha<sup>−1</sup> respectively, which were equivalent to 3.125, 6.250, and 12.5% of the recommended use rates (##UREF##17##PPPD, 2022##). The aforementioned rates of herbicides were applied when the sunflower reached 8-10 true leaves. Sunflower injury caused by these herbicide rates was recorded at 7, 14, and 28 DAT based on a scale of 0–100%, where 0% indicated no impact of herbicides, while 100% represented complete plant death. Plants were harvested for yield at the ripening stage. At harvest, five plants from the middle two rows of each plot were randomly selected and harvested by hand on September 05, 2021, and October 04, 2022. The heights of the selected plants, sunflower head diameter (SHD), 1000-seed weight (OTSW), and yield were measured (##UREF##23##Serim &amp; Maden, 2014##).</p>", "<title>Statistical analysis</title>", "<p>The data from the dose-response study was evaluated using a nonlinear regression model in R statistical software (##UREF##18##RStudio Team, 2023##). The DRC package was used to calculate the dose-response curve and parameters with a four-parameter log–logistic model (##FORMU##0##Equation (1)##). \n</p>", "<p>where <italic toggle=\"yes\">y</italic> represents seedling dry matter at herbicide treatment rate <italic toggle=\"yes\">x</italic>; b, c, d, and GR<sub>50</sub> represent slope, lower limit, upper limit, and herbicide rate that reduced seedling dry matter by 50%, respectively.</p>", "<p>The data from the field experiments were analyzed with analysis of variance for each herbicide and cultivar, and mean separation was performed with Fisher’s protected least significant difference test at the 5% level of probability. The agricolae package (##UREF##12##Mendiburu &amp; Yaseen, 2020##) was used in the R statistical software program (##UREF##18##RStudio Team, 2023##). The Pearson correlation test was used to find a relationship between quinclorac injuries measured at 4 separate times and the yield (and yield components). The test was not performed for FBP because higher rates of FBP killed sunflowers before harvest.</p>" ]
[ "<title>Results and Discussion</title>", "<title>Growth chamber experiment</title>", "<p>The sunflower cultivar ‘Tunca’ was severely injured when exposed to low rates of florpyrauxifen-benzyl + penoxsulam (##FIG##0##Fig. 1A##). However, the lowest rate of herbicide unexpectedly increased shoot length, probably due to the hormetic impact of auxinic herbicides, similar to what was previously reported by ##UREF##15##Mudge et al. (2021)##. Overall, shoot lengths decreased as the rates increased. The impact of the highest four rates of herbicide was the most destructive, and the growing points of cultivar ‘Bosfora’ were completely killed at these rates at 28 DAT (##FIG##0##Fig. 1B##). More than half of the leaf area of seedlings exposed to these rates became necrotic. The response of cultivar Bosfora to FBP was similar to that of cultivar Tunca, except at rates higher than 1.02 g ai ha<sup>−1</sup> where it was more injured (##FIG##0##Figs. 1A## and ##FIG##0##1B##).</p>", "<p>Injury from quinclorac in both cultivars increased over time, with a slight and gradual increase in sunflower injury with rate. By 28 DAT, the highest sunflower injury was caused by the highest rate of quinclorac (##FIG##0##Figs. 1C## and ##FIG##0##1D##). The response of these sunflower cultivars to lower rates of quinclorac was very similar; however, cultivar Bosfora was slightly more sensitive to quinclorac than cultivar Tunca at higher rates. As opposed to FBP, sunflower plants of both cultivars treated with quinclorac still had live growing points, and necrotic areas on the seedling leaves were relatively limited even at the highest rates. At the lowest rates, FBP resulted in no damage to these cultivars, while at the lowest dose of quinclorac growth reduction was still observed.</p>", "<p>The quantitative response of the cultivar Tunca and cultivar Bosfora to FBP and quinclorac was evaluated <italic toggle=\"yes\">via</italic> a dose-response assay using a log–logistic model. The GR<sub>50</sub> values of FBP for the cultivars Tunca and Bosfora were 1.07 and 0.75 g ai ha<sup>−1</sup>, respectively (##TAB##0##Table 1##), nearly 2.9% and 2% of the recommended rate of FBP (65 g ai ha<sup>−1</sup>). The GR<sub>50</sub> values of quinclorac for sunflower cultivars were 14.16 and 7.56 g ai ha<sup>−1</sup>, 3,8% and 2% of the recommended rate of quinclorac (375 g ai ha<sup>−1</sup>). Similar to the visual herbicidal impact of FBP and quinclorac, the results show that FBP was slightly more injurious than quinclorac to these cultivars (##FIG##1##Fig. 2##).</p>", "<title>Field experiment</title>", "<title>Crop injury</title>", "<p>Sunflower crops exposed to FBP and quinclorac responded to low-dose treatment, especially the leaves. FBP resulted in typical auxin symptoms, such as parallel veins, cupping, twisting, chlorosis, and distortion (##FIG##2##Fig. 3##). Quinclorac also caused these symptoms, except it did not demonstrate distortion (##FIG##3##Fig. 4##). The severity of symptoms was higher in cultivar Bosfora than in cultivar Tunca and increased as the rates increased. Stunting became apparent at 28 DAT for both cultivars. The highest rate of FBP prevented the establishment of sunflower heads in both cultivars (##FIG##4##Figs. 5B## and ##FIG##4##5D##).</p>", "<p>Quinclorac and FBP injury was low at 7 DAT, and no difference was found between cultivar Tunca and cultivar Bosfora (##FIG##5##Figs. 6A## and ##FIG##5##6B##). The response of sunflower cultivar to FBP increased as the rates rose and reached 75–77.5% at 8.13 g ai ha<sup>−1</sup> at 7 DAT. Similar to the growth chamber study, quinclorac injury in cultivar Bosfora (13.75–33.75%) and cultivar Tunca (17.5–30%) was lower than those of FBP. At 14 DAT, the response of the cultivars to the herbicides was generally similar to that at 7 DAT. Sunflower injury due to FBP increased with the increase in herbicide rates from 2.03 to 8.13 g ai ha<sup>−1</sup> at 14 DAT. Cultivar Bosfora exposure to quinclorac at 11.72, 23.44, and 46.88 g ai ha<sup>−1</sup>resulted in 15, 22.5, and 38.75% injury, respectively, while cultivar Tunca was less sensitive to these rates at 14 DAT.</p>", "<p>Sunflower injury at the highest rate of FBP was 100% by 28 DAT while the lowest FBP rate resulted in sunflower injury of 76.25–88.75% (##FIG##5##Figs. 6A## and ##FIG##5##6B##). Differences between sunflower cultivars became more apparent at 28 DAT. Although the phytotoxicity of quinclorac increased at 28 DAT, crop injury caused by the herbicide was nearly half that of FBP.</p>", "<p>Visible injury rates reached the greatest level at harvest. Sunflower injury at 2.03 g ai ha<sup>−1</sup> FBP was higher than &gt;90% for cultivar Bosfora and &gt;85% for cultivar Tunca. The highest FBP rate led to complete death of both sunflower cultivars. The injury increased in severity as the quinclorac rate increased in both cultivars. The injury on cultivar Bosfora and cultivar Tunca ranged from 42.5–57.5% and from 36.25–48.75%, respectively. The severity of injury due to quinclorac was limited to at 7 DAT because injury assessment was only performed on leaves, while this influence was more apparent in evaluations of other crop parameters, including plant height, stem structure, size of flower bud and head, were included in the evaluations. Compared to quinclorac, FBP was more phytotoxic to both cultivars, and this destructive impact was observed even from the first assessments at 7 DAT.</p>", "<p>The injury rate of auxin herbicides on sensitive row crops has been reported in some studies. For instance, ##UREF##19##Schroeder, Cole &amp; Dexter (1979)## stated that 2,4-D and dicamba resulted in tremendous injury to sunflowers. In another study, ##UREF##24##Snipes, Street &amp; Mueller (1992)## reported that 17 g ha<sup>−1</sup> or higher rates of quinclorac may injure cotton when applied at the cotyledon stage, and cotton injury increased with increasing herbicide rate, especially during late-stage applications, such as at the pin-head square stage. Overall, the cotton injury rate reached 59% at 140 g ha<sup>−1</sup> in their study, and sunflower injury caused by quinclorac at 46.88 ai g ha<sup>−1</sup> was 52-61.25% in our study. A study conducted by ##UREF##9##Lovelace et al. (2007)## revealed that tomato was also among quinclorac-sensitive crops, and crop injury caused by quinclorac applied at 42 g ai ha<sup>−1</sup> was 45% at 49 DAT. In our study, the third evaluation was done at 55 DAT, and crop injuries were 48.75–57.5% when herbicide was applied at 46.88 g ai ha<sup>−1</sup>. These data are consistent with the work of ##UREF##9##Lovelace et al. (2007)##. Comparing these results to those of ##UREF##24##Snipes, Street &amp; Mueller (1992)## and ##UREF##9##Lovelace et al. (2007)##, sunflower can be classified as a more sensitive crop than cotton.</p>", "<p>Florpyrauxifen-benzyl is a relatively new herbicide on the market; therefore, few drift studies are available in the literature. FBP containing 2.03 g ai ha<sup>−1</sup> florpyrauxifen-benzyl + penoxsulam caused 76 and 89% injury at 28 DAT on cultivar Tunca and cultivar Bosfora, respectively. In agreement with the results of our study, ##UREF##14##Miller &amp; Norsworthy (2018b)## stated that 3 g ai ha<sup>−1</sup> florpyrauxifen-benzyl applied to sunflowers at the three true-leaf stage resulted in 69% injury, at 28 DAT under greenhouse conditions.</p>", "<title>Plant height</title>", "<p>The heights of the sunflower cultivars constantly declined as the FBP and quinclorac rates increased. All FBP rates resulted in a significant decrease in the plant height of the sunflower cultivars; The lowest rate of FBP resulted in an 8–9% decrease while the highest resulted in a 32–44% decrease (##FIG##6##Fig. 7A##). Similarly, the height reduction from quinclorac ranged from 13–14% at the lowest rate to 25–32% at the highest. Similar to the findings of this study, ##UREF##14##Miller &amp; Norsworthy (2018b)## also stated that 3 g ai ha<sup>−1</sup> florpyrauxifen-benzyl applied to sunflowers at the three true-leaf stage resulted in a 66% plant height reduction at 28 DAT under greenhouse conditions. The lower plant height observed in our study may be due to the application time of herbicide or penoxsulam in the herbicide mixture.</p>", "<title>Sunflower head diameter and One thousand seed weight</title>", "<p>Increasing FBP and quinclorac rates caused sunflower head diameter (SHD) to decline regardless of the cultivar. The greatest percentage of decline resulting from FBP was recorded at 4.06 g ai ha<sup>−1</sup> with 76–77%. At the highest rate of FBP (8.13 g ai ha<sup>−1</sup>), no flower heads were observed (##FIG##4##Figs. 5B## and ##FIG##4##5D##). SHD reduction in cultivar Bosfora and cultivar Tunca from quinclorac ranged from 19–57% and 9–51%, respectively (##FIG##6##Fig. 7B##). A slight decrease was observed in One thousand seed weight (TSW) compared to plant height and SHD, but this decrease, even at the highest level of herbicide, was no more than 21% (##FIG##6##Fig. 7C##). Sunflower exposed to higher FBP rates, 4.06 and 8.13 g ai ha<sup>−1</sup>, could not produce mature seeds. The results showed that TSW was a less susceptible yield component to FBP drift rates than the others.</p>", "<title>Yield</title>", "<p>Associated with other yield components, significant sunflower yield loss was determined even at lower rates of quinclorac and FBP (##FIG##6##Fig. 7D##). The highest yield reduction was observed for sunflower cultivars treated with FBP at 4.06 and 8.03 g ai ha<sup>−1</sup>at 100%. The lowest FBP rate reduced cultivar Bosfora and cultivar Tunca yields by 79.8 and 74.3%, respectively. The yield reduction of cultivar Bosfora from quinclorac rates was 45.1–87.9%, while yield loss of cultivar Tunca caused these rates to range from 16.3–82.3%. Although no study was found in the literature related to the impact of low rates of quinclorac and FBP on sunflower yield and yield components such as SHD and TSW, previous research reported significant reductions in crop growth, yield, and some yield components in other systems. ##UREF##14##Miller &amp; Norsworthy (2018b)## found that soybean yield was reduced 71% when florpyrauxifen-benzyl was applied at 3 g ae ha<sup>−1</sup> rate. In another study, ##UREF##24##Snipes, Street &amp; Mueller (1992)## calculated that quinclorac at 50 g ha<sup>−1</sup> reduces cotton yield by 25%. On the other side, ##UREF##9##Lovelace et al. (2007)## indicated that quinclorac resulted in yield loss in tomatoes, but the crop may recover itself from the adverse impact of herbicides depending on the herbicide rate and application timing. Our results are consistent with those of the aforementioned studies that lower rates of florpyrauxifen-benzyl were more destructive to crop yields than quinclorac.</p>", "<p>Crop cultivars can have various genetic backgrounds depending on the breeding aim; therefore, it is not surprising that they respond differently to abiotic stressors, including drought stress, heat stress, and herbicides. Using herbicide-tolerant crops or less sensitive crop cultivars to herbicide are among the cost-effective and reliable solutions to prevent the injurious impact of herbicide drift on sensitive crops. This practice has been shown in other studies of by ##UREF##3##France et al. (2022)##, ##REF##34834805##Zangoueinejad et al. (2021)## and ##UREF##27##Warmund, Ellersieck &amp; Smeda (2022)##, who showed differences between the tolerance levels of soybean, melon, and tomato cultivars to synthetic auxin herbicides dicamba, 2.4-D, and 2.4-D or dicamba, respectively. The yield data clearly showed that cultivar Bosfora was more sensitive to FBP and quinclorac rates than cultivar Tunca. To reduce the impact of these off-target effects, more cultivars can be screened, and robust cultivars can be selected to reduce the risk of off-target herbicide damage.</p>", "<title>Correlation analysis</title>", "<p>Remarkably high Pearson correlation coefficients were found between quinclorac injury at 7 or 14 DAT and plant height (##TAB##1##Table 2##). The negative relations between quinclorac injury at 7 or 14 DAT and SHD, TSW, and/or yield were also high, but their significance was slightly below the relations between quinclorac injury at 7 or 14 DAT and plant height. At 28 DAT, plants treated with quinclorac began to recover from treatment; therefore, quinclorac injury at 28 DAT had a weaker correlation with yield and yield component compared to previous evaluation times. There was a strong positive relationship between plant height and yield, and the importance of the relationship was greater than that of other relationships.</p>", "<p>The correlation analysis performed in this research can be a powerful tool to estimate the injurious impact caused by drift rates of synthetic auxin herbicides on yield and yield components long before harvest. In our study, strong relationships between injury rates and yields (or yield components) were similar to those found in previous studies (##UREF##9##Lovelace et al., 2007##; ##UREF##11##Marple, Al-Khatib &amp; Peterson, 2008##; ##UREF##2##Daramola et al., 2023##). The ability to model injury rates and yield loss resulting from herbicides provide an opportunity for farmers to decide whether to stop or continue current agricultural practices. If herbicide damage reduces farmer’s income below the total expenses, farmers may wish to change their management in order to remain profitable; therefore, correlation analysis between herbicides and yield can be used as a decision-support tool for farmers.</p>" ]
[ "<title>Results and Discussion</title>", "<title>Growth chamber experiment</title>", "<p>The sunflower cultivar ‘Tunca’ was severely injured when exposed to low rates of florpyrauxifen-benzyl + penoxsulam (##FIG##0##Fig. 1A##). However, the lowest rate of herbicide unexpectedly increased shoot length, probably due to the hormetic impact of auxinic herbicides, similar to what was previously reported by ##UREF##15##Mudge et al. (2021)##. Overall, shoot lengths decreased as the rates increased. The impact of the highest four rates of herbicide was the most destructive, and the growing points of cultivar ‘Bosfora’ were completely killed at these rates at 28 DAT (##FIG##0##Fig. 1B##). More than half of the leaf area of seedlings exposed to these rates became necrotic. The response of cultivar Bosfora to FBP was similar to that of cultivar Tunca, except at rates higher than 1.02 g ai ha<sup>−1</sup> where it was more injured (##FIG##0##Figs. 1A## and ##FIG##0##1B##).</p>", "<p>Injury from quinclorac in both cultivars increased over time, with a slight and gradual increase in sunflower injury with rate. By 28 DAT, the highest sunflower injury was caused by the highest rate of quinclorac (##FIG##0##Figs. 1C## and ##FIG##0##1D##). The response of these sunflower cultivars to lower rates of quinclorac was very similar; however, cultivar Bosfora was slightly more sensitive to quinclorac than cultivar Tunca at higher rates. As opposed to FBP, sunflower plants of both cultivars treated with quinclorac still had live growing points, and necrotic areas on the seedling leaves were relatively limited even at the highest rates. At the lowest rates, FBP resulted in no damage to these cultivars, while at the lowest dose of quinclorac growth reduction was still observed.</p>", "<p>The quantitative response of the cultivar Tunca and cultivar Bosfora to FBP and quinclorac was evaluated <italic toggle=\"yes\">via</italic> a dose-response assay using a log–logistic model. The GR<sub>50</sub> values of FBP for the cultivars Tunca and Bosfora were 1.07 and 0.75 g ai ha<sup>−1</sup>, respectively (##TAB##0##Table 1##), nearly 2.9% and 2% of the recommended rate of FBP (65 g ai ha<sup>−1</sup>). The GR<sub>50</sub> values of quinclorac for sunflower cultivars were 14.16 and 7.56 g ai ha<sup>−1</sup>, 3,8% and 2% of the recommended rate of quinclorac (375 g ai ha<sup>−1</sup>). Similar to the visual herbicidal impact of FBP and quinclorac, the results show that FBP was slightly more injurious than quinclorac to these cultivars (##FIG##1##Fig. 2##).</p>", "<title>Field experiment</title>", "<title>Crop injury</title>", "<p>Sunflower crops exposed to FBP and quinclorac responded to low-dose treatment, especially the leaves. FBP resulted in typical auxin symptoms, such as parallel veins, cupping, twisting, chlorosis, and distortion (##FIG##2##Fig. 3##). Quinclorac also caused these symptoms, except it did not demonstrate distortion (##FIG##3##Fig. 4##). The severity of symptoms was higher in cultivar Bosfora than in cultivar Tunca and increased as the rates increased. Stunting became apparent at 28 DAT for both cultivars. The highest rate of FBP prevented the establishment of sunflower heads in both cultivars (##FIG##4##Figs. 5B## and ##FIG##4##5D##).</p>", "<p>Quinclorac and FBP injury was low at 7 DAT, and no difference was found between cultivar Tunca and cultivar Bosfora (##FIG##5##Figs. 6A## and ##FIG##5##6B##). The response of sunflower cultivar to FBP increased as the rates rose and reached 75–77.5% at 8.13 g ai ha<sup>−1</sup> at 7 DAT. Similar to the growth chamber study, quinclorac injury in cultivar Bosfora (13.75–33.75%) and cultivar Tunca (17.5–30%) was lower than those of FBP. At 14 DAT, the response of the cultivars to the herbicides was generally similar to that at 7 DAT. Sunflower injury due to FBP increased with the increase in herbicide rates from 2.03 to 8.13 g ai ha<sup>−1</sup> at 14 DAT. Cultivar Bosfora exposure to quinclorac at 11.72, 23.44, and 46.88 g ai ha<sup>−1</sup>resulted in 15, 22.5, and 38.75% injury, respectively, while cultivar Tunca was less sensitive to these rates at 14 DAT.</p>", "<p>Sunflower injury at the highest rate of FBP was 100% by 28 DAT while the lowest FBP rate resulted in sunflower injury of 76.25–88.75% (##FIG##5##Figs. 6A## and ##FIG##5##6B##). Differences between sunflower cultivars became more apparent at 28 DAT. Although the phytotoxicity of quinclorac increased at 28 DAT, crop injury caused by the herbicide was nearly half that of FBP.</p>", "<p>Visible injury rates reached the greatest level at harvest. Sunflower injury at 2.03 g ai ha<sup>−1</sup> FBP was higher than &gt;90% for cultivar Bosfora and &gt;85% for cultivar Tunca. The highest FBP rate led to complete death of both sunflower cultivars. The injury increased in severity as the quinclorac rate increased in both cultivars. The injury on cultivar Bosfora and cultivar Tunca ranged from 42.5–57.5% and from 36.25–48.75%, respectively. The severity of injury due to quinclorac was limited to at 7 DAT because injury assessment was only performed on leaves, while this influence was more apparent in evaluations of other crop parameters, including plant height, stem structure, size of flower bud and head, were included in the evaluations. Compared to quinclorac, FBP was more phytotoxic to both cultivars, and this destructive impact was observed even from the first assessments at 7 DAT.</p>", "<p>The injury rate of auxin herbicides on sensitive row crops has been reported in some studies. For instance, ##UREF##19##Schroeder, Cole &amp; Dexter (1979)## stated that 2,4-D and dicamba resulted in tremendous injury to sunflowers. In another study, ##UREF##24##Snipes, Street &amp; Mueller (1992)## reported that 17 g ha<sup>−1</sup> or higher rates of quinclorac may injure cotton when applied at the cotyledon stage, and cotton injury increased with increasing herbicide rate, especially during late-stage applications, such as at the pin-head square stage. Overall, the cotton injury rate reached 59% at 140 g ha<sup>−1</sup> in their study, and sunflower injury caused by quinclorac at 46.88 ai g ha<sup>−1</sup> was 52-61.25% in our study. A study conducted by ##UREF##9##Lovelace et al. (2007)## revealed that tomato was also among quinclorac-sensitive crops, and crop injury caused by quinclorac applied at 42 g ai ha<sup>−1</sup> was 45% at 49 DAT. In our study, the third evaluation was done at 55 DAT, and crop injuries were 48.75–57.5% when herbicide was applied at 46.88 g ai ha<sup>−1</sup>. These data are consistent with the work of ##UREF##9##Lovelace et al. (2007)##. Comparing these results to those of ##UREF##24##Snipes, Street &amp; Mueller (1992)## and ##UREF##9##Lovelace et al. (2007)##, sunflower can be classified as a more sensitive crop than cotton.</p>", "<p>Florpyrauxifen-benzyl is a relatively new herbicide on the market; therefore, few drift studies are available in the literature. FBP containing 2.03 g ai ha<sup>−1</sup> florpyrauxifen-benzyl + penoxsulam caused 76 and 89% injury at 28 DAT on cultivar Tunca and cultivar Bosfora, respectively. In agreement with the results of our study, ##UREF##14##Miller &amp; Norsworthy (2018b)## stated that 3 g ai ha<sup>−1</sup> florpyrauxifen-benzyl applied to sunflowers at the three true-leaf stage resulted in 69% injury, at 28 DAT under greenhouse conditions.</p>", "<title>Plant height</title>", "<p>The heights of the sunflower cultivars constantly declined as the FBP and quinclorac rates increased. All FBP rates resulted in a significant decrease in the plant height of the sunflower cultivars; The lowest rate of FBP resulted in an 8–9% decrease while the highest resulted in a 32–44% decrease (##FIG##6##Fig. 7A##). Similarly, the height reduction from quinclorac ranged from 13–14% at the lowest rate to 25–32% at the highest. Similar to the findings of this study, ##UREF##14##Miller &amp; Norsworthy (2018b)## also stated that 3 g ai ha<sup>−1</sup> florpyrauxifen-benzyl applied to sunflowers at the three true-leaf stage resulted in a 66% plant height reduction at 28 DAT under greenhouse conditions. The lower plant height observed in our study may be due to the application time of herbicide or penoxsulam in the herbicide mixture.</p>", "<title>Sunflower head diameter and One thousand seed weight</title>", "<p>Increasing FBP and quinclorac rates caused sunflower head diameter (SHD) to decline regardless of the cultivar. The greatest percentage of decline resulting from FBP was recorded at 4.06 g ai ha<sup>−1</sup> with 76–77%. At the highest rate of FBP (8.13 g ai ha<sup>−1</sup>), no flower heads were observed (##FIG##4##Figs. 5B## and ##FIG##4##5D##). SHD reduction in cultivar Bosfora and cultivar Tunca from quinclorac ranged from 19–57% and 9–51%, respectively (##FIG##6##Fig. 7B##). A slight decrease was observed in One thousand seed weight (TSW) compared to plant height and SHD, but this decrease, even at the highest level of herbicide, was no more than 21% (##FIG##6##Fig. 7C##). Sunflower exposed to higher FBP rates, 4.06 and 8.13 g ai ha<sup>−1</sup>, could not produce mature seeds. The results showed that TSW was a less susceptible yield component to FBP drift rates than the others.</p>", "<title>Yield</title>", "<p>Associated with other yield components, significant sunflower yield loss was determined even at lower rates of quinclorac and FBP (##FIG##6##Fig. 7D##). The highest yield reduction was observed for sunflower cultivars treated with FBP at 4.06 and 8.03 g ai ha<sup>−1</sup>at 100%. The lowest FBP rate reduced cultivar Bosfora and cultivar Tunca yields by 79.8 and 74.3%, respectively. The yield reduction of cultivar Bosfora from quinclorac rates was 45.1–87.9%, while yield loss of cultivar Tunca caused these rates to range from 16.3–82.3%. Although no study was found in the literature related to the impact of low rates of quinclorac and FBP on sunflower yield and yield components such as SHD and TSW, previous research reported significant reductions in crop growth, yield, and some yield components in other systems. ##UREF##14##Miller &amp; Norsworthy (2018b)## found that soybean yield was reduced 71% when florpyrauxifen-benzyl was applied at 3 g ae ha<sup>−1</sup> rate. In another study, ##UREF##24##Snipes, Street &amp; Mueller (1992)## calculated that quinclorac at 50 g ha<sup>−1</sup> reduces cotton yield by 25%. On the other side, ##UREF##9##Lovelace et al. (2007)## indicated that quinclorac resulted in yield loss in tomatoes, but the crop may recover itself from the adverse impact of herbicides depending on the herbicide rate and application timing. Our results are consistent with those of the aforementioned studies that lower rates of florpyrauxifen-benzyl were more destructive to crop yields than quinclorac.</p>", "<p>Crop cultivars can have various genetic backgrounds depending on the breeding aim; therefore, it is not surprising that they respond differently to abiotic stressors, including drought stress, heat stress, and herbicides. Using herbicide-tolerant crops or less sensitive crop cultivars to herbicide are among the cost-effective and reliable solutions to prevent the injurious impact of herbicide drift on sensitive crops. This practice has been shown in other studies of by ##UREF##3##France et al. (2022)##, ##REF##34834805##Zangoueinejad et al. (2021)## and ##UREF##27##Warmund, Ellersieck &amp; Smeda (2022)##, who showed differences between the tolerance levels of soybean, melon, and tomato cultivars to synthetic auxin herbicides dicamba, 2.4-D, and 2.4-D or dicamba, respectively. The yield data clearly showed that cultivar Bosfora was more sensitive to FBP and quinclorac rates than cultivar Tunca. To reduce the impact of these off-target effects, more cultivars can be screened, and robust cultivars can be selected to reduce the risk of off-target herbicide damage.</p>", "<title>Correlation analysis</title>", "<p>Remarkably high Pearson correlation coefficients were found between quinclorac injury at 7 or 14 DAT and plant height (##TAB##1##Table 2##). The negative relations between quinclorac injury at 7 or 14 DAT and SHD, TSW, and/or yield were also high, but their significance was slightly below the relations between quinclorac injury at 7 or 14 DAT and plant height. At 28 DAT, plants treated with quinclorac began to recover from treatment; therefore, quinclorac injury at 28 DAT had a weaker correlation with yield and yield component compared to previous evaluation times. There was a strong positive relationship between plant height and yield, and the importance of the relationship was greater than that of other relationships.</p>", "<p>The correlation analysis performed in this research can be a powerful tool to estimate the injurious impact caused by drift rates of synthetic auxin herbicides on yield and yield components long before harvest. In our study, strong relationships between injury rates and yields (or yield components) were similar to those found in previous studies (##UREF##9##Lovelace et al., 2007##; ##UREF##11##Marple, Al-Khatib &amp; Peterson, 2008##; ##UREF##2##Daramola et al., 2023##). The ability to model injury rates and yield loss resulting from herbicides provide an opportunity for farmers to decide whether to stop or continue current agricultural practices. If herbicide damage reduces farmer’s income below the total expenses, farmers may wish to change their management in order to remain profitable; therefore, correlation analysis between herbicides and yield can be used as a decision-support tool for farmers.</p>" ]
[ "<title>Conclusions</title>", "<p>Each new herbicide introduced into the market has been a new opportunity for rice farmers to control herbicide resistant weeds; however, these new rice synthetic auxin herbicides, FBP and quinclorac, can have destructive impacts on susceptible crops grown nearby such as sunflowers. While both FBP and quinclorac are both synthetic auxins, they work in slightly different ways and therefore have different impacts on crop response. In this study, quinclorac and FBP applied to sunflower cultivars resulted in different injury symptoms and yield losses from different cultivars of sunflowers. Crop injury and yield data clearly showed that cultivar Bosfora was more sensitive to FBP and quinclorac rates than cultivar Tunca, and both cultivars were more sensitive to FBP than quinclorac. The lowest rate of FBP resulted in a 74.3 and 79.8% yield reduction while the higher rates led to a 100% sunflower yield reduction. In our work, we also found that plant height reduction caused by quinclorac at early growth stages may be a valuable indicator to evaluate crop injury and yield loss. Rice growers should be attentive to weather conditions, application parameters, herbicides, herbicide properties, and the safety measurements given by pesticide advisors to prevent drift risk on sunflowers. Moreover, sunflower producers should be careful about the location of sunflower fields prior to sowing and closely communicate with rice producers who use these synthetic auxin herbicides and choose less sensitive sunflower cultivars to synthetic auxin herbicides instead of sensitive ones.</p>" ]
[ "<p>The agrochemical industry has launched several new synthetic auxin herbicides in rice to combat increasing numbers of herbicide resistant weeds to other modes of action. Excessive or inappropriate use of these herbicides has resulted in unintended consequences near the sites of application, such as herbicide drift. This study was conducted to determine the impact of drift of quinclorac and florpyrauxifen-benzyl+penoxsulam (FBP) on the yield and yield components of two sunflower cultivars. In a growth chamber experiment, quinclorac and FBP were applied to 2–4 true leaf stages at rates ranging from 2.93 to 93.75 and from 0.51 to 16.25 g ai ha<sup>−1</sup>, respectively. Nonlinear regression analyses indicated that the cultivar Bosfora was more sensitive to quinclorac and FBP than the cultivar Tunca. In field experiments, these sunflower cultivars were treated with drift rates of quinclorac (&lt;375 g ai ha<sup>−1</sup>) and FBP (&lt;65 g ai ha<sup>−1</sup>) when they were at the 8-10 true leaf stage. Quinclorac and FBP drift rates resulted in up to 52-61% and 85–100% injury and 82-88% and 100% yield loss, respectively. Crop injury and yield data clearly showed that cultivar Bosfora was more sensitive to FBP and quinclorac rates than cultivar Tunca, and both cultivars were more sensitive to FBP than quinclorac. In our work, we also found that plant height reduction caused by quinclorac at early growth stages may be a valuable indicator to evaluate crop injury and yield loss.</p>" ]
[ "<title> Supplemental Information</title>" ]
[ "<title>Additional Information and Declarations</title>" ]
[ "<fig position=\"float\" id=\"fig-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-1</object-id><label>Figure 1</label><caption><title>Response of Tunca (A and C) and Bosfora (B and D) cultivars to florpyrauxifen-benzyl + penoxsulam (A and B) and quinclorac (C and D) rates in the growth chamber.</title></caption></fig>", "<fig position=\"float\" id=\"fig-2\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-2</object-id><label>Figure 2</label><caption><title>Dose-response curves of florpyrauxifen-benzyl + penoxsulam (left) and quinclorac (right) applied to cultivar Bosfora and cultivar Tuna.</title><p>Florpyrauxifen-benzyl + penoxsulam rate for Tunca, y = 0.39 + (2.92 / (1 + (exp(1.66(log (Dose) – log (1.07)))))); florpyrauxifen-benzyl + penoxsulam rate for Bosfora, y = 0.39 + (2.92 / (1 + (exp(3.22(log (Dose) – log (0.75)))))); quinclorac rate for Tunca, y = 1.57 + (1.67 / (1 + (exp(4.34(log (Dose) – log (14.16)))))); quinclorac rate for Bosfora, y = 1.57 + (1.67 / (1 + (exp(1.91(log (Dose) – log (7.56)))))).</p></caption></fig>", "<fig position=\"float\" id=\"fig-3\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-3</object-id><label>Figure 3</label><caption><title>Impact of florpyrauxifen-benzyl + penoxsulam on sunflower (cultivar Bosfora) at 28 DAT in 2022 (left, lowest drift rate; middle, moderate drift rate; right, highest drift rate).</title></caption></fig>", "<fig position=\"float\" id=\"fig-4\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-4</object-id><label>Figure 4</label><caption><title>Impact of quinchlorac on sunflower (cultivar Bosfora) at 28 DAT in 2022 (left, lowest drift rate; middle, moderate drift rate; right, highest drift rate).</title></caption></fig>", "<fig position=\"float\" id=\"fig-5\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-5</object-id><label>Figure 5</label><caption><title>Efficacy of drift rates of florpyrauxifen-benzyl + penoxsulam (B and D) and quinclorac (A and C) on heads of Tunca (A and B) and Bosfora (C and D) cultivars (X: recommended rate).</title><p>*The recommended rate (X) of florpyrauxifen-benzyl + penoxsulam and quinclorac were 65 and 375 g ai ha<sup>−1</sup>, respectively.</p></caption></fig>", "<fig position=\"float\" id=\"fig-6\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-6</object-id><label>Figure 6</label><caption><title>Florpyrauxifen-benzyl + penoxsulam (FBP) and quinclorac (Q) injury on the sunflower cultivar Bosfora (A) and cultivar Tunca (B) at 7, 14, 28 DAT, and harvest (%).</title><p><sup>1</sup>The recommended rate (X) of FBP and quinclorac were 65 and 375 g ai ha<sup>−1</sup>, respectively.</p></caption></fig>", "<fig position=\"float\" id=\"fig-7\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/fig-7</object-id><label>Figure 7</label><caption><title>Yield and yield components of sunflower cultivars in response to FBP and quinclorac (a, Plant height; b, Sunflower head diameter (cm); c, One thousand seed weight (g); Yield (kg ha<sup>−1</sup>)).</title><p><sup>1</sup>Means followed by the same letter are not significantly different (<italic toggle=\"yes\">P</italic> ≤ 0.05). <sup>2</sup>FBP, florpyrauxifen-benzyl + penoxsulam. <sup>3</sup>The recommended rate (X) of FBP and quinclorac were 65 and 375 g ai ha<sup>−1</sup>, respectively.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"table-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/table-1</object-id><label>Table 1</label><caption><title>Nonlinear regression parameters of florpyrauxifen-benzyl + penoxsulam and quinclorac<xref rid=\"table-1fn1\" ref-type=\"table-fn\"><sup>a</sup></xref>.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Herbicide</th><th rowspan=\"1\" colspan=\"1\">b<sub>bosfora</sub></th><th rowspan=\"1\" colspan=\"1\">b<sub>tunca</sub></th><th rowspan=\"1\" colspan=\"1\">c</th><th rowspan=\"1\" colspan=\"1\">d</th><th rowspan=\"1\" colspan=\"1\">GR<sub>50bosfora</sub></th><th rowspan=\"1\" colspan=\"1\">GR<sub>50tunca</sub></th><th rowspan=\"1\" colspan=\"1\">Comp</th><th rowspan=\"1\" colspan=\"1\">Sig.</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">FBP</td><td rowspan=\"1\" colspan=\"1\">3.22</td><td rowspan=\"1\" colspan=\"1\">1.66</td><td rowspan=\"1\" colspan=\"1\">0.39</td><td rowspan=\"1\" colspan=\"1\">3.31</td><td rowspan=\"1\" colspan=\"1\">0.75</td><td rowspan=\"1\" colspan=\"1\">1.07</td><td rowspan=\"1\" colspan=\"1\">0.70</td><td rowspan=\"1\" colspan=\"1\">0.035</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Quinclorac</td><td rowspan=\"1\" colspan=\"1\">1.91</td><td rowspan=\"1\" colspan=\"1\">4.34</td><td rowspan=\"1\" colspan=\"1\">1.57</td><td rowspan=\"1\" colspan=\"1\">3.24</td><td rowspan=\"1\" colspan=\"1\">7.56</td><td rowspan=\"1\" colspan=\"1\">14.16</td><td rowspan=\"1\" colspan=\"1\">0.53</td><td rowspan=\"1\" colspan=\"1\">0.007</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-2\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/table-2</object-id><label>Table 2</label><caption><title>Pearson correlation coefficients among the evaluation times after quinclorac application and quinclorac dose, yield, yield components, plant height, sunflower head diameter, and one thousand seed weight.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\">\n<bold>Dose</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>PH</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>SHD</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>TSW</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Yield</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>I7DAT</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>I14DAT</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>I28DAT</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>IHarvest</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Dose</bold>\n</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>PH</bold>\n</td><td rowspan=\"1\" colspan=\"1\">−0.88<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>SHD</bold>\n</td><td rowspan=\"1\" colspan=\"1\">−0.95<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.87<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>TSW</bold>\n</td><td rowspan=\"1\" colspan=\"1\">−0.92<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.75<xref rid=\"table-2fn2\" ref-type=\"table-fn\"><sup>**</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.95<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Yield</bold>\n</td><td rowspan=\"1\" colspan=\"1\">−0.90<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.74<xref rid=\"table-2fn2\" ref-type=\"table-fn\"><sup>**</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.93<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.95<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>I7DAT</bold>\n</td><td rowspan=\"1\" colspan=\"1\">0.94<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.94<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.88<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.85<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.87<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>I14DAT</bold>\n</td><td rowspan=\"1\" colspan=\"1\">0.95<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.93<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.91<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.87<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.88<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.99<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>I28DAT</bold>\n</td><td rowspan=\"1\" colspan=\"1\">0.82<xref rid=\"table-2fn2\" ref-type=\"table-fn\"><sup>**</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.87<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.85<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.82<xref rid=\"table-2fn2\" ref-type=\"table-fn\"><sup>**</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.87<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.93<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.93<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>IHarvest</bold>\n</td><td rowspan=\"1\" colspan=\"1\">0.82<xref rid=\"table-2fn2\" ref-type=\"table-fn\"><sup>**</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.86<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.86<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.83<xref rid=\"table-2fn2\" ref-type=\"table-fn\"><sup>**</sup></xref></td><td rowspan=\"1\" colspan=\"1\">−0.87<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.92<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">0.93<xref rid=\"table-2fn3\" ref-type=\"table-fn\"><sup>***</sup></xref></td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">1</td></tr></tbody></table></alternatives></table-wrap>" ]
[ "<disp-formula id=\"eqn-1\"><label>(1)</label><alternatives><tex-math id=\"tex-eqn-1\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym}\n\\usepackage{amsfonts}\n\\usepackage{amssymb}\n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n\\begin{eqnarray*}Y=c+ \\frac{d-c}{1+\\exp \\nolimits (b(\\log \\nolimits \\left( x \\right) -\\log \\nolimits ({\\mathrm{GR}}_{50})))} \\end{eqnarray*}\\end{document}</tex-math><mml:math id=\"mml-eqn-1\" overflow=\"scroll\"><mml:mstyle displaystyle=\"true\"><mml:mi>Y</mml:mi><mml:mo>=</mml:mo><mml:mi>c</mml:mi><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mi>d</mml:mi><mml:mo>−</mml:mo><mml:mi>c</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mo class=\"qopname\"> exp</mml:mo><mml:mrow><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mi>b</mml:mi><mml:mrow><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mo class=\"qopname\">log</mml:mo><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mi>x</mml:mi></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:mo class=\"qopname\">log</mml:mo><mml:mrow><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">GR</mml:mi></mml:mrow><mml:mrow><mml:mn>50</mml:mn></mml:mrow></mml:msub></mml:mfenced></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced></mml:mrow></mml:mrow></mml:mfrac></mml:mstyle></mml:math></alternatives>\n</disp-formula>" ]
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[ "<supplementary-material id=\"supp-1\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/supp-1</object-id><label>Supplemental Information 1</label><caption><title>Dataset of Dose-response curves of florpyrauxifen-benzyl + penoxsulam (left) and quinclorac (right) applied to var. Bosfora and var. Tuna</title></caption></supplementary-material>", "<supplementary-material id=\"supp-2\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/supp-2</object-id><label>Supplemental Information 2</label><caption><title>Dataset of yield and yield components of sunflower varieties in response to various rates of FBP and quinclorac</title></caption></supplementary-material>", "<supplementary-material id=\"supp-3\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/supp-3</object-id><label>Supplemental Information 3</label><caption><title>Injury dataset of Florpyrauxifen-benzyl + penoxsulam and quinclorac on the sunflower var. Bosfora and var. Tunca</title></caption></supplementary-material>", "<supplementary-material id=\"supp-4\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16729/supp-4</object-id><label>Supplemental Information 4</label><caption><title>Quality Parameters Of Irrigation Water</title></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"table-1fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-1fn1\"><label>a</label><p>Abbreviations: FBP, Florpyrauxifen-benzyl + penoxsulam; b, slope of the curve at GR<sub>50</sub>; d, upper limit; GR<sub>50</sub>, herbicide rate that reduced seedling dry matter by 50%; Comp, comparison rate (GR<sub>50bosfora</sub>/GR<sub>50tunca</sub>); Sig, significance (<italic toggle=\"yes\">P</italic> &lt; 0.05).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-2fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-2fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-2fn2\"><label>**</label><p><italic toggle=\"yes\">P</italic> &lt; 0.05.</p></fn><fn id=\"table-2fn3\"><label>***</label><p><italic toggle=\"yes\">P</italic> &lt; 0.01.</p></fn></table-wrap-foot>", "<fn-group content-type=\"competing-interests\"><title>Competing Interests</title><fn id=\"conflict-1\" fn-type=\"COI-statement\"><p>Ahmet Tansel Serim is an Academic Editor for PeerJ.</p></fn></fn-group>", "<fn-group content-type=\"author-contributions\"><title>Author Contributions</title><fn id=\"contribution-1\" fn-type=\"con\"><p><xref rid=\"author-1\" ref-type=\"contrib\">Ahmet Tansel Serim</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-2\" fn-type=\"con\"><p><xref rid=\"author-2\" ref-type=\"contrib\">Eric L. Patterson</xref> analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Data Availability</title><fn id=\"addinfo-1\"><p>The following information was supplied regarding data availability:</p><p>The raw data is available in the <xref rid=\"supplemental-information\" ref-type=\"sec\">Supplementary Files</xref>.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"peerj-12-16729-s001.xlsx\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"peerj-12-16729-s002.xlsx\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"peerj-12-16729-s003.xlsx\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"peerj-12-16729-s004.pdf\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["Adams et al. (2017)"], "person-group": ["\n"], "surname": ["Adams", "Pelkki", "Ford", "Humphrey"], "given-names": ["JP", "MH", "VL", "A"], "year": ["2017"], "article-title": ["Initial effects of quinclorac on the survival and growth of high biomass tree species"], "source": ["Annals of Forest Research"], "volume": ["60"], "issue": ["1"], "fpage": ["75"], "lpage": ["87"], "pub-id": ["10.15287/afr.2016.734"]}, {"label": ["Altop et al. (2014)"], "person-group": ["\n"], "surname": ["Altop", "Mennan", "Streibig", "Budak", "Ritz"], "given-names": ["EK", "H", "JC", "U", "C"], "year": ["2014"], "article-title": ["Detecting ALS and ACCase herbicide tolerant accession of "], "italic": ["Echinochloa oryzoides", "Oryza sativa"], "source": ["Crop Protection"], "volume": ["65"], "fpage": ["202"], "lpage": ["206"], "pub-id": ["10.1016/j.cropro.2014.07.011"]}, {"label": ["Daramola et al. (2023)"], "person-group": ["\n"], "surname": ["Daramola", "Kharel", "Iboyi", "Devkota"], "given-names": ["OS", "P", "JE", "P"], "year": ["2023"], "article-title": ["Peanut response to 2 4-D plus glyphosate"], "source": ["Weed Technology"], "volume": ["37"], "issue": ["1"], "fpage": ["46"], "lpage": ["52"], "pub-id": ["10.1017/wet.2023.3"]}, {"label": ["France et al. (2022)"], "person-group": ["\n"], "surname": ["France", "Norsworthy", "Roberts", "Ross", "Barber", "Gbur"], "given-names": ["W", "J", "T", "J", "T", "E"], "year": ["2022"], "article-title": ["Effect of cultivar and planting date on soybean response to dicamba"], "source": ["International Journal of Agronomy"], "volume": ["2022"], "fpage": ["9479650"], "pub-id": ["10.1155/2022/9479650"]}, {"label": ["Greenshields & Putt (1958)"], "person-group": ["\n"], "surname": ["Greenshields", "Putt"], "given-names": ["JER", "ED"], "year": ["1958"], "article-title": ["The effects of 2,4-D spray drift on sunflowers"], "source": ["Canadian Journal of Plant Science"], "volume": ["38"], "issue": ["2"], "fpage": ["234"], "lpage": ["241"], "pub-id": ["10.4141/cjps58-037"]}, {"label": ["Haghnama & Mennan (2020)"], "person-group": ["\n"], "surname": ["Haghnama", "Mennan"], "given-names": ["K", "H"], "year": ["2020"], "article-title": ["Herbicide resistant barnyardgrass in Iran and Turkey"], "source": ["Planta Daninha"], "volume": ["38"], "elocation-id": ["e020227592"], "pub-id": ["10.1590/S0100-83582020380100060"]}, {"label": ["Kacan et al. (2020)"], "person-group": ["\n"], "surname": ["Kacan", "Tursun", "Ullah", "Datta"], "given-names": ["K", "N", "H", "A"], "year": ["2020"], "article-title": ["Barnyardgrass ("], "italic": ["Echinochloa crus-galli", "P. Beauv"], "source": ["Plant, Soil and Environment"], "volume": ["2020"], "fpage": ["357"], "lpage": ["365"], "pub-id": ["10.17221/92/2020-PSE"]}, {"label": ["Kaya et al. (2023)"], "person-group": ["\n"], "surname": ["Kaya", "Ba\u015faran", "\u00d6rnek", "Mennan"], "given-names": ["Y", "B", "H", "H"], "year": ["2023"], "article-title": ["Studies on the prevention of time-dependent water discharge methods and determination of phytotoxicities resulting from the use of quinclorac irrigation water used and discharged in rice planting fields in vegetable production fields"], "source": ["T\u00fcrkiye Turkish Journal of Weed Science"], "volume": ["26"], "issue": ["1"], "fpage": ["58"], "lpage": ["66"]}, {"label": ["Lanini & Carrithers (2000)"], "person-group": ["\n"], "surname": ["Lanini", "Carrithers"], "given-names": ["WT", "V"], "year": ["2000"], "article-title": ["Simulated drift of herbicides on grapes, tomatoes, cotton, and sunflower"], "conf-name": ["2000 Proceedings of the California Weed Science Society"], "fpage": ["107"], "lpage": ["110"], "pub-id": ["10.1145/200836.200838"]}, {"label": ["Lovelace et al. (2007)"], "person-group": ["\n"], "surname": ["Lovelace", "Talbert", "Scherder", "Hoagland"], "given-names": ["ML", "RE", "EF", "RE"], "year": ["2007"], "article-title": ["Effects of multiple applications of simulated quinclorac drift rates on tomato"], "source": ["Weed Science"], "volume": ["55"], "fpage": ["169"], "lpage": ["177"], "pub-id": ["10.1614/WS-06-054"]}, {"label": ["Mallory-Smith & Retzinger (2003)"], "person-group": ["\n"], "surname": ["Mallory-Smith", "Retzinger"], "given-names": ["C", "E"], "year": ["2003"], "article-title": ["Revised classification of herbicides by site of action for weed resistance management strategies"], "source": ["Weed Technology"], "volume": ["17"], "issue": ["3"], "fpage": ["605"], "lpage": ["619"], "pub-id": ["10.1614/0890-037X(2003)017[0605:RCOHBS]2.0.CO;2"]}, {"label": ["Marple, Al-Khatib & Peterson (2008)"], "person-group": ["\n"], "surname": ["Marple", "Al-Khatib", "Peterson"], "given-names": ["ME", "K", "DE"], "year": ["2008"], "article-title": ["Cotton injury and yield as affected by simulated drift of 2 4-D and dicamba"], "source": ["Weed Technology"], "volume": ["22"], "issue": ["4"], "fpage": ["609"], "lpage": ["614"], "pub-id": ["10.1614/WT-07-095.1"]}, {"label": ["Mendiburu & Yaseen (2020)"], "person-group": ["\n"], "surname": ["Mendiburu", "Yaseen"], "given-names": ["F", "M"], "year": ["2020"], "article-title": ["Agricolae: statistical procedures for agricultural research"], "version": ["R package version 1.4.0"], "comment": ["Available at: "], "ext-link": ["https://myaseen208.github.io/agricolae/", "https://cran.r-project.org/package=agricolae"]}, {"label": ["Miller & Norsworthy (2018a)"], "person-group": ["\n"], "surname": ["Miller", "Norsworthy"], "given-names": ["MR", "JK"], "year": ["2018a"], "article-title": ["Assessment of florpyrauxifen-benzyl potential to carryover to subsequent crops"], "source": ["Weed Technology"], "volume": ["32"], "issue": ["4"], "fpage": ["404"], "lpage": ["409"], "pub-id": ["10.1017/wet.2018.33"]}, {"label": ["Miller & Norsworthy (2018b)"], "person-group": ["\n"], "surname": ["Miller", "Norsworthy"], "given-names": ["MR", "JK"], "year": ["2018b"], "article-title": ["Row crop sensitivity to low rates of foliar-applied florpyrauxifen-Benzyl"], "source": ["Weed Technology"], "volume": ["32"], "issue": ["4"], "fpage": ["398"], "lpage": ["403"], "pub-id": ["10.1017/wet.2017.114"]}, {"label": ["Mudge et al. (2021)"], "person-group": ["\n"], "surname": ["Mudge", "Sartain", "Sperry", "Getsinger"], "given-names": ["CR", "BT", "BP", "KD"], "year": ["2021"], "article-title": ["Efficacy of florpyrauxifen-benzyl for eurasian watermilfoil control and nontarget Illinois pondweed, elodea, and coontail response"], "source": ["ERDC/TN APCRP CC-24"], "publisher-loc": ["Vicksburg"], "fpage": ["7"]}, {"label": ["OEC (2023)"], "person-group": ["\n"], "collab": ["Observatory of Economic Complexity (OEC)"], "year": ["2023"], "article-title": ["Sunflower seeds in Turkey"], "uri": ["https://oec.world/en/profile/bilateral-product/sunflower-seeds/reporter/tur?redirect=true"]}, {"label": ["PPPD (2022)"], "person-group": ["\n"], "collab": ["Republic of Turkey, Ministry of Food, Agriculture and Livestock"], "year": ["2022"], "article-title": ["Plant protection products database"], "uri": ["https://bku.tarim.gov.tr/Arama/Index"]}, {"label": ["RStudio Team (2023)"], "person-group": ["\n"], "collab": ["RStudio Team"], "year": ["2023"], "article-title": ["RStudio: integrated development environment for R. RStudio, PBC"], "uri": ["http://www.rstudio.com/"]}, {"label": ["Schroeder, Cole & Dexter (1979)"], "person-group": ["\n"], "surname": ["Schroeder", "Cole", "Dexter"], "given-names": ["GL", "DF", "AG"], "year": ["1979"], "article-title": ["Herbicide spray drift on sunflower"], "conf-name": ["Proc. North Cent. Weed Control Conf. 34"], "fpage": ["66"]}, {"label": ["Schwartz-Lazaro et al. (2017)"], "person-group": ["\n"], "surname": ["Schwartz-Lazaro", "Miller", "Norsworthy", "Scott"], "given-names": ["LM", "MR", "JK", "RC"], "year": ["2017"], "article-title": ["Comparison of simulated drift rates of common ALS-inhibiting rice herbicides to florpyrauxifen-benzyl on soybean"], "source": ["International Journal of Agronomy"], "volume": ["2017"], "fpage": ["9583678"], "pub-id": ["10.1155/2017/9583678"]}, {"label": ["Serim (2022)"], "person-group": ["\n"], "surname": ["Serim"], "given-names": ["AT"], "year": ["2022"], "article-title": ["Response of imidazolinone-resistant sunflower to various drift rates of glyphosate, glufosinate and indaziflam"], "source": ["Romanian Agricultural Research"], "volume": ["39"], "fpage": ["421"], "lpage": ["430"], "pub-id": ["10.59665/rar3939"]}, {"label": ["Serim et al. (2020)"], "person-group": ["\n"], "surname": ["Serim", "Asav", "T\u00fcrktemel", "\u00c7ak\u0131r Ar\u0131can", "Mennan"], "given-names": ["AT", "\u00dc", "\u0130", "N", "H"], "year": ["2020"], "article-title": ["Response of barnyard grass ("], "italic": ["Echinocloa"], "conf-name": ["II. International Agricultural, Biological Life Science Conference"], "fpage": ["1111"]}, {"label": ["Serim & Maden (2014)"], "person-group": ["\n"], "surname": ["Serim", "Maden"], "given-names": ["AT", "S"], "year": ["2014"], "article-title": ["Effects of soil residues of sulfosulfuron and mesosulfuron methyl + iodosulfuron methyl sodium on sunflower varieties"], "source": ["Journal of Agricultural Sciences"], "volume": ["20"], "issue": ["1"], "fpage": ["1"], "lpage": ["9"], "pub-id": ["10.1501/Tarimbil_0000001259"]}, {"label": ["Snipes, Street & Mueller (1992)"], "person-group": ["\n"], "surname": ["Snipes", "Street", "Mueller"], "given-names": ["CE", "JE", "TC"], "year": ["1992"], "article-title": ["Cotton ("], "italic": ["Gossypium hirsutum"], "source": ["Weed Science"], "volume": ["40"], "issue": ["1"], "fpage": ["106"], "lpage": ["109"], "pub-id": ["10.1017/S0043174500057040"]}, {"label": ["TUIK (2022)"], "person-group": ["\n"], "collab": ["TUIK"], "year": ["2022"], "article-title": ["Agricultural statistic in Turkey"], "uri": ["https://biruni.tuik.gov.tr/medas/?kn=92&locale=tr"]}, {"label": ["Wall (1996)"], "person-group": ["\n"], "surname": ["Wall"], "given-names": ["DA"], "year": ["1996"], "article-title": ["Effect of sublethal dosages of 2,4-D on annual broadleaf crops"], "source": ["Canadian Journal of Plant Science"], "volume": ["76"], "issue": ["1"], "fpage": ["179"], "lpage": ["185"], "pub-id": ["10.4141/cjps96-036"]}, {"label": ["Warmund, Ellersieck & Smeda (2022)"], "person-group": ["\n"], "surname": ["Warmund", "Ellersieck", "Smeda"], "given-names": ["MR", "MR", "RJ"], "year": ["2022"], "article-title": ["Sensitivity and recovery of tomato cultivars following simulated drift of dicamba or 2 4-D"], "source": ["Agriculture"], "volume": ["12"], "issue": ["9"], "fpage": ["1489"], "pub-id": ["10.3390/agriculture12091489"]}, {"label": ["Weed Science Society of America (2014)"], "person-group": ["\n"], "collab": ["Weed Science Society of America"], "year": ["2014"], "source": ["Herbicide handbook tenth edition"], "publisher-name": ["Weed Science Society of America"], "publisher-loc": ["Lawrence"]}]
{ "acronym": [ " PH", " SHD", " TSW", " I7DAT", " I14DAT", " I28DAT", " IHarvest" ], "definition": [ "plant height", "sunflower head diameter", "one thousand seed weight", "injury at 7 DAT", "injury at 14 DAT", "injury at 28 DAT", "injury at harvest" ] }
33
CC BY
no
2024-01-14 23:43:48
PeerJ. 2024 Jan 10; 12:e16729
oa_package/46/b7/PMC10787541.tar.gz
PMC10787542
0
[ "<title>Introduction</title>", "<p>According to the latest WHO Cancer Survey report for 2020, there are about 10 million cancer deaths and 19 million new cases worldwide (##REF##33538338##Sung et al., 2021##). At present, radiotherapy and chemotherapy have a certain killing effect on cancer cells, but the side effects are relatively obvious (##UREF##1##De Ruysscher et al., 2019##; ##REF##31236716##Johnston &amp; Beckman, 2019##; ##REF##33862002##Petit et al., 2021##; ##REF##33867827##Wang et al., 2021b##). With the spread of genetic testing and the development of targeted drugs, targeted therapies can be applied to cancer patients whose driver genes are mutated, amplified or rearranged (##REF##34108441##Wu et al., 2021##; ##REF##33028804##Yang et al., 2020##). This has significantly extended the survival time of cancer patients. However, over time, resistance to targeted drugs greatly limits their efficacy and application (##REF##33811122##Aldea et al., 2021##). Therefore, it is very important to deeply explore the pathological process of cancer and develop new therapeutic targets.</p>", "<p>YAP is a crucial downstream transcriptional coactivator in Hippo signaling pathway (##UREF##2##Driskill &amp; Pan, 2021##). YAP can bind to TEAD family protein to activate transcription of downstream targets which are implicated in cell growth and differentiation (##REF##29739703##Ardestani, Lupse &amp; Maedler, 2018##; ##REF##24325916##Lim et al., 2014##; ##REF##30037330##Pan et al., 2018##). Data from previous reports suggest the vital function of YAP/TAZ in transcription regulation of cancer cells (##REF##30401838##Chang et al., 2018##; ##UREF##0##Cordenonsi &amp; Piccolo, 2018##; ##REF##26439301##Galli et al., 2015##; ##UREF##3##Oh et al., 2014b##; ##REF##24613358##Skibinski et al., 2014##; ##REF##26295846##Stein et al., 2015##; ##REF##26258633##Zanconato et al., 2015##). Meanwhile, studies have also shown that the disturbance of YAP signal probably is the main mechanism of drug resistance to diversified targeting and chemotherapy, and the expression of PD-L1 and various cytokines mediated by YAP is very important for tumor immune escape (##REF##31174841##Nguyen &amp; Yi, 2019##). These studies imply that YAP may be used as a potential target for cancer and supply an effective idea for clinical treatment (##REF##35119068##Cunningham &amp; Hansen, 2022##).</p>", "<p>The ATP-dependent chromatin remodeling complexes play a crucial role in altering the structure of chromatin and controlling the accessibility of transcription factors to DNA, thereby facilitating the dynamic regulation of gene expression in the context of cancer progression. As a chromatin remodeling factor, chromatin accessibility complex subunit 1 (CHRAC1) has been involved in transcription and DNA replication (##REF##28512350##Clapier et al., 2017##; ##REF##31769422##Hasan &amp; Ahuja, 2019##). According to the result of a small interfering RNA screening, CHRAC1 was identified as an oncogenic driver gene (##REF##24148822##Mahmood et al., 2014##). In addition, CHRAC1 expression level was increased in cisplatin resistant ovarian cancer cell lines and lung cancer tumor tissues, and CHRAC1 disruption inhibited the migration and invasion of cisplatin resistant ovarian cancer cells and H1299 lung cancer cells (##UREF##4##Wang et al., 2021a##; ##REF##34030482##Yang et al., 2021##). Although previous studies have reported the significance of CHRAC1 in the progression of several cancers, the mechanism has not been well studied and the role of CHRAC1 in other cancers remains to be elucidated.</p>", "<p>Although it has been well established that YAP activates transcription by recruiting the transcription factor TEAD, whether chromatin remodeling complex is involved in this process remained unclear. In the current research, we identified CHRAC1 as a potential YAP interactor using the Bio-ID method and demonstrated that CHRAC1 regulated cell proliferation through promoting the oncogenic transcription of YAP target oncogenes in breast and cervical cancer. Together, our findings reveal that CHRAC1 plays a crucial role in enhancing YAP transcription activity and tumorigenesis, suggesting it may be a potential target for cancer therapy.</p>" ]
[ "<title>Material and Methods</title>", "<title>Cell line and transduction</title>", "<p>HEK293T, MDA-MB-231 and Hela cells were purchased from Procell (Wuhan, China) and cultured in DMEM (Gibco) with 10% FBS (Gibco). For CHRAC1 knockdown, cancer cell was transduced with pLKO.1-puro (shNC) or pLKO.1-puro-shCHRAC1 (1# or 2#). Interfering sequences were: shCHRAC1-1#: 5′-ACTCCACTGTCTCTAAGTAAA-3′; shCHRAC1-2#: 5′-ACTCCACTGTCTCTAAGTAAA-3′.</p>", "<title>Western blotting and Co-immunoprecipitation (Co-IP) assay</title>", "<p>Cells were lysed in Beyotine lysis buffer (Cat#: P1003) containing protease inhibitor and phenylmethylsulfonyl fluoride (PMSF) at 4 °C for 20 min and centrifuged at 4 °C, 12000 rpm for 15 min. Then cell lysate was denatured by boiling with SDS sample buffer at 95 °C for 10 min and separated by 10% SDS-PAGE gel. After that, the membranes were incubated with specific primary antibody (ABclonal rabbit CHRAC1: A14896, 1:500; ABclonal rabbit CHRAC17: A6469, 1:1000) overnight at 4 °C, secondary antibody for 1 h at room temperature and analyzed with electrogenerated chemiluminescence (ECL) system (Cat#: WBKLS0500; Millipore, Burlington, MA, USA).</p>", "<p>For Co-IP assays, cell lysates of 293T cells transfected with Flag-tagged CHARC1and HA-tagged YAP vectors were incubated with Agarose beads (Cat#: 16-266; Millipore, Burlington, MA, USA) and Flag antibody (E005; ABclonal) or IgG control overnight at 4 °C. The agarose beads/protein complex was washed 4 times with NP40 buffer and denatured at 95 °C for 15 min with SDS sample buffer and then subjected SDS-PAGE.</p>", "<title>Bio-ID</title>", "<p>Bio-ID experiment was conducted with reference to previous method (##REF##29729186##Zhang et al., 2018a##). Briefly, cells that stably expressed YAP-BirA* were cultured in complete medium containing 50 µM of biotin for 24 h, then washed with pre-cooled 1 x PBS 6-8 times, lysed with 1ml lysis buffer including protease inhibitor, and sonicated. The supernatant was obtained by centrifugation (4 °C, 15,000 rpm, 10 min) and incubated with 50 µl Streptavidin-conjugated beads (17511301; GE Healthcare, Chicago, IL, USA) overnight at 4 °C. After washing with 2% sodium dodecyl sulfate (SDS), 1% Triton X-100, 250 mM LiCl, 50 mM Tris buffer and 50 mM NH<sub>4</sub>HCO<sub>3</sub> buffer, the beads were stored at −20 °C for mass spectrometry or western blotting analysis. For western blotting detection, beads were denatured in 2X loading buffer at 100 °C for 10 min. Then supernatants were subjected SDS-PAGE. After blocking with 3% Bovine albumin (BSA) for 2 h at room temperature, the membranes were incubated with HRP-conjugated Streptavidin for 1 h at room temperature. After washing 4 times with TBST, the membranes were analyzed with electrogenerated chemiluminescence (ECL) system.</p>", "<title>Survival analysis of CHRAC1</title>", "<p>The Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) (<ext-link xlink:href=\"http://gepia2.cancer-pku.cn/\" ext-link-type=\"uri\">http://gepia2.cancer-pku.cn/</ext-link>) was used to investigate the overall survival (OS) and dis-ease-free survival (DFS) curves, as well as a survival map of CHRAC1 in The Cancer Genome Atlas (TCGA) tumor types. The expression threshold was set at 50% for high CHRAC1 expression and low CHRAC1 expression.</p>", "<title>CCK-8, wound-healing and colony formation assays</title>", "<p>Cell proliferation curves were measured by cell Counting Kit-8 (CCK-8) (BMU106-CN; Abbkine, Wuhan, China). Cells were seeded in 96-well plate with 4,000 cells in each well and cultured for 0, 24, 48, 72 and 96 h. Then the 450 nm absorbance was measured at each time point by adding CCK-8 reagent. For wound-healing assay, cells were inoculated in 6-well plates with 70% confluence and cultured for 36 h. Cell monolayer was scratched with the pipette tip to generate wound which was photographed every 12 h to monitor cell migration. For colony formation, cells were inoculated in 6-well plate with 1,000 (Hela) or 4,000 (MDA-MB-231) cells each well. After 10 days, the clones were stained with 0.05% crystal violet for imaging to monitor cell growth.</p>", "<title>Real-time quantitative PCR (RT-qPCR)</title>", "<p>RT-qPCR was used to verify the effect of CHRAC1 knockdown on YAP target genes in MDA-MB-231 and Hela cells. In this study, shNC was used as a control group and CHRAC1 shRNA (1# and 2#) was used as experimental group. Total RNA from cells was extracted according to the instructions of Vazyme RNA extraction Kit (Cat#: RC112-01; Vazyme, Beijing, China). RNA concentration and Purity were detected by Nanodrop spectrophotometer and 1µg RNA was applied to reverse transcription to synthesize complementary DNA (cDNA) using Reverse Transcription Kit (Cat#: RK20429, ABclonal) under the following conditions: 37 °C for 2 min, 55 °C for 15 min, 85 °C for 5 min. The reverse-transcribed cDNA was diluted 5-fold and RT-qPCR was conducted with the SYBR Green qPCR Mix reagent (Cat#: RK21203, ABclonal) on a Bio-Rad quantitative PCR instrument under the following conditions: 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference gene. The method to calculate the relative expression was 2<sup>−ΔΔCt</sup>. All of the RT-qPCR reactions were performed in duplicates. RT-qPCR primers sequences are available in supplementary materials.</p>", "<title>Immunofluorescence (IF)</title>", "<p>Cells were cultured for 36 h, fixed with 4% paraformaldehyde, and blocked with Image-iT signal enhancer (Thermo: <ext-link xlink:href=\"http://www.ncbi.nlm.nih.gov/nuccore/I36933\" ext-link-type=\"uri\">I36933</ext-link>). Thereafter, cells were incubated with primary antibody (Santa Cruz mouse YAP: sc-101199, 1:200; ABclonal rabbit CHRAC1: <ext-link xlink:href=\"http://www.ncbi.nlm.nih.gov/nuccore/A14896\" ext-link-type=\"uri\">A14896</ext-link>, 1:200) overnight at 4 °C and secondary antibody for 1 h. For Ki67 staining, freshly dissected tumor tissue was fixed and embedded in OCT reagent (Sakura) and cut to 4µm sections. Thereafter, sections were treated with citrate buffer for antigen retrieval and the following steps are similar to cell immunofluorescence staining.</p>", "<title>RNA-seq</title>", "<p>After the extraction of total RNA, the sequencing library was generated using the Prep Kit V2 (Illumina, San Diego, CA, USA) for RNA Library according to the manufacturer’s procedure. RNA-seq library was sequenced using the 75-nucleotide paired-end sequencing protocol on a NextSeq sequencer (Illumina). The technical replicates are duplicates for each sample and the RNAseq details are as follows: sequencing depth: 34.10939x; dispersions: 0.017797; effect 2; false positive rate: 0.05. The statistical power of this experimental design, calculated in RNASeqPower is 0.98. For bioinformatics analysis: genes whose Fold Change (padj &lt;= 0.05) is less than or equal to -2 were defined as down-regulated genes (CHRAC1 knockdown <italic toggle=\"yes\">versus</italic> control cell). Heat-map for different cancer hallmarks was created by TB-tools. The sequencing data have been deposited in the FigShare (DOI: <ext-link xlink:href=\"http://dx.doi.org/10.6084/m9.figshare.23989101\" ext-link-type=\"uri\">10.6084/m9.figshare.23989101</ext-link>).</p>", "<title>Mouse model</title>", "<p>Five-week-old male and female BALB/c nude mice (60 single), there was a lack of mature T lymphocytes, were purchased from Animal Center in Tongji Medical College, and subsequently housed under specific-pathogen-free conditions (Temperature: 22  ± 2 °C, Relative humidity: 60  ± 10%, 12 h light/dark cycle) at the Animal Center in Tongji Medical College. All the mice were given an unrestricted access of sterile food and water. Nine single male/female mice were randomly assigned to three separate groups (each group: three singles (sample size is determined according to the results of the preliminary experiment)): shNC group (control group) and CHRAC1 shRNA group (1# and 2#) (experimental group). 5 × 10<sup>6</sup> MDA-MB-231 or Hela cells treated with shNC or CHRAC1 shRNA were injected into the dorsal side of each nude mice (MDA-MB-231 cell: female mice; Hela cell: male mice). After about 2 weeks, tumor volume was monitored through double blind trials with vernier caliper every other day, and the size was measured as V = (L × W<sup>2</sup>) / 2 (L: length, W: width). Tumors were removed, imaged and weighed from mice with no significant weight change after 35 or 40 days. The tumor volume is generally no larger than 2000 mm<sup>3</sup>. The use and research design of animals was approved the Institutional Review Committee of Huazhong University of Science and Technology. The approval number: 2022 IACUC Number: 3148.</p>", "<title>Immunohistochemistry (IHC) staining</title>", "<p>Tissue sections of 48 human breast cancer and 48 cervical cancer specimens were purchased from Shanghai Wellbio Biotechnology company (Shanghai, China). Verbal informed consent was obtained and cancer specimens in this research have been approved by the Ethics Committee of of Huazhong University of Science and Technology and Shanghai Zhuoli Biotechnology Co., LTD (approval number: ZL2019-9-LL028, ZL2019-11-LL029). IHC staining was applied to detect the expression levels of CHRAC1 and YAP in breast and cervical cancer tissues. After deparaffinization and rehydration, antigen retrieval was performed. Endogenous peroxidase was blocked with 10% goat serum 1 h. Next, tissue microarrays were stained with primary antibodies (rabbit CHRAC1: Abclonal A14896, 1:200; rabbit YAP: Cell Signaling Technology 14074, 1:200) overnight at 4 °C and secondary antibody for 1 h. After washing, color development was performed with DAB kit (Maxim, DAB-0031) and the microarray was scanned with 3DHISTECH’s Slide Converter. The scanned images were opened with the Caseviewer software and enlarged to different magnifications. Click the “take snapshot” button on the software to take a picture. The images were saved in PNG format with 300 dpi resolution.</p>", "<title>Statistical analysis</title>", "<p>Statistical analysis was conducted with Prism 8 (GraphPad Software, La Jolla, CA, USA). Two-way analysis of variance (ANOVA) was used in CCK-8 and tumor volume detection assay. Student’s <italic toggle=\"yes\">t</italic>-test (two-tailed) was performed to compare CHRAC1 expression, wound-healing ability, colony formation ability, tumor weight and Ki67 staining. Linear regression analysis was performed to investigate the correlation between CHRAC1 and YAP in cancer tissues.</p>" ]
[ "<title>Results</title>", "<title>Interactome of YAP and CHRAC1 is a potential Bio-ID interactor of YAP</title>", "<p>To clarify YAP oncogenic regulators, we identified the spatial proximity interactome of YAP using the Bio-ID (proximity-dependent biotin identification) method (##FIG##0##Fig. 1A##). HEK293T cells expressing YAP-BirA*-HA were cultured in medium supplemented with or without biotin; much more biotinylated proteins were captured in total cell lysates cultured with biotin (##FIG##0##Figs. 1B## and ##FIG##0##1C##). Following mass spectrometry, we identified 254 potential interactors of YAP (YAP Bio-ID interactor). The Gene Ontology (GO) analysis on biological process for YAP Bio-ID interactors revealed a significant enrichment in Hippo pathway, cell division, cell–cell adhesion, DNA repair, DNA replication, mRNA processing and chromatin remodeling (##FIG##0##Fig. 1D##). In additional to numerous known YAP interactors (AMOT, LATS1 and AMOTL1), CHRAC1 attracted our attention. As a member of chromatin remodeling complex, CHRAC1 has been involved in transcription and DNA replication. However, the significance of CHRAC1 in cancer development has not been investigated extensively. Therefore, we focused on the study of CHRAC1.</p>", "<title>High expression of CHRAC1 predicts advanced pathological tumor stages and poor survival</title>", "<p>To explore the expression of CHRAC1 in cancer tissues, we analyzed the expression of CHRAC1 in cancer tissues including breast and cervical. Immunohistochemistry (IHC) result displayed that CHRAC1 was mainly expressed in the nucleus and the staining was extremely weak in para-tumor tissues, while breast and cervical cancer biopsies showed strong CHRAC1 signal (##FIG##1##Figs. 2A## and ##FIG##1##2B##).</p>", "<p>To evaluate the potential clinical value of CHRAC1, we explored the correlation between CHRAC1 expression and patient survival within different tumors in the TCGA dataset (##SUPPL##0##Fig. S1A##). High CHRAC1 mRNA level was correlated with decreased overall and disease-free survival in cases of CESC and other tumor types by using the Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) tool (##FIG##1##Figs. 2C##–##FIG##1##2E##, ##SUPPL##0##Figs. S1B##–##SUPPL##0##S1K##). Additionally, the CHRAC1 level was significantly associated with pathological stages of kidney renal clear cell carcinoma (KIRC), adrenocortical carcinoma (ACC) and PAAD patients (##SUPPL##1##Fig. S2##). Moreover, in breast cancer specimens (<italic toggle=\"yes\">n</italic> = 48), CHRAC1 expression increased with the progression of tumor stage (##FIG##1##Figs. 2F## and ##FIG##1##2G##). In cervical cancer specimens (<italic toggle=\"yes\">n</italic> = 48), patients with metastasis had elevated CHRAC1 protein expression compared to those patients with primary tumors (##FIG##1##Fig. 2H##). Together, these data manifest that high CHRAC1 expression may predict advanced pathological tumor stages and poor survival in breast and cervical cancer.</p>", "<title>Downregulation of CHRAC1 inhibits tumor growth</title>", "<p>To validate the effect of CHRAC1 on cancer progression, we silenced CHRAC1 (##FIG##2##Fig. 3A##) in breast and cervical cancer cell lines (MDA-MB-231 and Hela). The downregulation of CHRAC1 have been confirmed by immunoblot, the result showed that the knock down of CHRAC1 specifically inhibited the protein level of CHRAC1 but not another family member CHRAC17 (##SUPPL##0##Fig. S3A##). CCK-8 assay displayed that CHRAC1 silencing suppressed the activity of MDA-MB-231 and Hela cells (##FIG##2##Fig. 3B##). Wound-healing assay also indicated that CHRAC1 down-regulation restrained cancer cell migration (##FIG##2##Fig. 3C##). In addition, colony formation assay showed that cancer cells with ablated CHRAC1 demonstrated significantly decreased colony size and number (##FIG##2##Fig. 3D##). To make the knockdown data more compelling, we also investigated the effect of CHRAC1 over-expression on cancer cells. The results showed that CHRAC1 over-expression significantly promoted cell migration and proliferation both in MDA-MB-231 and Hela cells (##SUPPL##0##Figs. S3B##–##SUPPL##0##S3D##). Collectively, these data suggest that CHRAC1 silencing inhibits the proliferation of breast and cervical cancer cells.</p>", "<p>To verify the influence of CHRAC1 on tumorigenesis, we conducted a xenograft assay in nude mouse. Downregulation of CHRAC1 in Hela cells could significantly reduce tumor size, and tumor formation was not possible in MDA-MB-231 cells with CHRAC1 inhibition (##FIG##2##Figs. 3E## and ##FIG##2##3F##). The average weight of CHRAC1-silenced Hela xenografts was about one-third of the control group (##FIG##2##Fig. 3G##). To investigate the proliferation capacity of tumor cells, we performed immunofluorescence staining of Ki67 for tissue sections. The ratio of Ki67 positive cells was statistically reduced in CHRAC1-silenced tumors compared to control tumors (##FIG##2##Fig. 3H##). Collectively, these data display that CHRAC1 silencing suppresses tumor growth of breast and cervical cancer cells in nude mice.</p>", "<title>Inhibition of CHRAC1 suppresses the oncogenic transcription of YAP</title>", "<p>To illustrate the mechanism of CHRAC1 regulation on tumorigenesis, we performed RNA-seq assay in CHRAC1-silenced Hela cells. CHRAC1 ablation caused differential expression of 2,595 genes, of which 1,300 genes were increased and 1,295 genes were decreased (##FIG##3##Fig. 4A##). Gene ontology (GO) annotation of the 1,295 down-regulated genes indicated that they were highly enriched for GO terms linked to transcription, cell proliferation, cell apoptosis, cell migration and related signaling pathways (##FIG##3##Fig. 4B##). A heatmap of RNA-seq data clearly showed that knock-down of CHRAC1 suppressed the expression of representative cancer hallmarks, such as DNA repair, G2M checkpoint and the P53 pathway (##FIG##3##Fig. 4C##). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation displayed that CHRAC1 silencing resulted in significant downregulation (<italic toggle=\"yes\">p</italic> &lt; 0.01) of Hippo pathway-related genes (##FIG##3##Fig. 4D##). Then, we focused on the direct YAP target oncogenes previously identified (##UREF##5##Zanconato et al., 2018##; ##REF##26258633##Zanconato et al., 2015##). Gene set enrichment analysis (GSEA) displayed that YAP target gene signature was enriched in control Hela cells but not in CHRAC1 silenced cells, suggesting that CHRAC1 might trigger the oncogenic transcription program of YAP in Hela cells (##FIG##3##Fig. 4E##). Further, RT-qPCR results confirmed that depletion of CHRAC1 reduced the mRNA level of three classical YAP target genes (<italic toggle=\"yes\">CTGF</italic>, <italic toggle=\"yes\">CYR61</italic>, <italic toggle=\"yes\">ANKRD1</italic>) (##FIG##3##Fig. 4F##). We also performed immunofluorescence (IF) assay for CTGF in Hela and MDA-MB-231 cell lines to validate our finding at the protein level. The results showed that knock down of CHRAC1 significantly decreased the fluorescence intensity of CTGF both in Hela and MDA-MB-231 cells (##FIG##3##Fig. 4G##). Together, these results demonstrate that CHRAC1 indeed influences the transcriptional activation of YAP.</p>", "<title>CHRAC1 interacts with YAP and is positively correlated with YAP in breast and cervical cancer patients</title>", "<p>According to the Bio-ID results, CHRAC1 may be a potential interactor of YAP. To validate the interaction of CHRAC1 and YAP, we performed exogenous co-immunoprecipitation (Co-IP) assay. The result showed that flag-tagged CHARC1 was able to specifically precipitated HA-tagged YAP in 293T cells compared to the IgG control (##FIG##4##Fig. 5A##, left panel). In addition, Co-IP detection was also conducted in CHRAC1 overexpressed MDA-MB-231 and Hela cells, and it was found that YAP could specifically co-purify CHRAC1 in both cells (##FIG##4##Fig. 5A##, right panel). Furthermore, immunofluorescence assay displayed that CHRAC1 co-localized with YAP both in MDA-MB-231 and Hela cells (##FIG##4##Fig. 5B##). Therefore, there is indeed an interaction between YAP and CHRAC1. Then, we investigated the clinical correlation of CHRAC1 and YAP and found CHRAC1 was associated with YAP across Pan-cancer, including BRCA and CESC in TCGA database (##FIG##4##Figs. 5C##–##FIG##4##5E##). In addition, both breast and cervical cancer biopsies with high CHRAC1 expression showed stronger YAP staining (##FIG##4##Fig. 5F##). Consistent with this, there was significant correlation ( <italic toggle=\"yes\">p</italic> &lt; 0.0001) between YAP and CHRAC1 in these cancer biopsies (##FIG##4##Fig. 5F##). Thus, CHRAC1 is elevated in breast and cervical cancer and the upregulation correlates well with YAP.</p>", "<p>In summary, our study found that depletion of CHRAC1 suppresses cancer cell proliferation and tumor growth. The potential mechanism may be that CHRAC1 interacts with YAP to enhance the transcription of YAP down-stream oncogenes to promote tumor growth (##FIG##5##Fig. 6##). Moreover, CHRAC1 is frequently upregulated in diverse cancers and the upregulation is statistically associated with YAP activation and poor prognosis in cancer patients. These findings highlight CHRAC1 might be a promising candidate for cancer diagnosis and therapy.</p>" ]
[ "<title>Discussion</title>", "<p>CHRAC1, a component of the chromatin remodeling complex, is associated with poor prognosis of cancer patients (##REF##10880450##Poot et al., 2000##). In addition, researches have reported that abnormal expression of CHRAC1 is frequently associated with the occurrence and progression of human cancer (##UREF##4##Wang et al., 2021a##; ##REF##34030482##Yang et al., 2021##). However, the mechanism remains to be clarified and the function of CHRAC1 in various cancer types has not been fully studied.</p>", "<p>In this study, we identified CHRAC1 as a potential oncogene in multiple human tumors <italic toggle=\"yes\">via</italic> pan-cancer analyses and experimental verification. First of all, we discovered that CHRAC1 was upregulated in a variety of cancer tissues compared to relevant normal tissues. Meanwhile, CHRAC1 expression was statistically correlated with poor survival and pathological stages in various tumor types. Collectively, these findings indicate that elevated CHRAC1 may be a prognostic factor for cancer.</p>", "<p>Next, we verified the oncogenic function of CHRAC1 through experiments. In <italic toggle=\"yes\">in vitro</italic> study, we discovered that knockdown of CHRAC1 restrained the growth of breast and cervical cancer cells. However, there are some limitations to this research. For example, it may make the knock down data compelling to investigate the effects of CHRAC1 overexpression on cell growth. In <italic toggle=\"yes\">in vivo</italic> study, we demonstrated that CHRAC1 silencing decreased tumor weight and tumor size in MDA-MB-231 and Hela xenograft mouse model. However, whether CHRAC1 affects tumor metastasis needs further study. Additionally, development of drugs that target CHRAC1 may advance the findings to clinical applications.</p>", "<p>To clarify how CHRAC1 affects cancer development, we conducted RNA-seq assay and found that the transcription program of control cells and CHRAC1 knockdown cells was significantly different. The inhibition of CHRAC1 also affected the expression level of many cancer hallmarks. Moreover, many known YAP target genes were significantly downregulated in CHRAC1 knockdown groups. It has been well established that YAP target genes served as oncogenes in many cancer types (##REF##27300434##Zanconato, Cordenonsi &amp; Piccolo, 2016##; ##REF##30176928##Zhang et al., 2018b##). These indicate that CHRAC1 may promote tumor growth by enhancing the oncogenic transcription of YAP. However, in addition to YAP, whether CHRAC1 knockdown affects other transcription factors remains to be studied.</p>", "<p>Base on the Bio-ID results, CHRAC1 was a potential interactor of YAP and the interaction between these two proteins was verified by Co-IP and IF assays. In addition, numerous known YAP interactors (AMOT, LATS1 and AMOTL1) have been identified by Bio-ID method, which confirms the feasibility of this approach. According to previous reports, YAP is known to recruit transcription factors TEAD1-4, RNA polymerase, the mediator complex, and other factors to establish a transcription hub within the nucleus, facilitating oncogenic transcription during cancer progression (##REF##25017066##Oh et al., 2014a##; ##REF##26295846##Stein et al., 2015##; ##REF##26258633##Zanconato et al., 2015##). Notably, there are robust interactions between these components and YAP. Our findings demonstrate the presence of CHRAC1 in YAP complex, exhibiting strong co-localization with YAP within the nucleus. Moreover, the loss of CHRAC1 impedes the transcriptional activation of YAP. Consequently, we suggest that the interaction between YAP and CHRAC1 plays a crucial role in recruiting CHRAC1 to YAP transcription hub, thereby promoting the oncogenic transcription.</p>", "<p>To further investigate the clinical significance of CHRAC1, we conducted IHC analysis in human breast and cervical cancer biopsies. The expression of CHRAC1 in cancer tissues was significantly higher than that in the para-cancer group. Moreover, YAP was also highly expressed in breast and cervical cancer specimens with high expression of CHRAC1. Consistent with this, there was a statistical correlation between YAP and CHRAC1 (<italic toggle=\"yes\">p</italic> &lt; 0.0001). Despite this, specimens from different cancer types are required to verify the association between CHRAC1 and YAP. Therefore, the practical application of CHRAC1 in cancer prediction and treatment needs further experimental and clinical research.</p>" ]
[]
[ "<title>Background</title>", "<p>As a component of chromatin remodeling complex, chromatin accessibility complex subunit 1 (CHRAC1) is critical in transcription and DNA replication. However, the significance of CHRAC1 in cancer progression has not been investigated extensively. This research aimed to determine the function of CHRAC1 in breast and cervical cancer and elucidate the molecular mechanism.</p>", "<title>Methods</title>", "<p>The Bio-ID method was used to identify the interactome of transcriptional activator Yes-associated protein (YAP) and the binding between YAP and CHRAC1 was verified by immunofluorescence. CCK8, colony formation and subcutaneous xenograft assays were conducted to explore the function of CHRAC1 in cancer cell proliferation. RNA-seq analysis and RT-PCR were used to analyze the transcription program change after CHRAC1 ablation. The diagnostic value of CHRAC1 was analyzed by TCGA database and further validated by immunohistochemistry staining.</p>", "<title>Results</title>", "<p>In the current study, we found that the chromatin remodeler CHRAC1 was a potential YAP interactor. CHRAC1 depletion suppressed breast and cervical cancer cell proliferation and tumor growth. The potential mechanism may be that CHRAC1 interacts with YAP to facilitate oncogenic transcription of YAP target genes in Hippo pathway, thereby promoting tumorigenesis. CHRAC1 was elevated in cervical and breast cancer biopsies and the upregulation correlated with shorter survival, poor pathological stages and metastasis of cancer patients. Moreover, CHRAC1 expression was statistically associated with YAP in breast and cervical cancer biopsies.</p>", "<title>Conclusions</title>", "<p>These findings highlight that CHRAC1 contributes to cancer progression through regulating the oncogenic transcription of YAP, which makes it a potential therapeutic target for cancer treatment.</p>" ]
[ "<title> Supplemental Information</title>" ]
[ "<p>Thanks to Professor Sun Shuguo for his guidance and discussion of the manuscript.</p>", "<title>Additional Information and Declarations</title>" ]
[ "<fig position=\"float\" id=\"fig-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/fig-1</object-id><label>Figure 1</label><caption><title>Interactome of YAP and CHRAC1 is a potential Bio-ID interactor of YAP.</title><p>(A) Flowchart of Bio-ID approach. (B) Schematic illustration of YAP-BirA*-HA construct. (C) HEK293T cells stably expressing YAP-BirA*-HA construct were cultured with 50 µM/L biotin for 24 h. The biotinylated proteins were captured by streptavidin-agarose beads and detected by HRP-conjugated streptavidin. (D) GO analysis on biological process for YAP Bio-ID interactors.</p></caption></fig>", "<fig position=\"float\" id=\"fig-2\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/fig-2</object-id><label>Figure 2</label><caption><title>High expression of CHRAC1 predicts advanced pathological tumor stages and poor survival in breast and cervical cancer.</title><p>(A) Representative CHRAC1 IHC staining in breast and cervical cancer tumor tissues and para-tumor tissues. Scale bar, 20 µm and 5 µm. (B) Statistical analysis of CHRAC1 intensity was shown in diagram. (C–E) Overall survival curves of patients with high or low expression of CHRAC1 in CESC (C), KIRP (D) and LAML (E). Data were from TCGA database and were analyzed with GEPIA2 tool. (F) Representative IHC staining of CHRAC1 in different tumor stages of breast cancer biopsies. Scale bar, 50 µm and 5 µm. (G) Quantification of CHRAC1 staining in Fig. 2F was demonstrated in diagram. (H) Quantification of CHRAC1 staining in cervical cancer biopsies with metastasis was demonstrated in the histogram. * <italic toggle=\"yes\">P</italic> &lt; 0.05, *** <italic toggle=\"yes\">P</italic> &lt; 0.001. Two-tailed student’s <italic toggle=\"yes\">t</italic>-test.</p></caption></fig>", "<fig position=\"float\" id=\"fig-3\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/fig-3</object-id><label>Figure 3</label><caption><title>Downregulation of CHRAC1 inhibits tumor growth.</title><p>(A) Silencing of CHRAC1 in MDA-MB-231 and Hela cells detected by RT-qPCR. (B) CHRAC1 silencing restrains cell viability. (C) CHRAC1 silencing restrains cell migration. The statistical analysis was demonstrated in histogram. (D) The effect of CHRAC1 silencing on colony formation. The statistical analysis of clone numbers was demonstrated in histogram. *** <italic toggle=\"yes\">P</italic> &lt; 0.001, ** <italic toggle=\"yes\">P</italic> &lt; 0.01, * <italic toggle=\"yes\">P</italic> &lt; 0.05. (E) Typical tumor images in mice inoculated with control and CHRAC1-silenced cancer cells. (F) CHRAC1 silencing reduces tumor volume. **** <italic toggle=\"yes\">P</italic> &lt; 0.0001, two-way ANOVA. (G) CHRAC1 silencing reduces tumor weight. ** <italic toggle=\"yes\">P</italic> &lt; 0.01, Student’s <italic toggle=\"yes\">t</italic>-test (two-tailed). (H) Ki67 staining for proliferating cells in control and CHRAC1 silenced Hela xenografts. Quantification of Ki67-positive cells was demonstrated in the histogram. *** <italic toggle=\"yes\">P</italic> &lt; 0.001, ** <italic toggle=\"yes\">P</italic> &lt; 0.01, Student’s <italic toggle=\"yes\">t</italic>-test (two-tailed).</p></caption></fig>", "<fig position=\"float\" id=\"fig-4\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/fig-4</object-id><label>Figure 4</label><caption><title>Inhibition of CHRAC1 suppresses the oncogenic transcription of YAP.</title><p>(A) The volcano map displayed the differential expression of genes upon CHRAC1 inhibition. (B–D) GO annotation (B), heatmap (C), and KEGG analysis (D) of the down-regulated genes upon CHRAC1 silencing. (E) GSEA analysis of YAP targets in CHRAC1 knockdown Hela cells. (F) Effects of CHRAC1 knockdown on mRNA expression of YAP targets in MDA-MB-231 and Hela cells. (G) Effects of CHRAC1 knockdown on protein level of YAP target CTGF in MDA-MB-231 and Hela cells by IF staining.</p></caption></fig>", "<fig position=\"float\" id=\"fig-5\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/fig-5</object-id><label>Figure 5</label><caption><title>CHRAC1 interacts with YAP and is positively correlated with YAP in breast and cervical cancer patients.</title><p>(A) Exogenous Co-IP of HA-YAP and Flag-CHRAC1 in HEK293T cells (left panel). Co-IP detection of YAP and CHRAC1 in CHRAC1 overexpressed MDA-MB-231 and Hela cells (right panel). (B) CHRAC1 is co-localized with YAP in MDA-MB-231 (upper panel) and Hela (lower panel) cells. (C–E) Correlation analysis between YAP and CHRAC1 in pan-cancer (C), BRCA (D) and CESC (E) according to the TCGA database. (F) Typical IHC images of breast and cervical cancer specimens with low and high YAP/CHRAC1 expression. Scale bar, 50 µm and 5 µm. Correlation analysis of YAP and CHRAC1 in breast and cervical cancer tissues was shown in diagram.</p></caption></fig>", "<fig position=\"float\" id=\"fig-6\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/fig-6</object-id><label>Figure 6</label><caption><title>Mechanism of CHRAC1 promoting cancer cell proliferation.</title><p>The potential mechanism may be that CHRAC1 interacts with YAP to enhance the transcriptional activation of YAP downstream target genes, such as <italic toggle=\"yes\">CTGF</italic>, <italic toggle=\"yes\">CYR61</italic> and <italic toggle=\"yes\">ANKRD1</italic>, and thus promotes the tumor growth.</p></caption></fig>" ]
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[ "<supplementary-material id=\"supp-1\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/supp-1</object-id><label>Supplemental Information 1</label><caption><title>Supplementary materials and figures</title></caption></supplementary-material>", "<supplementary-material id=\"supp-2\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/supp-2</object-id><label>Data S1</label><caption><title>Raw Data</title></caption></supplementary-material>", "<supplementary-material id=\"supp-3\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/supp-3</object-id><label>Supplemental Information 3</label><caption><title>Original Western blot data</title></caption></supplementary-material>", "<supplementary-material id=\"supp-4\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/supp-4</object-id><label>Supplemental Information 4</label><caption><title>ARRIVE 2.0 Checklist</title></caption></supplementary-material>", "<supplementary-material id=\"supp-5\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16752/supp-5</object-id><label>Supplemental Information 5</label><caption><title>MIQE checklist</title></caption></supplementary-material>" ]
[ "<fn-group content-type=\"competing-interests\"><title>Competing Interests</title><fn id=\"conflict-1\" fn-type=\"COI-statement\"><p>The authors declare there are no competing interests.</p></fn></fn-group>", "<fn-group content-type=\"author-contributions\"><title>Author Contributions</title><fn id=\"contribution-1\" fn-type=\"con\"><p><xref rid=\"author-1\" ref-type=\"contrib\">Shasha Li</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-2\" fn-type=\"con\"><p><xref rid=\"author-2\" ref-type=\"contrib\">Lulu Wang</xref> performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-3\" fn-type=\"con\"><p><xref rid=\"author-3\" ref-type=\"contrib\">Jing Shi</xref> analyzed the data, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-4\" fn-type=\"con\"><p><xref rid=\"author-4\" ref-type=\"contrib\">Yi Chen</xref> performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.</p></fn><fn id=\"contribution-5\" fn-type=\"con\"><p><xref rid=\"author-5\" ref-type=\"contrib\">Ang Xiao</xref> performed the experiments, prepared figures and/or tables, and approved the final draft.</p></fn><fn id=\"contribution-6\" fn-type=\"con\"><p><xref rid=\"author-6\" ref-type=\"contrib\">Bingyue Huo</xref> performed the experiments, prepared figures and/or tables, and approved the final draft.</p></fn><fn id=\"contribution-7\" fn-type=\"con\"><p><xref rid=\"author-7\" ref-type=\"contrib\">Wenjing Tian</xref> performed the experiments, prepared figures and/or tables, and approved the final draft.</p></fn><fn id=\"contribution-8\" fn-type=\"con\"><p><xref rid=\"author-8\" ref-type=\"contrib\">Shilu Zhang</xref> performed the experiments, prepared figures and/or tables, and approved the final draft.</p></fn><fn id=\"contribution-9\" fn-type=\"con\"><p><xref rid=\"author-9\" ref-type=\"contrib\">Gang Yang</xref> analyzed the data, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-10\" fn-type=\"con\"><p><xref rid=\"author-10\" ref-type=\"contrib\">Wensheng Gong</xref> analyzed the data, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-11\" fn-type=\"con\"><p><xref rid=\"author-11\" ref-type=\"contrib\">Huixia Zhang</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn id=\"addinfo-1\"><p>The following information was supplied relating to ethical approvals (<italic toggle=\"yes\">i.e.</italic>, approving body and any reference numbers):</p><p>Cancer specimens involved in this study were approved by the Ethics Committee of of Huazhong University of Science and Technology and Shanghai Zhuoli Biotechnology Co., LTD (approval number: ZL2019-9-LL028, ZL2019-11-LL029).</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn id=\"addinfo-2\"><p>The following information was supplied relating to ethical approvals (<italic toggle=\"yes\">i.e.</italic>, approving body and any reference numbers):</p><p>The animal experiments have been approved by the Ethics Committee of Huazhong University of Science and Technology (Approval number: 2022 IACUC Number: 3148).</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>DNA Deposition</title><fn id=\"addinfo-3\"><p>The following information was supplied regarding the deposition of DNA sequences:</p><p>The RNA-seq data are available at FigShare: Li, Shasha (2023). Hela shCHRAC1. figshare. Online resource. <ext-link xlink:href=\"https://doi.org/10.6084/m9.figshare.23989101.v1\" ext-link-type=\"uri\">https://doi.org/10.6084/m9.figshare.23989101.v1</ext-link> and at the National Genomics Data Center: HRA004198.</p><p>\n<ext-link xlink:href=\"https://ngdc.cncb.ac.cn/gsa-human/browse/HRA004198\" ext-link-type=\"uri\">https://ngdc.cncb.ac.cn/gsa-human/browse/HRA004198</ext-link>\n</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Data Availability</title><fn id=\"addinfo-4\"><p>The following information was supplied regarding data availability:</p><p>The raw data and original immunoblots are available in the <xref rid=\"supplemental-information\" ref-type=\"sec\">Supplemental Files</xref>.</p></fn></fn-group>" ]
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[{"label": ["Cordenonsi & Piccolo (2018)"], "person-group": ["\n"], "surname": ["Cordenonsi", "Piccolo"], "given-names": ["M", "S"], "year": ["2018"], "article-title": ["Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4"], "source": ["Nature Medicine"], "volume": ["24"], "issue": ["10"], "fpage": ["1599"], "lpage": ["1610"], "pub-id": ["10.1038/s41591-018-0158-8"]}, {"label": ["De Ruysscher et al. (2019)"], "person-group": ["\n"], "surname": ["De Ruysscher", "Niedermann", "Burnet", "Siva", "Lee", "Hegi-Johnson"], "given-names": ["D", "G", "NG", "S", "AWM", "F"], "year": ["2019"], "article-title": ["Radiotherapy toxicity"], "source": ["Nature Reviews Disease Primers"], "volume": ["5"], "fpage": ["13"], "pub-id": ["10.1038/s41572-019-0064-5"]}, {"label": ["Driskill & Pan (2021)"], "person-group": ["\n"], "surname": ["Driskill", "Pan"], "given-names": ["JH", "D"], "year": ["2021"], "article-title": ["The hippo pathway in liver homeostasis and pathophysiology"], "source": ["Annual Review of Pathology: Mechanisms of Disease"], "volume": ["16"], "fpage": ["299"], "lpage": ["322"], "pub-id": ["10.1146/annurev-pathol-030420-105050"]}, {"label": ["Oh et al. (2014b)"], "person-group": ["\n"], "surname": ["Oh", "Slattery", "Ma", "White", "Mann", "Irvine"], "given-names": ["H", "M", "LJ", "KP", "RS", "KD"], "year": ["2014b"], "article-title": ["Yorkie promotes transcription by recruiting a histone methyltransferase complex"], "source": ["Cell Reports"], "volume": ["8"], "fpage": ["448"], "lpage": ["458"], "pub-id": ["10.1016/j.celrep.2014.06.017"]}, {"label": ["Wang et al. (2021a)"], "person-group": ["\n"], "surname": ["Wang", "Li", "Guo", "Wang", "Huang", "Zhuo", "Lai", "Liao", "Ge", "Nie", "Jin", "Wang", "Zhang", "Liu", "Li", "Zhang"], "given-names": ["MW", "SS", "WC", "LL", "JX", "JZ", "BT", "CQ", "TL", "YX", "S", "MX", "YGL", "YM", "XW", "HX"], "year": ["2021a"], "article-title": ["CHRAC1 promotes human lung cancer growth through regulating YAP transcriptional activity"], "source": ["Carcinogenesis"], "volume": ["43"], "fpage": ["264"], "lpage": ["276"], "pub-id": ["10.1093/carcin/bgab103"]}, {"label": ["Zanconato et al. (2018)"], "person-group": ["\n"], "surname": ["Zanconato", "Battilana", "Forcato", "Filippi", "Azzolin", "Manfrin", "Quaranta", "Di Biagio", "Sigismondo", "Guzzardo", "Lejeune", "Haendler", "Krijgsveld", "Fassan", "Bicciato", "Cordenonsi", "Piccolo"], "given-names": ["F", "G", "M", "L", "L", "A", "E", "D", "G", "V", "P", "B", "J", "M", "S", "M", "S"], "year": ["2018"], "article-title": ["Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4"], "source": ["Nature Medicine"], "volume": ["24"], "fpage": ["1599"], "lpage": ["1610"], "pub-id": ["10.1038/s41591-018-0158-8"]}]
{ "acronym": [ " CHRAC1", " YAP", " IF", " IHC", " CCK-8", " GO", " KEGG", " GSEA", " OS", " DFS", " CESC", " BRCA", " KIRC", " ACC" ], "definition": [ "chromatin accessibility complex subunit 1", "Yes-associated protein", "Immunofluorescence", "Immunohistochemistry", "Cell Counting Kit-8", "Gene Ontology", "Kyoto Encyclopedia of Genes and Genomes", "Gene set enrichment analysis", "overall survival", "disease-free survival", "Cervical squamous cell carcinoma and endocervical adenocarcinoma", "Breast invasive carcinoma", "Kidney renal clear cell carcinoma", "Adrenocortical carcinoma" ] }
32
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2024-01-14 23:43:48
PeerJ. 2024 Jan 10; 12:e16752
oa_package/51/03/PMC10787542.tar.gz
PMC10787543
0
[ "<title>Introduction</title>", "<p>Due to the tactical changes and the success-oriented structure in today’s soccer, it is necessary for the players to develop various characteristics (##REF##33592641##Padrón-Cabo et al., 2021##). Today, most of the motor skills, depending on basic features and characteristics, are more prominent in soccer (##REF##28658072##Clark et al., 2019##). While change of direction, anaerobic power, and ability to maintain that power at high-speed gain importance in soccer, endurance is needed even more (##REF##30988855##Rivilla-García et al., 2019##). The systems of this game, which requires a high tackling power, force the players to frequently use their technical skills and aerobic-anaerobic power. Success in soccer demands superior physical, motor, and physiological performance levels as well as skill and ability (##REF##23332540##Buchheit et al., 2013##).</p>", "<p>In movements (walking, running, sprinting, jumping, and endurance) in soccer, many motoric performance characteristics must be developed due to the need for speed and frequent direction changes and the nature of the movements in the field (##REF##24476783##Söhnlein, Müller &amp; Stöggl, 2014##). High intensity running and repetitive sprint activities are considered to be very important indicators for successful performance (##REF##35795177##Clemente et al., 2022##). The most important parameters for a soccer player are to be able to run faster than the opponent with or without the ball during the match and to move faster than the opponent while tackling to jump higher (##REF##24751658##Ramírez-Campillo et al., 2014##). It is known that soccer players have aerobic base as they cover an average of 8.6–14.2 km during a match (##UREF##5##Dardouri et al., 2014##). However, considering that each player performs 1,000–1,400 short-term movements that last 4–6 s and each one performs sprints that end in an average of 2–4 s every 90 s, anaerobic performance is believed to be extremely important (##UREF##10##Köklü, Özkan &amp; ve Ersöz, 2009##).</p>", "<p>Anaerobic performance is mainly achieved through explosive type training that achieves results in the least amount of time. For this reason, the energy source that needs to be completed urgently is obtained from creatine phosphate (CP), adenosine triphosphate (ATP) and anaerobic glycolysis (##REF##27139591##Asadi et al., 2016##). In anaerobic-based games such as football, boosts made by more than one method are obtained from the anaerobic energy system (##REF##24700201##Zamparo et al., 2014##). Providing functional performance development of players towards the formation of anaerobic power also reveals the purpose of strength training. One of the factors affecting anaerobic performance and strength is muscles. Explosive muscle contractions, especially those produced by the knee extensors, have a very important role in the anaerobic performance measures of football players (##REF##29176387##Beato et al., 2018##). During a football match, muscle power and strength are critical physical factors for successful participation. Plyometric training are widely implemented as training methodology for enhancing functional sports performance (##REF##23176602##Stewart, Turner &amp; Miller, 2014##).</p>", "<p>Plyometric training, which can be performed by soccer players to increase explosive power is one of the best the training practises for the development of anaerobic power in soccer, and it can be applied with different arrangements aiming to accelerate the muscle response in combining strength and speed and increase in the number and quality of high-intensity movements (##REF##19910813##Meylan &amp; Malatesta, 2009##). These trainings in soccer meet the explosive strength requirement enables the eccentric muscle contractions and increase in tension of the muscle. This increase in intramuscular tension increase muscle strength and thus the speed of movement (##UREF##2##Bompa, 2013##).</p>", "<p>Soccer clubs, coaches, and soccer players are employing specific training models for parameters such as speed, agility, and strength, which are among the physical components of soccer players (##REF##26390150##Loturco et al., 2015##). Research show that plyometric training is one of the most effective specific training methods that can directly affect match performance and develop the strength/power (##UREF##14##William &amp; Kirubakar, 2018##). Additionally, if the aim of this success-indexed game is to increase the speed for athletic performance, anaerobic performance, jump height, explosive strength and maximal strength in the match, it has been proven that plyometric training comes first (##REF##23717351##Váczi et al., 2013##).</p>", "<p>Strength, and explosive strenght training have been reported to result in various physiological adaptations that enhance athletic performance (##REF##18080951##Ferrari et al., 2008##). ##UREF##6##Edwin &amp; Gordon (2000)## reported that 8 weeks of a plyometric training improved sprint times, strength and anaerobic power (##REF##18080951##Ferrari et al., 2008##). On the other hand, in order to increase the physical performance of football players, it is necessary to determine the physiological profiles of football players. Explosive power, force etc. when the training is based on physiological foundations for this profile, it is possible to increase the performance of the player (##REF##16802248##Herrero et al., 2006##). Accordingly, it can be seen that all these physical characteristics can only reach the desired level thanks to a well-programmed training program. Preseason training is important in order to properly regulate the intensity and intensity of the training during the preparation season and to monitor the players’ physical and physiological parameters well (##REF##23332540##Buchheit et al., 2013##).</p>", "<p>Preseason training is designed to develop players’ physical capacities and prepare them for the various demands of the competitive season. For example, semi-professional and amateur soccer players who increased their aerobic capacity across a 8–10-week pre-season period were less likely to be injured during the subsequent in-season period (##REF##30539123##Eliakim et al., 2018##). Preseason team training sessions were counted for each team, covering the period from the first training session of the season until the first competitive match (in the national league and international league). Pre-season team training sessions include a large variety of training types, for instance, match play, running, and fitness training (##REF##28640152##Colby et al., 2017##).</p>", "<p>Considering the relationship between regular soccer training (RT) and high-intensity plyometric training (HIPT) in the study, research show that there are links between high- intensity plyometric training and speed-jump performance. There are also limited number of studies that investigate this relationship. The problem statement of our research is whether high-intensity plyometric training makes a difference compared to regular training in the selected parameters. Our hypothesis is that high-intensity training makes a difference compared to regular training. Therefore, the aim of the study was to examine the effect of high-intensity plyometric training on some parameters in elite male soccer players in the U17.</p>" ]
[ "<title>Method</title>", "<title>Participants</title>", "<p>A total of 28 male soccer players playing in the Elite Academy (U17) league of the Youth Development League of a team in the 1st league of Turkey participated in the study voluntarily (training experience; playing soccer in the elite academy team for at least 4 years). Each player spent ∼7 h in training plus one official match per week, and the team competed at the highest level for this age group. Each group consisted of five defenders, five midfielders and four attackers. The participation rate from the training of the RT and HIPT were 92.6% and 94.5% respectively.</p>", "<p>In ##TAB##0##Table 1##, it can be seen that the mean age of the soccer players in the RT group was 16.71 ± 0.47 years, the mean stature was 163.46 ± 3.31 cm, and the mean body weight was 61.04 ± 1.59 kg. Furthermore, the mean age of the HIPT group was 16.64 ± 0.50 years, the mean stature was 165.60 ± 3.03 cm, and the mean body weight was 59.76 ± 1.92 kg.</p>", "<title>Study design</title>", "<p>In the study, randomized parallel matched-group design was adopted, and 28 volunteers were divided into two groups each including equal numbers: a regular training group (RT) and a high-intensity plyometric c training group (HIPT). The participants from the same team were randomly assigned to the groups. This study was conducted during the pre-season period of 2022–2023. It lasted ten weeks and consisted of a week of pre-testing, eight weeks of training interventions, and a week of post-testing (##TAB##1##Table 2##).</p>", "<p>Data collection occured from June 2022–August 2022. For context, the study began after one weeks of pre-season commencement. The training intervention lasted eight weeks and we present the study’s timeline in ##FIG##0##Fig. 1##. Players were assessed 2 times over the period. Before each assessment, 48 h of rest were guaranteed regarding the last training session/match. The assessments were always performed on the same day of the week (Monday–Tuesday–Wednesday). The assessments started at 10 a.m.–12 p.m. The first assessment’s average temperature and relative humidity were 27 °C and 2%, respectively. The second assessment’s average temperature and relative humidity were 33 °C and 5%, respectively. Assessments occurred in full sun (##REF##37340051##Nobari et al., 2023##).</p>", "<p>As well as the regular training all the participants did, HIPT group also performed training interventions three days a week except for the first and last week. After completing the interventions, they did regular soccer training during the 8 week period. The regular training lasted 90 min, and the training intervention session lasted approximately 20–25 min. All participants and their parents were informed about the procedures, requirements, benefits, and risks of the study beforehand and written consent was obtained before the study started. Moreover, this study was approved (approval code: E-37077861-200-38769) by the Iǧdır University Ethics Committee (2021/21) and was conducted under the ethical guidelines of the Declaration of Helsinki for studying humans. In this study, we have followed all the Helsinki guidelines at all stages for human studies.</p>", "<title>Detailed schedule of training interventions</title>", "<title>Context of training intervention</title>", "<p>The preseason consisted of twenty four sessions per week for 8 weeks. Microcycles of weekly training are presented in ##TAB##1##Table 2##. In the preseason, most training sessions included (in this order): submaximal aerobic training (65–75% HRmax), interval runs (80–95% HRmax), high/ medium interval training. Also, the intensity of exercise was selected in the range between 70 to 95%) and strength training. The strength training performed in the pre-season was circuit based and was the same for all players. In addition to regular training, players participated in twenty four HIPT during the preseason. During the season, participants trained five times per week, along with tactical one match. ##TAB##1##Table 2## and ##FIG##0##Fig. 1## shows the details of each training session during the in pre-season period and the scheduling of the research protocol within the training schedule.</p>", "<p>Consequently, all training sessions were held on natural grass with stock shoes and soccer training uniforms. The pattern of formation of the names of movements is by the study of ##UREF##11##Loturco et al. (2020)##, but in this study, the intensity, rest time, the number of repetitions and duration of training intervention vary. Movement intensity, volume and speed were considered the same during the research period.</p>", "<p>##FIG##0##Figure 1## shows the detailed schedule of the training interventions which were performed in a 8 weeks period, the participants had five training sessions each week, and in three of these sessions they also performed the training interventions. These sessions were on Mondays, Wednesdays, and Fridays. First, the participants started the training with a standard warm up targeting (##FIG##1##Fig. 2##). The HIPT group performed a change of direction and sprint training (three sets of three short shuttle runs with four change of direction each, for an amount of thirty change of direction) and 4 × 5 drop jumps over 60 cm height followed by a subsequent jump over two obstacles (15 cm height), 4 × 6 horizontal jumps, as well as 4 × 6 jumps over 15 cm obstacles.</p>", "<title>Specific warm up protocol</title>", "<p>The players underwent a 20-min warm up session led by a licensed strength and conditioning coach involving stretching, the classical warm-up procedure applied in the study includes 5 minute-jogging with 40–50% of the maximum heart rate, followed by five frontal-lateral hopping, five mobilization, five upper extremity static stretching exercises and familiarization with the test, respectively (##FIG##1##Fig. 2##). The movements were applied for two sets and 15 s.</p>", "<p> In addition, athletes did a standard warm-up focusing on lower limbs which consisted of self- paced low-intensity running, lower-limb dynamic stretching, and reactive strength exercises before the tests.</p>", "<title>Data collection methods</title>", "<title>Body weight</title>", "<p>Body weight measurements of the participants were obtained in kg using the Direct Segmental Multi-frequency–Bioelectrical Impedance Analysis Method (DSM-BIA) (Inbody 270; Biospace, Cerritos, CA, USA), with the electronic scale integrated into the Bioelectrical impedance analyzer, while they were barefoot and in anatomical posture wearing only shorts and a T-shirt (##REF##14618471##Lukaski, 2003##).</p>", "<title>Stature</title>", "<p>Body height of the participants was obtained in cm with a wall-mounted stadiometer (Holtain Ltd, Crymych, UK) while they were in anatomical posture, bare feet, heels together, holding their breath, head in the frontal plane, with the headboard touching the vertex point.</p>", "<title>Sprint tests</title>", "<p>The <italic toggle=\"yes\">10 m-30 m-40 m</italic> straight sprint tests were performed by the participants, and the performance was measured using the Fusion SmartSpeed Timing Gates System (Fusion Sport, Coopers Plains, QLD, Australia) connected to a wireless computer. The system is used to measure the sprint time and consists of gates, each of which has a photocell with an infrared transmitter and a light reflector, and a radio frequency identification reader to identify the athlete.</p>", "<p>There were eight gates in 40-meter run over a straight-line test. The distance between the photocell and the light reflector was 2 m, and the gates were placed at 10 m, 30 m, and 40 m away from the start line, and the 40 m gate indicated the finish line. When the athletes crossed these gates, with the help of the IR beam their times were recorded. The athletes started from a standing position after the signal was given (##UREF##13##Sporiš et al., 2011##).</p>", "<title>Standing long jump test</title>", "<p>In the standing long jump (SLJ) test, athletes jumped horizontally from a standing still position and tried to move their body forward as far as possible. The total jump distance from the take-off line to the mark made on landing by the heel of the athlete was recorded in cm with a tape measure. The atheltes were not allowed to take steps backward or perform preparatory hops/runs (##REF##30208780##Sgrò et al., 2017##).</p>", "<title>Triple hop test</title>", "<p>Three triple hops were performed by the participants on artificial turf, and they did the test for each leg and were allowed to swing their arms during. The mean of each test score for each leg was calculated and recorded for data analysis. The sideline of a soccer pitch used for the test, and each yard of a testing area was outlined by paint. The athletes were instructed to put their foot behind the start line and hop forwards three times as far and quickly as possible to decrease contact time with ground (GCT). On the third and the final hop, they were asked to stay in that position for 3 s. When they could not ‘stick’, the attempt was declared void, and the athlete was asked to repeat the jumps after a 90 s rest. There was a 90 s break between each trial, and the trials were performed alternating the leg (<italic toggle=\"yes\">i.e.,</italic> 1st trial = left leg, 2nd trial = right leg, 3rd trial = left leg and so on) (##REF##33010748##Lloyd et al., 2020##).</p>", "<title>505 Change-of-direction speed test</title>", "<p>The methodology used for the 505 change-of-direction speed test (##FIG##2##Fig. 3##) was used as per established methods, with one timing gate (Fusion Sports, Coopers Plain, QLD, Australia) positioned to record time. The modified 5-0-5 test protocol was employed to measure the players’ COD time and COD deficit. The test consists of starting in a standing position (foot split) and accelerating over a 10-m distance before performing two COD of 180° (from A to C point was 5+5 m). The time from the final 10-m (5+5 m) is recorded using two pairs of photocells (Fusion Sports, Coopers Plain, QLD, Australia). The photocell height was adjusted based on the height of the player’s hip. The players were allowed to use the preferred leg for braking and turning movements. However, they were always asked to use the same leg. The same instruction was used for the foot in front at the starting position. Each participant performed two trials, with a rest period of three minutes. The COD time (s) was obtained for each trial. The smallest time was used for further statistical procedures (##REF##33138002##Nobari et al., 2020##; ##REF##26982972##Nimphius et al., 2016##).</p>", "<title>Test schedule</title>", "<p>Pre-test and post-test measurements were completed on the first and the last week from Monday to Wednesday. In the pre-test in pre-season, age, stature, body weight measurements were taken in the morning at 10:00. On the second day at 10:00 in the morning, long jump test and triple hop test were conducted for right and left limbs separately. On the third day at 10:00 am, the athletes performed sprint and change of direction (COD) tests. The same procedure was also completed on the last week. Before the tests started, the participants were asked to not to change their usual dietary intake on the assessment days, and they were informed about the test procedures and divided into two groups (##FIG##3##Fig. 4##).</p>", "<title>Research model</title>", "<p>Quasi-experimental design was used in the study. Since the process of determining unbiased sampling is generally difficult in sports and health fields, the quasi-experimental design, which is considered within experimental designs, is more preferred (##UREF##12##McMillan &amp; Schumacher, 2010##). The main difference of the quasi-experimental design from the full experimental design is that the sample is not randomly determined in the quasi-experimental design (##UREF##4##Çoban, 2017##).</p>", "<title>Data analysis</title>", "<p>Whether the existing data are normally distributed was determined by applying skewness and kurtosis tests. Skewness and kurtosis values between +3 and −3 are generally accepted for normal distribution (##UREF##8##Kalaycı, 2009##). Since the skewness (+1,31 to −1,29) and kurtosis (+2,35 to −1,32) values of all analyzed measurements were within the above ranges, It was accepted to be normally distributed. For the purposes of the study, the Mixed Measure Two-Way ANOVA Test was used to determine the difference between the pre-test and post-test. In the statistical analysis of the data obtained in the study, the margin of error was taken as <italic toggle=\"yes\">p</italic> &lt; .05. For the purposes of the study, the mixed measure two-way ANOVA test was used to determine the difference between the pre-test and post-test. In the statistical analysis of the data obtained in the study, the margin of error was taken as <italic toggle=\"yes\">p</italic> &lt; .05.</p>" ]
[ "<title>Results</title>", "<p>In this section, after giving the descriptive information (##TAB##2##Table 3##) of the athletes participating in our study, statistical results regarding the parameters 10 m sprint (sec) (##TAB##3##Table 4##), 30 m sprint (sec) (##TAB##4##Table 5##), 40 m sprint (sec) (##TAB##5##Table 6##), long jump (cm) (##TAB##6##Table 7##), triplehop right (m) (##TAB##7##Table 8##), triplehop left (m) (##TAB##8##Table 9##), 505 cod test (sec) (##TAB##9##Table 10##) scores are given.</p>", "<p>It can be seen in ##TAB##2##Table 3## that there is a statistically significant difference between the groups in the 40 m sprint (sec) and long jump (cm) parameters (<italic toggle=\"yes\">p</italic> &lt; 0.05). There is no statistically significant difference in other parameters (<italic toggle=\"yes\">p</italic> &gt; 0.05). When looking at the improvement rates, there is a significant improvement (15%) in the HIPT group compared to the RT group in the long jump parameter. There is also a significant improvement in the 40m sprint parameter in the RT group compared to the HIPT group (5%, 9% respectively).</p>", "<p>In ##TAB##3##Table 4##, a significant difference is found between the athletes in the RT (football training) and HIPT (high-intensity plyometric training) groups in terms of their 10-metre running performance scores [F(1. 26) = 37.72, <italic toggle=\"yes\">p</italic> &lt; .05]. It was determined that the difference between the pre-test and post-test 10-metre running performance scores of the athletes included in the study was significant [F(1. 26) = 226.26, <italic toggle=\"yes\">p</italic> &lt; .05]. As seen in the table, it was determined that the measurement*group joint effect of the test results of the athletes’ 10-metre running performance scores was not significant [F(1. 26) = .093, <italic toggle=\"yes\">p</italic> &gt; .05].</p>", "<p>A significant difference is found in ##TAB##4##Table 5## between the athletes in the RT and HIPT groups in terms of the score they received from the 30 m sprint (sec) performance [F(1. 26) = 8.74, p&lt;.05]. It was determined that the difference between the pre-test and post-test 30 m sprint (sec) run performance scores of the athletes included in the study was significant [F(1. 26) = 66.60, p&lt;.05]. This result shows that the sprint 30 m sprint (sec) performance scores of the athletes increased in the process. As seen in the table, it was determined that the measurement*group joint effect of the test results of the athletes’ 30 m sprint (sec).</p>", "<p>A significant difference is found in ##TAB##5##Table 6## between the athletes in the RT and HIPT groups in terms of their 40 m sprint (sec) performance scores [F(1. 26) = 59.12, <italic toggle=\"yes\">p</italic> &lt; .05]. It was determined that the difference between the pre-test and post-test 40 m sprint (sec) performance scores of the athletes included in the study was significant [F(1. 26) = 176.28, <italic toggle=\"yes\">p</italic> &lt; .05]. This result shows that the 40 m sprint (sec) performance scores of the athletes increased in the process. As seen in the table, it was determined that the measurement*group joint effect of the test results of the athletes’ forty metre run performance scores was significant [F(1. 26) = 18.61, <italic toggle=\"yes\">p</italic> &lt; .05]. Therefore, it is seen that the training applied to the athletes has an effect on the 40 m sprint (sec) performance scores of the athletes.</p>", "<p>In ##TAB##6##Table 7##, no significant difference is found between the athletes in the RT and HIPT groups in terms of their long jump (cm) performance scores [F(1. 26) = .520, <italic toggle=\"yes\">p</italic> &gt; .05]. It was determined that the difference between the pre-test and post-test long jump (cm) performance scores of the athletes included in the study was significant [F(1. 26) = 195.97, <italic toggle=\"yes\">p</italic> &lt; .05]. This result shows that the long jump (cm) performance scores of the athletes increased in the process. As seen in the table, it was determined that the measurement*group joint effect of the test results of the long jump (cm) performance scores of the athletes was significant [F(1. 26) = 84.94, <italic toggle=\"yes\">p</italic> &lt; .05]. Therefore, it is seen that the training applied to the athletes has an effect on the long jump (cm) performance scores of the athletes.</p>", "<p>##TAB##7##Table 8## shows a significant difference was found between the athletes in the RT and HIPT groups in terms of their triplehop right (m) performance scores [F(1. 26) = 22.12, <italic toggle=\"yes\">p</italic> &lt; .05]. It was determined that the difference between the pre-test and post-test triplehop right (m) metre running performance scores of the athletes included in the study was significant [F(1. 26) = 70.67, <italic toggle=\"yes\">p</italic> &lt; .05]. This result shows that the triplehop right (m) performance scores of the athletes increased in the process. As seen in the table, it was determined that the measurement*group joint effect of the test results of the triplehop right (m) running performance scores of the athletes was not significant [F(1. 26) = 2.03, <italic toggle=\"yes\">p</italic> &gt; .05]. Therefore, it is seen that the training applied to the athletes had no effect on the triplehop right (m) performance scores of the athletes.</p>", "<p>In ##TAB##8##Table 9##, a significant difference was found between the athletes in the RT and HIPT groups in terms of their triplehop left (m) performance scores [F(1. 26) = 56.64, <italic toggle=\"yes\">p</italic> &lt; .05]. It was determined that the difference between the pre-test and post-test triplehop left (m) metre running performance scores of the athletes included in the study was significant [F(1. 26) = 78.82, <italic toggle=\"yes\">p</italic> &lt; .05]. This result shows that the triplehop left (m) performance scores of the athletes increased in the process. As seen in the table, it was determined that the measurement*group joint effect of the test results of the triplehop left (m) running performance scores of the athletes was not significant [F(1. 26) = 6.87, <italic toggle=\"yes\">p</italic> &gt; .05]. Therefore, it is seen that the training applied to the athletes had no effect on the triplehop left (m) performance scores of the athletes.</p>", "<p>In ##TAB##9##Table 10##, a significant difference was found between the athletes in the RT and HIPT groups in terms of the score they received from the 505 COD test (sec) performance [F(1. 26) = 24.66, p&lt;.05]. It was determined that the difference between the pre-test and post-test 505 COD test (sec) running performance scores of the athletes included in the study was significant [F(1. 26) = 193.53, p&lt;.05]. This result shows that the athletes’ 505 COD test (sec) performance scores increased in the process. As seen in the table, it was determined that the measurement*group joint effect of the test results of the athletes’ 505 COD test (sec) performance scores was not significant [F(1. 26) = .620, <italic toggle=\"yes\">p</italic> &gt; .05]. Therefore, it is seen that the training applied to the athletes has no effect on the 505 COD test (sec) performance scores of the athletes.</p>" ]
[ "<title>Discussion</title>", "<p>It is known that athletic performance measurements and different training models are important factors for youth soccer players in elite level who are in their development stages to achieve success. This study was conducted to reveal the effect of 8-week high-intensity plyometric training on sprint, jump and change of direction parameters in young male (U17 Elite A-league) soccer players. The results indicated that this training provided more improvement in measured performance parameters than routine soccer training training (##TAB##2##Tables 3##–##TAB##9##10##). This shows that high-intensity plyometric training can be included in the necessary training units as a specific training unit, starting from the lower age groups, taking into account the developmental stages of children. To collect data for the study, pre-test and post-test design was used to measure the parameters, and the results were discussed taking the literature into account.</p>", "<title>Sprint performance</title>", "<p>Sprinting ability is accepted as the main determining factor in sportive performance in soccer. When we analyze soccer matches, the ability of continually sprint and do so by changing direction is considered as a determinant of high-level performance for coaches and researchers, especially in team sports such as soccer. Moreover, this is also accepted as an indicator of the fitness level of the athletes (##REF##22067248##Wong, Chan &amp; Smith, 2012a##).</p>", "<p>According to the results obtained from the study, when the pre-test and post-test values between the high-intensity plyometric training group and the regular training group were examined, it was determined that there was a significant difference between all sprint values except for the 30 m sprint value of the post-test. When the mean values are compared, it is shown that the post-test values of the high-intensity plyometric training group were better. In addition, similar improvement rate was observed in the 10m sprint performance (6%; 6%), while RT showed greater improvement in the 30 m (10%; 8%) and 40 m (9%; 5%) sprint performances (##TAB##2##Tables 3##–##TAB##5##6##).</p>", "<p>##UREF##3##Boraczyński &amp; Urniaz (2008)## examined the effect of plyometric training for 8 weeks on strength and speed parameters and observed a positive increase in the leg strength and speed characteristics of the athletes. ##REF##26252503##Fernandez-Fernandez et al. (2016)## reported that 8-week plyometric training with young tennis players had a positive and significant improvement in the speed characteristics. ##REF##26390150##Loturco et al. (2015)## stated that there was an improvement and statistical difference between the 30 m pre- and post-test values of 15 athletes who participated only in plyometric training for 8 weeks. In a study in which mixed method adopted, it was seen that plyometric exercises combined with maximal strength and high-intensity resistance training improved the sprint performance of athletes (##REF##19897415##Villarreal, Requena &amp; Newton, 2010##). Furthermore, ##REF##19910813##Meylan &amp; Malatesta (2009)## concluded that 6-week plyometric exercises improve 30 m sprint performance of soccer players. ##REF##29611771##Asadi et al. (2018)## also stated that plyometric training should be applied together with other training programs in order to improve sprint speed. ##REF##22510799##Wong et al. (2012b)## stated that plyometric training combined with weight training significantly increased the speed of athletes. In their study ##REF##11533565##Diallo et al. (2001)## reported that sprint performance significantly increased in the training group using a sprint bike compared to the control group. As can be seen from the above study results, which are similar to our study findings, 6–8 weeks of plyometric training has positive effects on the sprint parameter.</p>", "<p>The results showed that the increase in the values of the high-intensity training group affects the improvement of the speed characteristics of plyometric soccer training. Considering that the speed characteristics is the least improvable feature among the biomotor abilities that can be improved, it can be accepted that developing strength with the help of plyometric training makes a positive contribution to the existing speed potential. Furthermore, it can be said that plyometric training can improve short-distance sprint speed.</p>", "<title>Long jump performance</title>", "<p>The standing long jump is a test that requires the involvement of multiple joints and is widely used to evaluate the explosive power of the leg muscles (##REF##21834392##Moresi et al., 2011##). In this study, explosive leg strength of young soccer players was evaluated with dominant, non-dominant, and two-footed standing long jump tests.</p>", "<p>The results of this study present that while there is a statistically significant difference in the pre-test values between the groups, there is no difference in the post-test values (##TAB##6##Table 7##). When the mean values are compared, it is seen that the HIPT post-test measurement values were better. In addition, HIPT showed much more improvement than RT (3% and 15% respectively) (##TAB##4##Table 5##). When the literature related to this parameter is examined, in a study in which 6-week mixed training program was applied to the soccer players during in-season period, no significant difference was found in terms of the standing long jump values (##REF##28566807##Chtara et al., 2017##). There are studies with similar results regarding the results we obtained, and in a study comparing individual athletes and team, no significant difference was found in standing long jump values (##UREF##9##Koç &amp; Aslan, 2010##). ##REF##27446242##Wang &amp; Zhang (2016)## obtained similar results to our research in their study on plyometric training in elite soccer players.</p>", "<p>Moreover, it is thought that high-intensity plyometric training led to an increase in jump distance in soccer players in this study. Many studies have used at least one jump test to determine the effects of plyometric training on athletic performance. For example, in the study conducted by ##REF##20224449##Buchheit et al. (2010)##, a statistically significant difference was observed in counter movement jumping (CMJ) in the plyometric training group compared to the sprint training group. Similarly, ##REF##19910813##Meylan &amp; Malatesta (2009)## found that there was a statistically significant improvement in the CMJ training group compared to the control group. ##REF##22450257##Michailidis et al. (2013)## stated that when compared to the control group, there was a significant improvement in tests such as SJ, CMJ, and depth jump (DJ) in the training group, and ##REF##19966586##Rubley et al. (2011)## reported that the training group improved significantly in vertical jump (VJ) performance compared to the control group. ##REF##19002073##Thomas, French &amp; Hayes (2009)## examined the effects of both DJ and CMJ plyometrics training on strength and agility in young soccer players and reported that both training groups showed significant improvement in CMJ.</p>", "<p>##REF##20844458##Chelly et al. (2010)## determined that an 8-week plyometric training period improved the standing long jump distance in the experimental group consisting of active healthy individuals compared to the control group. Furthermore, it was stated that compared to the control group, a 10-week plyometric training had a 2.8% increase in standing long jump distance performance in the experimental group, which included active healthy individuals (##REF##17530960##Markovic et al., 2007##). In our study, when the mean values are compared, it is shown that the post-test values were better and showed an increase, and the results obtained correlated with the literature. According to the results obtained from this research, it was determined that high- intensity soccer training improved the standing long jump performance compared to routine training. In addition, the fact that both research groups were in the developmental age may have ensured that there was no significant difference in the post-test values between the groups.</p>", "<title>Triple hop (right and left) performance</title>", "<p>The triple jump test requires a combination of muscle strength, power, and balance (##REF##1962720##Noyes, Barber &amp; Mangine, 1991##). This test reflects the lower extremity muscle strength and power of soccer players (##REF##18345338##Hamilton et al., 2008##).</p>", "<p>According to the results obtained from the study, there is a statistically significant difference between the groups in term of the pre-test and the post-test values (##TAB##7##Tables 8## and ##TAB##8##9##). When the mean values are compared, it is shown that the RT post-test values were better. However, HIPT showed much more improvement than RT (right: 4% and 1%; left: 4% and 2%, respectively) (##TAB##4##Table 5##). ##REF##24852255##Ozbar, Ates &amp; Agopyan (2014)## found that in female athletes in the plyometric training (PT) group, dominant leg increased by 12.1%, the non-dominant leg increased by 15.7%, and in the control group, the dominant leg increased by 4.3% and the non-dominant leg increased by 6.6% in terms of the triple hop for distance (THD) performance. This situation reveals the effect of PT from different perspectives. It was observed that PT made a greater difference in the jump distance of the non-dominant leg compared to the dominant leg, especially in the THD test. It is reported that this may be related to the significant increase in performance in all jump parameters and the common technical and conditional exercises performed on other days in the control group (##REF##24852255##Ozbar, Ates &amp; Agopyan, 2014##). In this study, which is similar to our results, the higher percentage of development in this study may be due to gender. Furthermore, inter-group analysis after a 12- week plyometric training of professional female soccer players by ##REF##32519832##Nonnato et al. (2022)## reported a positive change in terms of the triple jump test results of dominant limb (<italic toggle=\"yes\">p</italic> = 0.031; medium effect size) and non-dominant limb (<italic toggle=\"yes\">p</italic> = 0.021; medium effect size). ##REF##29176387##Beato et al. (2018)## reported that there were improvements between the groups in the triple jump test results after 6 weeks of plyometric training, but there was no significant correlation. According to the results obtained in this study, it is shown that there was no correlation with the literature in terms of this parameter. In addition, it is seen that further studies are needed to be conducted to eliminate these contradictions and to determine the effects of plyometric training on triple jump performance.</p>", "<title>505 COD performance</title>", "<p>Agility, which is defined as a rapid change in the speed and direction of movement in response to an external stimulus (##REF##30189647##Tabacchi et al., 2018##), is a concept based athletic performance and the change in decision-perception and frontal speed in soccer and it is affected by these factors when the quality of a player is evaluated (##REF##16882626##Sheppard &amp; Young, 2006##). Change-of-direction time (##FIG##2##Fig. 3##), namely the 5-0-5 test time, is nearly perfectly correlated with linear acceleration in small distances such as 5-, 10-m, and 20-m (##REF##37340051##Nobari et al., 2023##).</p>", "<p>According to the results obtained from this study, there was a statistically significant difference between the groups in terms of the pre-test and the post-test values (##TAB##9##Table 10##). When the mean values are compared, it is seen that the HIPT post-test measurement values were better. In addition, HIPT and RT showed similar improvement rates (6% and 5%, respectively) (##TAB##2##Table 3##).</p>", "<p>Similar to our study results, in a study conducted with different age groups, it was stated that there was a significant difference in favor of the experimental group in the mean values of agility in the pre- and post-tests (##UREF##1##Atacan, 2010##). In another study conducted with 15–16 year-old soccer players, it was determined that the post-test mean values of agility were better in favor of the experimental group (##UREF##7##Hazır, Mahir &amp; Açıkada, 2010##). In addition, in a study similar to the results of our research, they found that after particularly arranged plyometric training, agility post-test values increased in favor of the experimental group compared to the control group (##REF##22531616##Pienaar &amp; Coetzee, 2013##). In a study conducted with soccer players playing in development leagues, it was determined that there was a significant increase in the agility values of male soccer players who participated in plyometric training during the preparation period (##REF##33592641##Padrón-Cabo et al., 2021##). In their study, ##UREF##14##William &amp; Kirubakar (2018)## examined the relationship between the plyometric and agility values of soccer players also obtained similar results with our study.</p>", "<p>Taking these results into consideration, it is thought that the increase in agility performance of the high-intensity plyometric training group compared to the regular training group is due to the application of plyometric training in different combinations together with soccer training. However, it can be said that the positive improvement in the regular training group and the soccer training program have a positive effect on the selected parameter.</p>", "<p>The present study contains limitations that need to be taken into account. First, the food consumption records and hydration levels of the participants were not monitored throughout the 10-week study period. To address this limitation, a 24-hour retrospective food record could have been employed to track the participants’ food consumption for two weekdays and one day on the weekend, spanning a total of three days. This would have allowed for the calculation of their energy intake. Another limitation of our study is that it was exclusively conducted with young male athlete participants. This narrow focus on young male athlete participants makes it challenging to generalize the results, particularly in relation to the adult male athlete population. Future research should consider including both male and female participants to obtain a more comprehensive understanding of the findings and their applicability to a broader population.</p>" ]
[ "<title>Conclusions</title>", "<p>Jumping and speed are directly linked with leg and hip strength, and various methods have been developed to improve particularly leg strength. The most commonly used one of these is the plyometric training (##REF##28182510##Chaabene &amp; Negra, 2017##). When plyometric training is performed regularly and correctly, it contributes to an increase in performance in sports branches (<italic toggle=\"yes\">i.e.,</italic> soccer, handball, volleyball) where jump and speed parameters are very important (##UREF##0##Aksović et al., 2021##). In addition, it is thought that the studies examining the effect of plyometric training designed for lower and upper extremities using various techniques or on different floors/surfaces can provide new information to sports sciences (sand, land, water) on biomotor abilities in different age groups. In addition, the relationship between body composition and jump in young male soccer players is an area open to research.</p>", "<p>As a result, when the data obtained is examined, it can be said that HIPT had better values in sprint, jump, and change of direction parameters, so plyometric training was more beneficial for anaerobic parameters than routine soccer training in children. It is recommended that the trainers make their plans considering these results.</p>" ]
[ "<p>The aim of this study was to examine the effects of high-intensity plyometric training (HIPT) on some parameters in elite soccer players in the U17. Volunteer soccer players were randomly divided into two groups according to their positions: the regular training (RT) group (age: 16.71 ± 0.47 years; stature: 163.46 ± 3.31 cm; body weight: 61.04 ± 1.59 kg) and the HIPT group (age: 16.64 ± 0.50 years; stature: 165.60 ± 3.03 cm; body weight: 59.76 ± 1.92 kg), and each group included five defenders, five midfielders, and four attackers. While the RT group did only routine soccer training, the HIPT group performed high-intensity plyometric training three days a week in addition to routine soccer training. In the study, body weight, stature, sprint (10 m, 30 m, and 40 m), jump (standing long jump, right and left-limb triple hop) and 5-0-5 change of direction speed test measurements of the soccer players were taken. Skewness and Kurtosis values obtained from the pre-test and post-test measurements were calculated to test whether the normality assumption of the study was met. A mixed measure two-way ANOVA test was used to determine the difference between the pre-test and post-test. The significance was set as <italic toggle=\"yes\">p</italic> &lt; 0.05. The results indicated that 8-week high- intensity plyometric training provided more improvement in measured performance parameters than routine soccer training. As a result, when the data obtained is examined, it can be said that HIPT had better values in sprint, jump, and change of direction parameters, so plyometric training was more beneficial for anaerobic parameters than routine soccer training in children. It is recommended that the trainers make their plans considering these results.</p>" ]
[ "<title>Supplemental Information</title>" ]
[ "<p>We would like to thank all the athletes and coaching staff for their support during the study measurements.</p>", "<title>Additional Information and Declarations</title>" ]
[ "<fig position=\"float\" id=\"fig-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/fig-1</object-id><label>Figure 1</label><caption><title>A detailed weekly training schedule.</title></caption></fig>", "<fig position=\"float\" id=\"fig-2\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/fig-2</object-id><label>Figure 2</label><caption><title>Spesific warm up protocol.</title></caption></fig>", "<fig position=\"float\" id=\"fig-3\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/fig-3</object-id><label>Figure 3</label><caption><title>Change of direction speed test.</title></caption></fig>", "<fig position=\"float\" id=\"fig-4\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/fig-4</object-id><label>Figure 4</label><caption><title>Test schedule.</title></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"table-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-1</object-id><label>Table 1</label><caption><title>Descriptive characteristics of the participants.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">\n<bold>Variables</bold>\n</th><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\">\n<bold>n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold> x¯</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"2\" colspan=\"1\">\n<bold>Age (year)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT Group</td><td rowspan=\"1\" colspan=\"1\">14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16.71</td><td rowspan=\"1\" colspan=\"1\">0.47</td></tr><tr><td rowspan=\"1\" colspan=\"1\">HIPT Group</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">16.64</td><td rowspan=\"1\" colspan=\"1\">0.50</td></tr><tr><td rowspan=\"2\" colspan=\"1\">\n<bold>Stature (cm)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT Group</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">163.46</td><td rowspan=\"1\" colspan=\"1\">3.31</td></tr><tr><td rowspan=\"1\" colspan=\"1\">HIPT Group</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">165.60</td><td rowspan=\"1\" colspan=\"1\">3.03</td></tr><tr><td rowspan=\"2\" colspan=\"1\">\n<bold>Body weight (kg)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT Group</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">61.04</td><td rowspan=\"1\" colspan=\"1\">1.59</td></tr><tr><td rowspan=\"1\" colspan=\"1\">HIPT Group</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">59.76</td><td rowspan=\"1\" colspan=\"1\">1.92</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-2\" orientation=\"landscape\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-2</object-id><label>Table 2</label><caption><title>Weekly training schedule.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">\n<bold>Days</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>1st week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>2nd week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>3rd week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>4th week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>5th week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>6th week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>7th week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>8th week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>9th week</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>10th week</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Monday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Pre-test data collection</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">Post-test data collection</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Tuesday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Pre-test data collection</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">Post-test data collection</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Wednesday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Pre-test data collection</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">Cancelled training</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">Post-test data collection</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Thursday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">ST</td><td rowspan=\"1\" colspan=\"1\">Resting day</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Friday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">ST + HIPT</td><td rowspan=\"1\" colspan=\"1\">Resting day</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Saturday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Sunday</bold>\n</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Day off</td><td rowspan=\"1\" colspan=\"1\">Friendly match</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-3\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-3</object-id><label>Table 3</label><caption><title>Mean and standard deviation values of the measurement scores of the athletes participating in the study according to groups.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">\n<bold>Variables</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Groups</bold>\n</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Pre-Test</bold>\n</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Post test</bold>\n</th><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\">\n<bold>Improvement rate %</bold>\n</th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\">\n<bold> x¯</bold>\n</th><th rowspan=\"1\" colspan=\"1\">sd</th><th rowspan=\"1\" colspan=\"1\">\n<bold> x¯</bold>\n</th><th rowspan=\"1\" colspan=\"1\">sd</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Sprint 10 m (sec)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">1.87</td><td rowspan=\"1\" colspan=\"1\">0.041</td><td rowspan=\"1\" colspan=\"1\">1.77</td><td rowspan=\"1\" colspan=\"1\">0.049</td><td rowspan=\"1\" colspan=\"1\">0.763</td><td rowspan=\"1\" colspan=\"1\">66</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">1.79</td><td rowspan=\"1\" colspan=\"1\">0.031</td><td rowspan=\"1\" colspan=\"1\">1.69</td><td rowspan=\"1\" colspan=\"1\">0.031</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Sprint 30 m (sec)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">4.77</td><td rowspan=\"1\" colspan=\"1\">0.250</td><td rowspan=\"1\" colspan=\"1\">4.31</td><td rowspan=\"1\" colspan=\"1\">0.292</td><td rowspan=\"1\" colspan=\"1\">0.206</td><td rowspan=\"1\" colspan=\"1\">108</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">4.47</td><td rowspan=\"1\" colspan=\"1\">0.246</td><td rowspan=\"1\" colspan=\"1\">4.13</td><td rowspan=\"1\" colspan=\"1\">0.192</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Sprint 40 m (sec)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">5.89</td><td rowspan=\"1\" colspan=\"1\">0.147</td><td rowspan=\"1\" colspan=\"1\">5.36</td><td rowspan=\"1\" colspan=\"1\">0.178</td><td rowspan=\"1\" colspan=\"1\">0.000*</td><td rowspan=\"1\" colspan=\"1\">95</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">5.40</td><td rowspan=\"1\" colspan=\"1\">0.156</td><td rowspan=\"1\" colspan=\"1\">5.13</td><td rowspan=\"1\" colspan=\"1\">0.099</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Long jump (cm)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">2.30</td><td rowspan=\"1\" colspan=\"1\">0.167</td><td rowspan=\"1\" colspan=\"1\">2.36</td><td rowspan=\"1\" colspan=\"1\">0.190</td><td rowspan=\"1\" colspan=\"1\">0.000*</td><td rowspan=\"1\" colspan=\"1\">315</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">2.13</td><td rowspan=\"1\" colspan=\"1\">0.077</td><td rowspan=\"1\" colspan=\"1\">2.46</td><td rowspan=\"1\" colspan=\"1\">0.065</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Triple hop right</bold>\n<bold> (m)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">7.12</td><td rowspan=\"1\" colspan=\"1\">0.057</td><td rowspan=\"1\" colspan=\"1\">7.33</td><td rowspan=\"1\" colspan=\"1\">0.112</td><td rowspan=\"1\" colspan=\"1\">0.166</td><td rowspan=\"1\" colspan=\"1\">14</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">6.84</td><td rowspan=\"1\" colspan=\"1\">0.204</td><td rowspan=\"1\" colspan=\"1\">7.14</td><td rowspan=\"1\" colspan=\"1\">0.199</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Triple hop left</bold>\n<bold> (m)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">7.08</td><td rowspan=\"1\" colspan=\"1\">0.084</td><td rowspan=\"1\" colspan=\"1\">7.22</td><td rowspan=\"1\" colspan=\"1\">0.066</td><td rowspan=\"1\" colspan=\"1\">0.014</td><td rowspan=\"1\" colspan=\"1\">24</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">6.79</td><td rowspan=\"1\" colspan=\"1\">0.117</td><td rowspan=\"1\" colspan=\"1\">7.05</td><td rowspan=\"1\" colspan=\"1\">0.118</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>505 COD test (sec)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">RT</td><td rowspan=\"1\" colspan=\"1\">5.15</td><td rowspan=\"1\" colspan=\"1\">0.119</td><td rowspan=\"1\" colspan=\"1\">4.85</td><td rowspan=\"1\" colspan=\"1\">0.000</td><td rowspan=\"1\" colspan=\"1\">0.438</td><td rowspan=\"1\" colspan=\"1\">65</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">HIPT</td><td rowspan=\"1\" colspan=\"1\">4.98</td><td rowspan=\"1\" colspan=\"1\">0.087</td><td rowspan=\"1\" colspan=\"1\">4.71</td><td rowspan=\"1\" colspan=\"1\">0.125</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-4\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-4</object-id><label>Table 4</label><caption><title>ANOVA results of 10 m sprint (sec) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>Sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\"> 0.154</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">0.091</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.091</td><td rowspan=\"1\" colspan=\"1\">37.72</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-4fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.063</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.002</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">6.592</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">0.156</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.156</td><td rowspan=\"1\" colspan=\"1\">226.26</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-4fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">6.42</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">6.429</td><td rowspan=\"1\" colspan=\"1\">0.093</td><td rowspan=\"1\" colspan=\"1\">0.763</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.018</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.001</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-5\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-5</object-id><label>Table 5</label><caption><title>ANOVA results of sprint 30 m sprint (sec) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\">3.1</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">0.780</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.780</td><td rowspan=\"1\" colspan=\"1\">8.74</td><td rowspan=\"1\" colspan=\"1\">0.007<xref rid=\"table-5fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">2.32</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.089</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">3.21</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">2.26</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">2.26</td><td rowspan=\"1\" colspan=\"1\">66.60</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-5fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">0.057</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.057</td><td rowspan=\"1\" colspan=\"1\">1.68</td><td rowspan=\"1\" colspan=\"1\">0.206</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.885</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.34</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-6\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-6</object-id><label>Table 6</label><caption><title>ANOVA results of 40 m sprint (sec) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\">2.68</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">1.86</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">1.86</td><td rowspan=\"1\" colspan=\"1\">59.12</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-6fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.819</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.031</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">2.76</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">2.20</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">2.20</td><td rowspan=\"1\" colspan=\"1\">176.28</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-6fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">0.233</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.233</td><td rowspan=\"1\" colspan=\"1\">18.61</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-6fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.325</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.013</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-7\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-7</object-id><label>Table 7</label><caption><title>ANOVA results of long jump (cm) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\">0.912</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">0.018</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.018</td><td rowspan=\"1\" colspan=\"1\">0.520</td><td rowspan=\"1\" colspan=\"1\">0.477</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.894</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.034</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">0.883</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">0.564</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.564</td><td rowspan=\"1\" colspan=\"1\">195.97</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-7fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">0.244</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.244</td><td rowspan=\"1\" colspan=\"1\">84.94</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-7fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.075</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.003</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-8\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-8</object-id><label>Table 8</label><caption><title>ANOVA results of triplehop right (m) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\">1.70</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">0.785</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.785</td><td rowspan=\"1\" colspan=\"1\">22.12</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-8fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.923</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.035</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">1.31</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">0.939</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.939</td><td rowspan=\"1\" colspan=\"1\">70.67</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-8fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">0.027</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.027</td><td rowspan=\"1\" colspan=\"1\">2.03</td><td rowspan=\"1\" colspan=\"1\">0.166</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.345</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.013</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-9\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-9</object-id><label>Table 9</label><caption><title>ANOVA results of triplehop left (m) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\">1.07</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">0.734</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.734</td><td rowspan=\"1\" colspan=\"1\">56.64</td><td rowspan=\"3\" colspan=\"1\">0.000<xref rid=\"table-9fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.337</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.013</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">0.751</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">0.530</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.530</td><td rowspan=\"1\" colspan=\"1\">78.82</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-9fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">0.046</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.046</td><td rowspan=\"1\" colspan=\"1\">6.87</td><td rowspan=\"1\" colspan=\"1\">0.014</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.175</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.007</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap position=\"float\" id=\"table-10\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/table-10</object-id><label>Table 10</label><caption><title>ANOVA results of 505 cod test (sec) performance scores of the athletes participating in the study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Source of variance</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ss</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>sd</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>ms</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">f</italic>\n</bold>\n</th><th rowspan=\"1\" colspan=\"1\">\n<bold>\n<italic toggle=\"yes\">p</italic>\n</bold>\n</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Between groups</td><td rowspan=\"1\" colspan=\"1\">0.663</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">0.323</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.323</td><td rowspan=\"1\" colspan=\"1\">24.66</td><td rowspan=\"3\" colspan=\"1\">0.000<xref rid=\"table-10fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.340</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.013</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Within groups</td><td rowspan=\"1\" colspan=\"1\">0.751</td><td rowspan=\"1\" colspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement (PreTest-PostTest)</td><td rowspan=\"1\" colspan=\"1\">1.28</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">1.12</td><td rowspan=\"1\" colspan=\"1\">193.53</td><td rowspan=\"1\" colspan=\"1\">0.000<xref rid=\"table-10fn2\" ref-type=\"table-fn\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Measurement*Group</td><td rowspan=\"1\" colspan=\"1\">0.004</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0.004</td><td rowspan=\"1\" colspan=\"1\">0.620</td><td rowspan=\"1\" colspan=\"1\">0.438</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Error</td><td rowspan=\"1\" colspan=\"1\">0.152</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">0.006</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>" ]
[ "<inline-formula><alternatives><tex-math id=\"tex-ieqn-2\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym}\n\\usepackage{amsfonts}\n\\usepackage{amssymb}\n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n$\\mathrm{ı}$\\end{document}</tex-math><mml:math id=\"mml-ieqn-2\" overflow=\"scroll\"><mml:mi>ı</mml:mi></mml:math></alternatives></inline-formula>" ]
[]
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[]
[ "<supplementary-material id=\"supp-1\" position=\"float\" content-type=\"local-data\"><object-id pub-id-type=\"doi\">10.7717/peerj.16648/supp-1</object-id><label>Supplemental Information 1</label><caption><title>Data Files</title></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"table-1fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-1fn1\" fn-type=\"other\"><p>\n</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-2fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-2fn1\" fn-type=\"other\"><p>\n</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-3fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-3fn1\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-4fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-4fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-4fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-5fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-5fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-5fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-6fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-6fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-6fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-7fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-7fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-7fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-8fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-8fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-8fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-9fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-9fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-9fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"table-10fn\"><p>\n<bold>Notes.</bold>\n</p></fn><fn id=\"table-10fn1\" fn-type=\"other\"><p>\n</p></fn><fn id=\"table-10fn2\"><label>*</label><p><italic toggle=\"yes\">p</italic> &lt; .05.</p></fn></table-wrap-foot>", "<fn-group content-type=\"competing-interests\"><title>Competing Interests</title><fn id=\"conflict-1\" fn-type=\"COI-statement\"><p>The authors declare there are no competing interests.</p></fn></fn-group>", "<fn-group content-type=\"author-contributions\"><title>Author Contributions</title><fn id=\"contribution-1\" fn-type=\"con\"><p><xref rid=\"author-1\" ref-type=\"contrib\">Mehmet Söyler</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-2\" fn-type=\"con\"><p><xref rid=\"author-2\" ref-type=\"contrib\">Raif Zileli</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-3\" fn-type=\"con\"><p><xref rid=\"author-3\" ref-type=\"contrib\">Yunus Emre Çingöz</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-4\" fn-type=\"con\"><p><xref rid=\"author-4\" ref-type=\"contrib\">Gökmen Kılınçarslan</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-5\" fn-type=\"con\"><p><xref rid=\"author-5\" ref-type=\"contrib\">İdris Kayantaş</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-6\" fn-type=\"con\"><p><xref rid=\"author-6\" ref-type=\"contrib\">Tolga Altuğ</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-7\" fn-type=\"con\"><p><xref rid=\"author-7\" ref-type=\"contrib\">Selim Asan</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-8\" fn-type=\"con\"><p><xref rid=\"author-8\" ref-type=\"contrib\">Musa Şahin</xref> conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn id=\"contribution-9\" fn-type=\"con\"><p><xref rid=\"author-9\" ref-type=\"contrib\">Alper Cenk Gürkan</xref> conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn id=\"addinfo-1\"><p>The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):</p><p>In the meeting of Iğdır University Scientific Research and Publication Ethics Committee dated 07.07.2021 and numbered 2021/21, this study was carried out in accordance with the 10/1 of the Iğdır University Scientific Research and Publication Ethics Directive. It has been decided that it is in compliance with scientific research and publication ethics (Approval Code: E-37077861-200-38769).</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Data Availability</title><fn id=\"addinfo-2\"><p>The following information was supplied regarding data availability:</p><p>The raw data are available in the <xref rid=\"supplemental-information\" ref-type=\"sec\">Supplemental Files</xref>.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"peerj-12-16648-s001.xlsx\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["Aksovi\u0107 et\u00a0al. (2021)"], "person-group": ["\n"], "surname": ["Aksovi\u0107", "Bjelica", "Milanovi\u0107", "Jovanovi\u0107", "Zelenovi\u0107"], "given-names": ["N", "B", "F", "N", "M"], "year": ["2021"], "article-title": ["Plyometric training effects on explosive power, sprint and direction change speed in basketball: a review"], "source": ["Turkish Journal of Kinesiology"], "volume": ["7"], "issue": ["2"], "fpage": ["73"], "lpage": ["79"], "pub-id": ["10.31459/turkjkin.929325"]}, {"label": ["Atacan (2010)"], "person-group": ["\n"], "surname": ["Atacan"], "given-names": ["B"], "year": ["2010"], "article-title": ["Effect of an 8-week specially arranged plyometric training on the power and agility of young male soccer players"], "source": ["Master\u2019s thesis"], "institution": ["K\u0131r\u0131kkale University, K\u0131r\u0131kkale, T\u00fcrkiye"]}, {"label": ["Bompa (2013)"], "person-group": ["\n"], "surname": ["Bompa"], "given-names": ["TO"], "year": ["2013"], "source": ["Plyometr\u0131c periodic training theory and method"], "publisher-name": ["Dumat Ofset"], "publisher-loc": ["Ankara"], "fpage": ["365"], "lpage": ["337"], "edition": ["2nd edition"]}, {"label": ["Boraczy\u0144ski & Urniaz (2008)"], "person-group": ["\n"], "surname": ["Boraczy\u0144ski", "Urniaz"], "given-names": ["T", "J"], "year": ["2008"], "article-title": ["The effect of plyometric training on strength-speed abilities of basketball players"], "source": ["Research Yearbook"], "volume": ["14"], "issue": ["1"], "fpage": ["14"], "lpage": ["19"]}, {"label": ["\u00c7oban (2017)"], "person-group": ["\n"], "surname": ["\u00c7oban"], "given-names": ["M"], "year": ["2017"], "article-title": ["The effect of earthquake education with three-dimensional game on academic achievement and motivation of primary school students and students\u2019 opinions"], "source": ["D. Phil. Thesis"], "institution": ["Atat\u00fcrk University, Erzurum, T\u00fcrkiye"]}, {"label": ["Dardouri et\u00a0al. (2014)"], "person-group": ["\n"], "surname": ["Dardouri", "Selmi", "Sassi", "Gharbi", "Rebhi", "Moalla"], "given-names": ["W", "MA", "RH", "Z", "A", "W"], "year": ["2014"], "article-title": ["Reliability and discriminative power of soccer-specific field tests and skill index in young soccer players"], "source": ["Science & Sports"], "volume": ["29"], "issue": ["2"], "fpage": ["88"], "lpage": ["94"], "pub-id": ["10.1016/j.scispo.2013.06.004"]}, {"label": ["Edwin & Gordon (2000)"], "person-group": ["\n"], "surname": ["Edwin", "Gordon"], "given-names": ["R", "S"], "year": ["2000"], "article-title": ["Effect of a plyometric intervention program on sprint performance"], "source": ["The Journal of Strength and Conditioning Research"], "volume": ["14"], "fpage": ["295"], "lpage": ["301"]}, {"label": ["Haz\u0131r, Mahir & A\u00e7\u0131kada (2010)"], "person-group": ["\n"], "surname": ["Haz\u0131r", "Mahir", "A\u00e7\u0131kada"], "given-names": ["T", "\u00d6F", "C"], "year": ["2010"], "article-title": ["Relationship between agility and body composition, anaerobic power in young soccer players"], "source": ["Hacettepe Journal of Sport Sciences"], "volume": ["21"], "issue": ["4"], "fpage": ["146"], "lpage": ["153"]}, {"label": ["Kalayc\u0131(2009)"], "person-group": ["\n"], "surname": ["Kalayc\u0131"], "given-names": ["\u015e"], "year": ["2009"], "source": ["SPSS applied multivariate statistical techniques"], "publisher-name": ["Asil Publication Distribution"], "publisher-loc": ["Ankara"], "edition": ["4th edition"]}, {"label": ["Ko\u00e7 & Aslan (2010)"], "person-group": ["\n"], "surname": ["Ko\u00e7", "Aslan"], "given-names": ["H", "CS"], "year": ["2010"], "article-title": ["The comparison of male handball and volleyball players\u2019 selected physical and motor skills"], "source": ["Sel\u00e7uk University Journal of Physical Education and Sport Science"], "volume": ["12"], "issue": ["3"], "fpage": ["227"], "lpage": ["231"]}, {"label": ["K\u00f6kl\u00fc, \u00d6zkan & ve\u00a0Ers\u00f6z (2009)"], "person-group": ["\n"], "surname": ["K\u00f6kl\u00fc", "\u00d6zkan", "ve\u00a0Ers\u00f6z"], "given-names": ["Y", "A", "G"], "year": ["2009"], "article-title": ["Assessment and improvement of endurance performance in soccer"], "source": ["Celal Bayar University Journal of Sport Science"], "volume": ["4"], "fpage": ["142"], "lpage": ["150"]}, {"label": ["Loturco et\u00a0al. (2020)"], "person-group": ["\n"], "surname": ["Loturco", "Bishop", "Freitas", "Pereira", "Jeffreys"], "given-names": ["I", "C", "TT", "LA", "I"], "year": ["2020"], "article-title": ["Vertical force production in soccer: mechanical aspects and applied training strategies"], "source": ["Strength & Conditioning Journal"], "volume": ["42"], "issue": ["2"], "fpage": ["6"], "lpage": ["15"]}, {"label": ["McMillan & Schumacher (2010)"], "person-group": ["\n"], "surname": ["McMillan", "Schumacher"], "given-names": ["JH", "S"], "year": ["2010"], "source": ["Research in Education: Evidence-Based Inquiry"], "publisher-loc": ["Upper Saddle River"], "publisher-name": ["Pearson Education, Inc"]}, {"label": ["Spori\u0161 et\u00a0al. (2011)"], "person-group": ["\n"], "surname": ["Spori\u0161", "Milanovi\u0107", "Trajkovi\u0107", "Joksimovi\u0107"], "given-names": ["G", "Z", "N", "A"], "year": ["2011"], "article-title": ["Correlation between speed, agility and quickness (SAQ) in elite young soccer players"], "source": ["Acta Kinesiologica"], "volume": ["5"], "issue": ["2"], "fpage": ["36"], "lpage": ["41"]}, {"label": ["William & Kirubakar (2018)"], "person-group": ["\n"], "surname": ["William", "Kirubakar"], "given-names": ["RR", "SG"], "year": ["2018"], "article-title": ["Impact of various plyometric training with selcted asana practices on explosive strength, agility and balance of male soccerers"], "source": ["Asian Journal of Multidimensional Research (AJMR)"], "volume": ["7"], "issue": ["2"], "fpage": ["937"], "lpage": ["943"]}]
{ "acronym": [ " x¯", " sd", " HIPT", " ST", " ss", " ms", " f" ], "definition": [ "Mean", "Standart deviation", "High intensity plyometric training", "Soccer training", "Sum of squares", "Mean of squares", "Variance value" ] }
62
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2024-01-14 23:43:48
PeerJ. 2024 Jan 10; 12:e16648
oa_package/8b/9e/PMC10787543.tar.gz
PMC10787544
0
[ "<title>Introduction</title>", "<p>The newly discovered CKLF-like MARVEL transmembrane domain-containing (CMTM) family comprises CMTM1–8 and CKLF, which are related to the chemokine family and the transmembrane 4 superfamily (TM4SF). Of these, the comparison between CKLF1, CMTM1 and CMTM2 and chemokine reveals a higher similarity, while CMTM8 exhibits a higher level of sequence homology with TM4SF11, reaching up to 39% at the overall amino acid level. The characteristics of the remaining members of the CMTM family exhibit an intermediate nature between CMTM1 and CMTM8 (##REF##32195671##Duan et al., 2020##). The proteins encoded by the CMTM1–8 genes possess structural characteristics that are commonly found in typical chemokines and TM4SF. CMTM family members have important roles in not only tumor pathogenesis but also reproductive and immune systems (##REF##31754330##Wu et al., 2019##). They can activate and chemotactically attract immune cells, which impact tumor cell proliferation and invasion. This suggests that CMTM family members play a significant role in tumorigenesis and may be promising therapeutic targets (##REF##32944388##Wu et al., 2020##). Studies have shown that CMTM expression is associated with pancreatic, breast, gastric, and liver cancer (##REF##23893243##Li et al., 2014a##, ##REF##33282744##2020b##), and its mechanism of action is also related to gene methylation. Methylation of promoter CpG leads to silencing of the CMTM3 gene, which inhibits its function (##REF##19509237##Wang et al., 2009##). Additionally, CMTM overexpression can activate the apoptosis pathway, which leads to induce tumor cell death in renal cancer carcinoma (##REF##32944388##Wu et al., 2020##, ##REF##35220395##2022##). This review discusses the roles of CMTM family members in various cancers and the underlying mechanisms of action. Finally, we discuss the potential of the CMTM family members as therapeutic targets for tumors to improve patient outcomes.</p>" ]
[ "<title>Survey methodology</title>", "<p>The existing literature was extensively investigated using Google Scholar and PubMed to analyze the role of CMTM families in different cancers, focusing on the mechanisms of action of different CMTM family members and several targeted therapies in clinical application in recent years. The scientific literature reviewed was not refined by journal type, author or date of publication. We also considered the previous published literature on CMTM cross-references to identify other appropriate relevant resources.</p>" ]
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[ "<title>Conclusions</title>", "<p>CMTM family proteins perform critical functions in many biological processes during tumor development and metastasis, including triggering proliferation, resisting cell death, and activating invasion and metastasis. These characteristics all belong to “Hallmarks of cancer: the next generation” proposed by Douglas Hanahan and Robert A. Weinberg in 2011 (##REF##21376230##Hanahan &amp; Weinberg, 2011##). Immunotherapy is a relatively new therapeutic modality that has emerged in the last decade or so in addition to conventional surgical resection, radiotherapy and chemotherapy, and in particular, targeting PD-1/PD-L1 has achieved remarkable results in clinical trials in many tumors. CMTM family proteins are involved in the regulation of immune cells and key molecules such as PD-L1, which can be potential targets for immunotherapy.</p>", "<p>In this review, we briefly summarize recent findings on the roles of CMTM proteins in tumor growth and their mechanisms, as they relate to the cell cycle, tumor immunity, signaling pathways and their potential as therapeutic targets. CMTM3, 4, 5 and 7 play important roles in the cell cycle, leading to cell cycle arrest, tumor growth and migration inhibition. Blocking PD-1/PD-L1 <italic toggle=\"yes\">via</italic> monoclonal antibodies has shown outstanding clinical efficacy in patients with various tumors; however, there are still some nonresponsive individuals with poor outcomes. Notwithstanding the significance of PD-L1 expression by cells within the tumor microenvironment, our understanding of the regulation of the PD-L1 protein is limited. Current studies have focused on the mechanisms of action of CMTM proteins as PD-L1 protein regulators. Multiple studies have reported that PD-L1 is dependent on CMTM4 and 6 to efficiently carry out its inhibitory function, suggesting that they are potentially new targets to improve the efficacy of immune checkpoint therapy. CMTM family proteins also exert their biological functions by signaling pathways that correlate with cell growth and migration, including the EGFR, WNT and JAK2/STAT3 signaling pathways, indicating that CMTM proteins are potential targets for altering tumor progression and metastasis. Although we have consolidated a large amount of research to argue our point, our study does have some limitations that need to be addressed in the future. First, the current research on the CMTM family is inherently limited; the role of the CMTM family in tumor development has been validated, but the exact mechanisms remain unclear, and there are no robust experimental data to validate this claim. More studies are needed in the future to further explore the expression of CMTM in tumors, the related molecular mechanisms and signaling pathways and to provide new strategies for tumor immunotherapy.</p>", "<p>Recently, some studies have focused on the CMTM family and its role in cancer biological processes (##REF##27356683##Lu et al., 2016##; ##REF##32944388##Wu et al., 2020##; ##REF##36792080##Xie, Cheng &amp; Zhang, 2023##). However, this review offers unique advantages compared to existing research. Firstly, this review provides a more comprehensive analysis by examining various different types of cancers, including most of the abdominal, thoracic and oral tumors, whereas the review by ##REF##36792080##Xie, Cheng &amp; Zhang (2023)## focuses more on NSCLC. Furthermore, this review individually introduces each member of the CMTM family, followed by a detailed description of their specific mechanisms in specific cancers, rather than providing broad generalizations, and finally highlight the therapeutic potential of CMTM family. Additionally, considering the expanding clinical applications of tumor immunology, our review offers a more comprehensive understanding of the correlation between CMTM family and immunity, encompassing various mechanisms, including PD-L1/PD-1, EGFR, WNT, and JAK2/STAT3 pathways. Moreover, our review provides updated information and insights into the molecular pathways through which CMTM influences different types of cancer to ensure innovation.</p>", "<p>As described in this article, CMTM proteins play an important regulatory role in tumor development as well as in tumor immunity and have the potential to become novel targets for immunotherapy. In addition, the expression of CMTM proteins can also be used to characterize the immunological environment of patients, especially CMTM6, which is closely related to PD-L1, to appropriately estimate patient prognosis and help physicians identify subgroups of patients sensitive to immunotherapy for individualized treatment.</p>" ]
[ "<p>The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing (CMTM) family includes CMTM1–8 and CKLF, and they play key roles in the hematopoietic, immune, cardiovascular, and male reproductive systems, participating in the physiological functions, cancer, and other diseases associated with these systems. CMTM family members activate and chemoattract immune cells to affect the proliferation and invasion of tumor cells through a similar mechanism, the structural characteristics typical of chemokines and transmembrane 4 superfamily (TM4SF). In this review, we discuss each CMTM family member’s chromosomal location, involved signaling pathways, expression patterns, and potential roles, and mechanisms of action in pancreatic, breast, gastric and liver cancers. Furthermore, we discuss several clinically applied tumor therapies targeted at the CMTM family, indicating that CMTM family members could be novel immune checkpoints and potential targets effective in tumor treatment.</p>" ]
[ "<title>Cmtm family</title>", "<p>The CMTM family consists of nine members, namely, CKLF and CMTM1-CMTM8, which are located on various chromosomes (##TAB##0##Table 1##). CMTM1–4 and CKLF are clustered on chromosome 16, while CMTM6–8 form gene clusters on chromosome 3. Additionally, CMTM5 is located on chromosome 14. Most of CMTM family members exhibit alternative RNA splicing events, resulting in the production of at least one splicing isoform containing MARVEL transmembrane domain protein (##REF##33282744##Li et al., 2020b##). The functional attributes of CMTM gene products lie in an intermediate position between classical chemokines and TM4SF, indicating a hybrid nature. CMTM proteins exert an impact on tumor growth by attracting and activating immune cells (##REF##35252165##Wang et al., 2022b##). Furthermore, CMTM proteins are capable of regulating the cell cycle, influencing the EGFR-associated pathway and the EMT process, subsequently affecting the proliferation, invasion, and metastasis of tumor cells, indicating their crucial role in tumorigenesis and potential as therapeutic targets (##REF##32944388##Wu et al., 2020##). In addition to participating in tumorigenesis, the CMTM family also plays crucial roles in other hematopoietic, immune, cardiovascular, and male reproductive systems conditions (##REF##28428220##Chrifi et al., 2017##; ##REF##21351585##Li et al., 2010##, ##REF##16343975##2006##; ##REF##28457985##Zhang et al., 2017a##, ##UREF##13##2016a##). Several members of the CMTM family are either overexpressed or inhibited in various tumors, such as pancreatic and gastric cancer, which affect cell proliferation and patient survival, suggesting their involvement in tumorigenesis and prognostic value (##REF##34012634##Liang et al., 2021##; ##REF##34475993##Zhou et al., 2021##). Gene methylation is the underlying mechanism of CMTM5, as promoter methylation could silence CMTM5, and the restoration of CMTM5 overexpression can activate the apoptosis pathway to induce tumor cell death (##REF##19577543##Guo et al., 2009##; ##REF##34475993##Zhou et al., 2021##).</p>", "<title>CMTM1</title>", "<p>CMTM1 is located on q22.1 of human chromosome 16, and cDNA sequencing has revealed at least 23 selectively spliced subtypes, among which CMTM1_V17 is the most expressed form (##REF##36633525##Siegel et al., 2023##). The upregulation of CMTM1_V17 can confer chemoresistance to non-small cell lung cancer (NSCLC) patients who receive neoadjuvant chemotherapy, while high expression of this subtype in NSCLC tissues is associated with significantly poor prognosis in NSCLC patients (##REF##28129775##Si et al., 2017##). Furthermore, overexpression of CMTM1 in the glioma cell line A172 promotes tumor cell proliferation and migration, possibly through activation of epidermal growth factor receptor (EGFR), SRC kinase, and WNT signaling pathways (##REF##31754330##Wu et al., 2019##).</p>", "<title>CMTM2</title>", "<p>CMTM2 is located on q22.1 of human chromosome 16. It is widely expressed in normal tissues, with a high level of expression in testicular tissues, followed by pancreatic tissue, bone marrow and other tissues (##REF##31362196##Kang et al., 2019##). CMTM2 is located in the endoplasmic reticulum near the Golgi apparatus. The MARVEL structure of CMTM2 is related to its function as a carrier protein, as well as its role in cell membranes, which are related to the transport of intracellular substances during steroid synthesis. It also plays an important role in the production of testosterone (##REF##32944388##Wu et al., 2020##). CMTM2 can inhibit HIV-1 transcription to some extent by targeting the AP-1 and CREB pathways, as well as affect sperm and testosterone production (##REF##31990600##Guo et al., 2020##).</p>", "<title>CMTM3</title>", "<p>CMTM3, located on human chromosome 16 q22.1, has a leucine zipper structure and is widely expressed in normal tissues. It participates in cell proliferation, differentiation and development. It can also inhibit excessive cell proliferation, regulate cell migration and suppress tumorigenesis (##REF##36865551##Li, Yu &amp; Feng, 2023##; ##REF##36593526##Zhang et al., 2023b##). CMTM3 is often downregulated or completely silenced in tumor cells, which is closely related to CpG islands and methylation of its promoter (##REF##22487682##Ogawa et al., 2012##; ##REF##19509237##Wang et al., 2009##). CMTM3 can promote the degradation of EGFR to reduce its expression, activate caspase-3 to induce cell apoptosis, and partially inhibit the JAK2/STAT3 signaling pathway to suppress tumor cell proliferation. Thus, it is a potential target for tumor therapy (##REF##27629543##Li &amp; Zhang, 2017##; ##REF##29345297##Lu et al., 2018##; ##REF##19509237##Wang et al., 2009##; ##REF##27121055##Yuan et al., 2016##).</p>", "<title>CMTM4</title>", "<p>CMTM4, also located on chromosome 16 q22.1, is the most conserved chemokine with three selectively spliced subtypes. It can induce cell accumulation at the G2/M phase and inhibit the proliferation of HeLa cells without inducing apoptosis, revealing its important role in cell proliferation and cell cycle regulation (##REF##30097810##Chrifi et al., 2019##; ##REF##31435638##Xue et al., 2019##). CMTM4 has a synergistic and protective relationship with PD-L1 in tumor tissues. CMTM4 can effectively protect programmed death-ligand 1 (PD-L1) as a target for lysosomal degradation and prevent the clearance of tumor cells by immune cells, suggesting that CMTM4 plays an important role in tumor immunotherapy (##REF##34558800##Chui et al., 2022##; ##REF##28813410##Mezzadra et al., 2017##; ##REF##35958549##Zhang et al., 2022a##).</p>", "<title>CMTM5</title>", "<p>In contrast to the other family members, CMTM5 is independently located on human chromosome 14 q11.2, and there are at least six selectively spliced subtypes, with CMTM5_V1 as the main expressed form (##REF##33282744##Li et al., 2020b##). Methylation-mediated promoter silencing can inhibit the expression of CMTM5, but demethylation drugs can restore its expression in tumor cells, thereby effectively inhibiting the cloning, proliferation, adhesion, migration and invasion of tumor cells. CMTM5 overexpression has a significant inhibitory effect on the formation of tumor cell line colonies (##REF##34726254##Li et al., 2022a##; ##REF##32328181##Yuan et al., 2020##; ##REF##28457985##Zhang et al., 2017a##). Furthermore, CMTM5 induces apoptosis of pancreatic cancer cells by activating caspases 3, 8, and 9 (##REF##32568178##Wu, 2020##). Thus, CMTM5 is involved in several signaling pathways related to tumorigenesis and may be a potential target for tumor therapy (##REF##30704230##Ma et al., 2019##; ##REF##23286412##Yuan et al., 2012##).</p>", "<title>CMTM6</title>", "<p>CMTM6, a noncharacteristic protein on the cell surface, is located in a region rich in tumor suppressor genes. CMTM6 acts as a key regulator of PD-L1 in various tumor cells, binding to PD-L1 and maintaining its expression on the cell surface. CMTM6 can prevent PD-L1 from becoming a target of lysosomal-mediated degradation. In addition, CMTM6 participates in immune function by regulating T lymphocyte-mediated antitumor immunity, which indicates that CMTM6, as a specific therapeutic target, can enhance antitumor immunity to a certain extent (##REF##35024247##Wang et al., 2022a##; ##REF##30562063##Zhu et al., 2019##).</p>", "<title>CMTM7</title>", "<p>CMTM7 is widely expressed in various tissues but is downregulated or not expressed in some malignant tumors, such as pancreatic and esophageal cancer, suggesting that CMTM7 may participate in tumor development and metastasis, play a tumor suppressive role, and behave as an inhibitory site for many tumors (##REF##36597146##Chen et al., 2023##; ##REF##36792080##Xie, Cheng &amp; Zhang, 2023##). SOX10 is a transcriptional regulator of CMTM7, which is weakly expressed in breast and pancreatic cancer, where it behaves as a tumor suppressor gene (##REF##30392914##Jin, Qin &amp; Jia, 2018##; ##REF##23893243##Li et al., 2014a##; ##REF##34294040##Lu et al., 2021##; ##REF##34475993##Zhou et al., 2021##). There are typical CpG islands in the promoter region of CMTM7. Promoter methylation is the mechanism by which CMTM7 expression is downregulated, and CMTM7 expression can affect cell proliferation and tumor development (##REF##30392914##Jin, Qin &amp; Jia, 2018##). CMTM7 can reduce AKT phosphorylation and suppress ERK activation, thereby inhibiting PI3K/AKT downstream targets in KYSE180 cells (##REF##32568178##Wu, 2020##). Further analysis has shown that CMTM7 can inhibit AKT signaling and induce cell cycle arrest (##REF##30903681##Huang et al., 2019##). It can also inhibit the G1/S phase transition by upregulating P27 and downregulating CDK2 and CDK6 (##REF##23893243##Li et al., 2014a##).</p>", "<title>CMTM8</title>", "<p>CMTM8 is a novel chemotactic cytokine composed of 173 amino acids (##REF##33813507##Ge, Duan &amp; Deng, 2021##). It is notable that MARVEL transmembrane domain protein expression is required for CMTM8 to enhance the ligand-induced clearance of EGFR from the cell surface, thereby reducing ERK phosphorylation and affecting the EGFR-mediated signaling pathway (##REF##32268840##H’ng et al., 2020##). CMTM8 overexpression causes a change in mitochondrial caspase expression, ultimately inducing tumor cell apoptosis. Furthermore, CMTM8 enhances the migration and invasion of pancreatic cancer cells, possibly through β-catenin signaling activation in pancreatic cancer cells, which in turn promotes pancreatic cancer invasiveness. Notably, reducing CMTM8 expression can lead to the inhibition of pancreatic cancer metastasis, thus establishing CMTM8 as a potential therapeutic target for pancreatic cancer (##REF##23508014##Gardner et al., 2013##; ##REF##33553335##Shi et al., 2021##).</p>", "<title>Association of the cmtm family with several cancers</title>", "<p>CMTM proteins are associated with several cancers, such as pancreatic, breast, gastric, liver cancer, and head and neck squamous cell carcinoma (##FIG##0##Fig. 1##).</p>", "<title>CMTM proteins in pancreatic cancer</title>", "<p>Pancreatic cancer is a type of gastrointestinal malignancy that mainly originates from the pancreatic ductal epithelium and acinar cells. It can be categorized as either endocrine cancer or exocrine cancer. This disease is characterized by insidious occurrence, rapid progression, poor therapeutic effects, and poor prognosis (##REF##33160014##Chen et al., 2021##; ##REF##27956793##Ilic &amp; Ilic, 2016##; ##UREF##3##Jin et al., 2019##). The mortality rate of pancreatic cancer patients ranks sixth among all deaths caused by malignant tumors in China (##REF##30620402##Siegel, Miller &amp; Jemal, 2019##). Pancreatic cancer, whose prevalence is increasing, is expected to overtake breast, prostate, and colon cancer by 2030 to become the second leading cause of cancer death worldwide, only second to lung cancer (##REF##31832601##Li et al., 2019##). The occurrence and development of pancreatic cancer is accompanied by a large number of gene mutations, including K-ras, TP53, SMAD4 and CDKN2A, and more than 90% of pancreatic intraepithelial neoplasia cases show K-ras mutations and EGFR overexpression, while approximately 10% of cases are inherited (##REF##33347393##Gu et al., 2020##; ##REF##33423164##Khan et al., 2021##; ##UREF##6##Liu et al., 2023##). Pancreatic cancer has a low early diagnostic rate and a low responsiveness to drugs. In recent years, with the in-depth study of tumor immunity and tumor therapy, immunotherapy has become a potential treatment modality after surgery, radiotherapy and chemotherapy (##REF##32247999##Schizas et al., 2020##).</p>", "<p>Studies on the role of the CMTM family in pancreatic cancer are limited, but some preliminary explorations have been made. CMTM4 can negatively regulate PAK4 to inhibit the PI3K/AKT pathway, which in turn inhibits cell proliferation, and high CMTM4 expression can inhibit pancreatic cancer (##REF##32585413##Li et al., 2020a##). Ectopic expression of CMTM8 enhances the migration and invasive ability of pancreatic cancer cells (##REF##33553335##Shi et al., 2021##). CMTM5 and TNF-α synergistically induce apoptosis in pancreatic cancer cells (##REF##32568178##Wu, 2020##). Metastatic pancreatic cancer is likely to spread to the liver, whose cells express CTLA-4, programmed death receptor-1 (PD-1), and PD-L1, all of which inhibit T-cell functions and result in limited treatment options. Immune checkpoint inhibitors can be used to reactivate the body’s anti-tumor immune response, while chemokines, such as cytokines, play important roles in tumorigenesis and autoimmune diseases. CMTM1–8 participate in the occurrence and development of the disease, suggesting that new insights remain to be discovered and applied to pancreatic cancer immunotherapy (##REF##25931111##Delic et al., 2015##; ##REF##27867015##Yuan et al., 2017##). CMTM6 is also involved in preventing PD-L1 lysosomal degradation. LTX-315 is an oncolytic peptide that exhibits PD-L1 inhibition-induced anti-pancreatic cancer immunity effect <italic toggle=\"yes\">via</italic> its potential target ATP11B. CMTM6 mediates the interaction between ATP11B and PD-L1 (##REF##35288467##Tang et al., 2022##).</p>", "<title>CMTM proteins in gastric cancer</title>", "<p>Gastric cancer is a major global disease, with more than one million new cases every year. It is the fifth most diagnosed malignancy in the world. Due to the lack of biomarkers for early diagnosis, most patients are diagnosed at an advanced stage of the disease; therefore, it has a high mortality rate, making it the third most common cause of cancer-related death (##REF##35130500##Ajani et al., 2022##; ##REF##30207593##Bray et al., 2018##; ##UREF##10##Smyth et al., 2020##). Approximately 10% of gastric cancer cases have familial aggregation, and 1–3% of gastric cancer patients have germline mutations (##REF##36627197##Zhang et al., 2023a##). Studies have shown that CMTM expression is closely related to the occurrence of gastric cancer. According to the analysis obtained by bioinformatics methods, CMTM1, 3, 6, 7, and 8 mRNA levels were up-regulated in GC tissues, whereas CMTM2, 4, and 5 did not differ from normal tissues (##REF##34012634##Liang et al., 2021##). CMTM1 consists of 23 isoforms, of which the CMTM1-v17 protein is up-regulated in expression in various cancers. Although there are no studies related to its association with GC, analysis of clinical data shows that CMTM1 levels correlate with various clinical parameters. Thus, CMTM1 can be used as a biomarker for GC.</p>", "<p>A significant mutation in CMTM2 was identified after whole exome sequencing of 23 cases of diffuse-type gastric cancer and non-cancerous paired tissues, and CMTM2 expression predicted prognostic outcome in diffuse-type gastric cancer but not intestinal-type GC (##UREF##2##Choi et al., 2018##). CMTM3 expression is silenced or downregulated in gastric cell lines and primary tumors; it could inhibit EMT by upregulating E-calcineurin expression and downregulating N-calcineurin, waveform protein, and Twist1 expression, suggesting that CMTM3 inhibits gastric cancer metastasis by regulating the STAT3/Twist1/EMT signaling pathway (##REF##30903681##Huang et al., 2019##; ##REF##27121055##Yuan et al., 2016##). CMTM2 and CMTM3 have also been reported to mediate the effects of non-coding RNAs on GC. CMTM2 is involved in the LINC01391/miR-12116/CMTM2 axis and inhibits GC aerobic glycolysis and tumorigenesis (##REF##33033510##Qian et al., 2020##). CMTM3, on the other hand, mediates the promoting effects of miR‑135b‑5p on GC proliferation (##REF##29345297##Lu et al., 2018##). The predominant variant of CMTM5, CMTM5-v1, is widely expressed in average human adult and fetal tissues but is undetectable or down-regulated in most cancer cell lines. Like CMTM3, promoter methylation was detected in almost all silenced or down-regulated cell lines (##REF##33813507##Ge, Duan &amp; Deng, 2021##). CMTM5 has tumor suppressor activity but is frequently inactivated by promoter methylation (##REF##17908965##Shao et al., 2007##).</p>", "<p>Recently, studies have found that CMTM6 and PD-L1 expression correlate and are associated with poor prognosis and immune tolerance in GC (##REF##34759159##Nishi et al., 2021##). The co-detection of CMTM6 and PD-L1 can be a prognostic indicator for patients diagnosed with GC (##REF##33509216##Zhang, Zhao &amp; Wang, 2021##). CMTM6 and CMTM4 are critical regulators of PD-L1 that protect it from lysosomal explication. Thus, they have the potential to serve as indicators to guide the administration of PD-1/PD-L1 therapies or to be used in pre-immunotherapy screening (##REF##34680324##Wang et al., 2021##). CMTM7, widely expressed in normal gastric tissues, is weakly expressed in gastric cancer tissues (##REF##33662423##Abadi et al., 2021##; ##REF##23893243##Li et al., 2014a##). SOX10, a transcriptional regulator of CMTM7, mediates CMTM7 expression in gastric cancer. SOX10 overexpression in cell lines with silenced CMTM7 expression can significantly inhibit cell proliferation and gastric cancer progression. Thus, SOX10 can regulate cell proliferation and gastric cancer progression by regulating the expression of CMTM7 (##REF##30392914##Jin, Qin &amp; Jia, 2018##). CMTM8 protein is down-regulated in GC tissues, correlates with GC metastasis and patient prognosis, and is an independent protective factor for overall survival (##REF##33978454##Yan et al., 2021##).</p>", "<title>CMTM proteins in breast cancer</title>", "<p>Currently, breast cancer is the most frequently diagnosed cancer, and the sixth leading cause of cancer-related death among Chinese women (##REF##27645897##Richman et al., 2017##). The causes are related to reproductive and hormonal factors such as long menstrual lifespan (early-onset menstruation and later-onset menopause), nutrition, advanced maternal age and limited breastfeeding (##UREF##0##Bayraktar &amp; Arun, 2019##; ##UREF##1##Builder, 2021##; ##UREF##8##Moore, 2022##; ##UREF##12##Yi, 2021##). The prognostic challenge with breast cancer occurs when patients wait too long before starting treatment, especially if the delay leads to stage and disease progression or leads to more treatment complications. A 2013 study (##REF##23615681##Smith, Ziogas &amp; Anton-Culver, 2013##) reported that wait times of more than 6 weeks for starting surgical treatment resulted in a 5-year survival of 80%, while wait times of less than 2 weeks resulted in a 5-year survival of 90% (##REF##24872111##Fan et al., 2014##). The CMTM family is associated with the occurrence and development of breast cancer. CMTM5 and seven are biomarkers and prognostic factors of breast cancer. CMTM1_V17 is the most studied CMTM family member in breast cancer, and it is highly expressed in a variety of tumors, including breast cancer (##UREF##4##Kanathezath et al., 2021##). CMTM4, as a PD-L1 protein regulator, can also be a potential surgical target for breast cancer (##REF##35252165##Wang et al., 2022b##). In breast cancer patients, the expression level of CMTM5 and CMTM7 is decreased, while the expression level of CMTM6 is increased. Thus, high expression of CMTM7 may have a better prognostic effect. Elevated expression of CMTM6 in human epidermal growth factor receptor 2-positive (HER2+) BC stabilizes the HER2 protein by impeding the ubiquitination process. This heightened stability of the HER2 protein contributes to trastuzumab resistance, thus underscoring CMTM6 as a promising therapeutic target for overcoming resistance (##REF##36627608##Xing et al., 2023##). CMTM5 is positively correlated with longer survival time in triple-negative breast cancer (TNBC), a more aggressive type with poor prognosis, which is consistent with the overexpression of the tumor suppressor gene p53 in TNBC patients in earlier studies (##REF##32305967##Chen et al., 2020a##; ##REF##28393224##Dimas-González et al., 2017##). The expression of CMTM7 is higher in TNBC patients, and they show a higher response to chemotherapy and immunotherapy. Additionally, high expression of CMTM7 is positively correlated with immunomodulators, tumor-infiltrating immune cells (TIICs), and immune checkpoint activation (##REF##36568362##Jiang et al., 2022##).</p>", "<p>Epithelial-mesenchymal translation (EMT) confers aggressive and evasive characteristics to tumor cells. Activation of the EMT process in breast cancer cells can upregulate the expression of CMTM6, which is an essential protein for the cell surface expression of PD-L1. Studies have shown a positive correlation between EMT markers and two other members of the CMTM family, namely, CMTM3 and CMTM7 (##UREF##14##Zhu et al., 2020##). Compared with epithelial cells, CMTM6 and PD-L1 are both overexpressed in mammary mesenchymal cells. The expression level of CMTM1, 2, 4 and 8 in breast cancer are decreased, while the expression level of CMTM5 is not different between healthy individuals and patients with metastatic breast cancer. MiR-182-5p within the extracellular vesicles of BC can specifically target and suppress the expression of CMTM7, activating the CMTM7/EGFR/AKT pathway and facilitating the progression of breast cancer (##REF##34294040##Lu et al., 2021##). EMT inhibitors can be used in combination with PD-L1 blockers to improve the response rate (##REF##33803139##Xiao et al., 2021##). Modulators of the EMT process, as well as modulators of CMTM6 or CMTM7, can be used in combination with the anti-PD-L1 antibody in patients with highly aggressive breast cancer. In mesenchymal breast cancer cells, CMTM6 silencing decreases PD-L1 expression on the cell surface, while dual targeting of CMTM6 and CMTM7 significantly reduces PD-L1 expression (##REF##33803139##Xiao et al., 2021##), suggesting that CMTM-targeted molecular therapy can improve the prognosis of breast cancer patients.</p>", "<title>CMTM proteins in liver cancer</title>", "<p>Liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma, is the sixth most common malignancy and the third most common cause of cancer death worldwide (##REF##37173972##Lazzaro &amp; Hartshorn, 2023##; ##UREF##7##Ma et al., 2021##), and its incidence is on the rise (##REF##36139633##Liu &amp; Liu, 2022##). High rates of invasion and metastasis are the main factors leading to the poor prognosis of HCC patients, and EMT is believed to be the main mechanism underlying cancer cell migration and invasion (##REF##35426224##Zhang et al., 2022b##). In HCC, there is a notable increase in the expression of CMTM1, CMTM3, CMTM4, CMTM7, and CMTM8, indicating the pro-tumorigenesis effect of them. Conversely, the expression of CMTM2, CMTM5, and CMTM6 exhibited a significant decrease (##REF##36975415##Li et al., 2023a##). <italic toggle=\"yes\">In vivo</italic> study, it has been elucidated that the pro-tumorigenesis effect of CMTM3 is attributed to its ability to upregulate peroxisome proliferator‑activated receptor γ (PPARγ) and subsequently activate the PPAR pathway (##UREF##11##Wang et al., 2023##).</p>", "<p>CMTM3 inhibits the migration and invasion of HCC cells by inhibiting the EMT phenotype and the JAK2/STAT3 signaling pathway. According to one study, targeted therapy may be an effective modality for the treatment of liver cancer. First, the low expression of CMTM3 in HCC cell lines can significantly inhibit HCC cell proliferation and invasion as well as the EMT process. In addition, CMTM3 overexpression significantly downregulates the phosphorylation levels of JAK2 and STAT3 in HepG2 cells, which suggests that CMTM3 suppresses HCC cell proliferation and tumorigenesis by inhibiting the JAK2/STAT3 signaling pathway. Thus, CMTM3 may be a potential target for the prevention and treatment of HCC (##REF##27629543##Li &amp; Zhang, 2017##).</p>", "<p>Moreover, immune evasion also contributes to the difficulty of treating liver cancer, and immunotherapy developed to target the tumor immune microenvironment (TIME) kills cancer cells by activating their own immune system. The immune microenvironment plays an important role in the development and treatment of cancer, with tumor-infiltrating T cells, as its key members, recognizing and killing cancer cells (##REF##37006257##Chi et al., 2023##), and exosomes, which carry a variety of molecules, acting as a bridge between the various cells in the TIME that communicate with each other (##REF##35126514##Chen et al., 2022##). A full understanding of the TIME facilitates the development of new therapeutic targets, of which PD-1/PD-L1 is a relatively representative immune detection site, and in liver cancer, CMTM6 can stabilize PD-L1 and enhance the efficacy of immunotherapy (##REF##32770259##Liu et al., 2021##). In HCC, CKLF was found to be involved in the infiltration of immune cells and acted as an independent prognostic biomarker (##REF##36975415##Li et al., 2023a##). Furthermore, genes in the CMTM family, such as rs3811178 of CMTM5 and rs164207 of CMTM6, may regulate the HCC risk individually or in combination (##REF##29990439##Bei et al., 2018##). CMTM4, located near human chromosome 16q22.1, is often deleted in various tumors, including those in the liver and breast, suggesting the presence of a TSG at this site. The role of TSG is closely associated with tumor occurrence and development and can be inactivated through genetic and epigenetic mechanisms, including promoter CpG methylation and histone modification (##REF##34681626##Desaulniers et al., 2021##; ##UREF##5##Li et al., 2023b##; ##UREF##9##Pal, 2022##). CMTM7 expression is significantly decreased in HCC tissues and negatively correlated with TNM staging. Forced expression of CMTM7 can inhibit the growth and migration of HCC cells by inhibiting AKT signaling and inducing cell cycle arrest at the G0/G1 phase (##REF##30903681##Huang et al., 2019##). This may be possibly due to decreased expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and CDK6 and increased expression of p27, indicating that CMTM7 acts as a tumor suppressor by inhibiting cell cycle progression in HCC.</p>", "<title>CMTM proteins in oral cancers</title>", "<p>It is worth mentioning that CMTM3, as an oncogene, is expressed at low levels in cancer, yet its potential promoter has high methylation levels, and aberrant promoter methylation may be an early signal of tumor development. In laryngeal squamous cell carcinoma (LSCC), hypermethylation of the CMTM3 promoter was found to increase the risk of LSCC in men, especially in the smoking population and in older adults over 55 years of age (##REF##27521994##Shen et al., 2016##). The role of CMTM4 is difficult to define, as it exhibits different functions in different tumors, for example, it inhibits the growth of pancreatic cancer cells but its expression level is elevated in type I renal clear cell carcinoma. In head and neck squamous cell carcinoma (HNSCC), CMTM4 has a tendency to be overexpressed and unsurprisingly, it can stabilize PD-L1 expression, while CMTM4 also promotes the process of EMT and affects its related molecules. In addition, CMTM4 can regulate the cancer stem cell -like phenotype through the AKT pathway, which would enhance the significance of CMTM4 as a therapeutic target (##REF##34061408##Li et al., 2021##).</p>", "<title>Functions of cmtm proteins in tumor growth and immunity</title>", "<title>CMTM and its regulation of the cell cycle</title>", "<p>The cell cycle comprises the G1, S, G2, and M phases. This complex process is regulated by cyclins and cyclin-dependent kinases (CDKs). Moreover, there are checkpoints at G1/S and G2/M phase transitions; thus, DNA can be replicated accurately. CDKs can target these checkpoints and regulate the cell cycle. Researchers can also control the proliferation of tumor cells by regulating cell cycle-related proteins (##FIG##1##Fig. 2A##) (##REF##29275292##Wenzel &amp; Singh, 2018##). CMTM3 is frequently downregulated or silenced in testicular cancer cell lines and tumor tissues but highly expressed in normal testis tissues. In a human seminoma cell line, upon the re-expression of CMTM3 <italic toggle=\"yes\">via</italic> adenovirus delivery (Ad-CMTM3), the infected cells expressed a high level of p21, which led to arrest of the cell cycle at the G2 phase and inhibition of cell growth and migration (##REF##24586462##Li et al., 2014b##). Similarly, ##REF##20213316##Plate et al. (2010)## revealed that CMTM4 expression led to the accumulation of HeLa cells at the G2/M phase, subsequently inhibiting cell proliferation. In addition, CMTM5 resulted in G0/G1 arrest (##REF##28789377##Cai et al., 2017##), and CMTM7 caused G1/S phase arrest by upregulating p27 and downregulating CDK2 and 6 (##REF##30903681##Huang et al., 2019##; ##REF##23893243##Li et al., 2014a##).</p>", "<title>CMTM and tumor immunity</title>", "<p>Tumor immunity refers to how the immune system responds to tumor antigens and eliminates tumor cells. The tumor genome drives tumor immunity due to gene mutation and transcriptional aberration-generated T-cell neoepitopes. In addition, memory T cells play a vital positive role in tumor immunity (##REF##28102259##Chen &amp; Mellman, 2017##). Of note, tumor cells evolved to express immune checkpoint ligands, thus inhibiting the cytotoxicity of the immune system. As one of the classical ligands, PDL-1 interacts with the checkpoint PD-1 on T cells and inhibits T cell activity (##REF##35031777##Kornepati, Vadlamudi &amp; Curiel, 2022##).</p>", "<p>Beyond PD-L1 inhibitory therapy, the CMTM family may also have other functions in tumor immunity. For instance, CMTM6 is involved in immune functions by modulating T lymphocyte-mediated antitumor immunity. In a bioinformatics study that conducted gene set variation analysis of transcriptome data from 1,862 glioma samples available from CGGA RNA sequencing, TCGA RNA sequencing, CGGA microarrays, GSE16011 data, and IVY GBM databases, CMTM6 expression was positively correlated with immunosuppressive factors such as induced T-cell tolerance, cytokine synthesis and secretion, and regulatory T-cell differentiation (##REF##30131308##Guan et al., 2018b##). This study also revealed that CMTM6 expression may be positively correlated with inflammatory responses and somatic mutations that promote cancer progression.</p>", "<p>Furthermore, CKLF1 expression is upregulated in activated CD4<sup>+</sup> and CD8<sup>+</sup> lymphocytes, indicating that its expression is regulated in various autoimmune diseases (##REF##32195671##Duan et al., 2020##). CMTM7 regulates B-cell function in terms of tumor immunity. In CMTM7 knockout mice, decreased expression level of BCR on the B1a cell surface and decreased serum IgM levels were observed compared to normal mice (##REF##32305130##Liu et al., 2020##). With the rapid development of immunotherapy, a number of new therapeutic directions have emerged; IL-7 plays an integral role in the growth, development and even maintenance of memory of T cells, demonstrating its potential as an adjuvant in combination with cancer vaccines (##REF##36389666##Zhao et al., 2022##). Exosomes have great potential in cancer immunotherapy as a bridge of communication, capable of loading various molecules to deliver information; they can be used as potential markers to aid diagnosis, and targeted drugs can be investigated to target tumor exosomes, thus opening up more possibilities for immunotherapy (##REF##36741380##Gong et al., 2022##).</p>", "<title>Cmtm and tumor immunotherapy</title>", "<p>PD-L1 is expressed on the surface of various cells, including tumor cells and myeloid cells (##REF##34815353##Munoz et al., 2021##). Anti-PD-1 therapy aims to dampen the interactions between tumor-reactive T cells and tumor cells by blocking PD-1 ligand/PD-1 signaling (##REF##25858804##Topalian, Drake &amp; Pardoll, 2015##). In different tumors, blocking PD1 and PD-L1 interactions has shown great clinical benefits, except for some nonresponsive cases. To promote the clinical efficacy of immune checkpoint therapy, it is necessary to investigate the intrinsic mechanisms related to PD-L1 expression and then to identify potential targets for clinical research (##REF##22658127##Topalian et al., 2012##). However, our understanding of the regulation of PD-L1 is limited.</p>", "<p>CMTM6 is an uncharacterized protein expressed on the cell surface. To investigate this potentially new regulator of PD-L1 expression, ##REF##28813417##Burr et al. (2017)## performed a whole-genome CRISPR–Cas9 deletion library screen using the pancreatic cancer cell line BxPC-3 and observed that IFN-γ can indeed stimulate the expression of endogenous PD-L1. Subsequently, the authors demonstrated that CMTM6 colocalizes with PD-L1 on the cell surface with or without IFN-γ stimulation and that this interaction is only maintained under conditions of complete membrane integrity. Notably, among more than 5,000 quantified proteins on the cell surface, CMTM6 is one of four proteins that decreased more than twofold by plasma membrane profiling and other unbiased analytic methods, indicating that CMTM6 is required for PD-L1 expression. Furthermore, after examining PD-L1 maturation in wild-type and CMTM6-knockout cells, they found that CMTM6 maintains stable expression in the plasma membrane but not during translocation from the endoplasmic reticulum to the cell surface. They also demonstrated that a large proportion of surface PD-L1 is continuously internalized and recycled, and CMTM6 plays a role in this recycling by preventing PD-L1 from being targeted for lysosome-mediated degradation (##REF##28813417##Burr et al., 2017##). ##REF##28813410##Mezzadra et al. (2017)## showed that this function is shared by the closest family member, namely, CMTM4, in HAP1 cells. They also demonstrated that CMTM4 and 6 increased PD-L1 expression without affecting PD-L1 transcript levels but prolonged the half-life of PD-L1.</p>", "<p>CMTM6 knockdown leads to decreased expression of PD-L1 in 12 human tumor lines of melanoma, thyroid cancer, colorectal cancer, lung cancer and CML, as well as in three short-term melanoma xenografts (##REF##28813410##Mezzadra et al., 2017##). CMTM6 stabilizes PD-L1 expression by preventing lysosome-mediated degradation of PD-L1 (##REF##28813410##Mezzadra et al., 2017##). Another study demonstrated that CMTM6 knockdown reduced the expression of PD-L1 and increased the infiltration of CD8<sup>+</sup> and CD4<sup>+</sup> T cells, which enhanced antitumor immunity in head and neck squamous cell carcinoma (##REF##31771985##Chen et al., 2020b##). CMTM6 expression was also upregulated in advanced malignant glioma, and it was responsible for the poor prognosis of patients with glioma (##REF##30131308##Guan et al., 2018b##).</p>", "<p>In lung cancer patients, CMTM6 expression acts as a predictor of PD-1 inhibitor therapy; that is, patients with higher CMTM6 expression respond well to PD-1 inhibitors (##REF##31646201##Gao et al., 2019##; ##REF##31646074##Koh et al., 2019##). In addition, CMTM6 expression has been detected in several healthy tissues, and it may have functions other than triggering immune evasion by tumor cells (##FIG##1##Fig. 2B##).</p>", "<p>In a retrospective NSCLC cohort (438 individuals, in which 69 were treated with immunotherapy, 258 were untreated, and a collection of EGFR and K-ras genotyped tumors), high coexpression of CMTM6 and PD-L1, particularly in the stroma (<italic toggle=\"yes\">e.g</italic>., macrophages), predicted the outcomes of PD-1 blockade immunotherapy (##REF##31605795##Zugazagoitia et al., 2019##).</p>", "<p>In many other cancer types, CMTM6 is an indicator of clinical outcome and prognosis. Recently, a pancancer data analysis of 33 cancer types revealed a close correlation between CMTM6 and PD-L1 in many cancers as well as a correlation with overall survival (##REF##33552963##Zhao et al., 2020##). In 185 gastric cancer specimens, CMTM6 and PD-L1 were mainly expressed both on the cell surface and in the nucleus of tumor cells (expression rates were 78.38% and 75.68% respectively), and they may be independent indicators for overall survival (##REF##33509216##Zhang, Zhao &amp; Wang, 2021##). In metastatic melanoma, high CMTM6 and PD-L1 coexpression in the stromal compartment was highly correlated with longer survival in treated patients, while PD-L1 expression showed prognostic value in control patients (##REF##33457084##Martinez-Morilla et al., 2020##). Likewise, in TNBC, the expression of CMTM6 was correlated with progression-free survival (##REF##33569433##Tian et al., 2021##).</p>", "<p>Taken collectively, these findings reveal that PD-L1 is dependent on the expression of CMTM4 and six to efficiently carry out its inhibitory function, suggesting that they are potentially new targets to improve the efficacy of immune checkpoint therapy.</p>", "<title>Modulation of antitumor immunity by cmtm</title>", "<p>CMTM family proteins exert their biological functions by regulating signaling pathways related to cell growth and migration, including the EGFR, WNT, and JAK2/STAT3 signaling pathways, suggesting that CMTM proteins may serve as potential targets for modifying tumor development (##FIG##1##Fig. 2C##).</p>", "<title>Regulation of the EGFR signaling pathway</title>", "<p>EGFR signaling maintains cell homeostasis and regulates epithelial tissue development; therefore, it can be a biomarker of resistance in tumors (##REF##22785351##Yarden &amp; Pines, 2012##). EGFR is phosphorylated after stimulation by EGF, triggering the intracellular signaling cascade to control cell proliferation and differentiation. Several CMTMs can regulate this critical signaling pathway (##REF##29124875##Sigismund, Avanzato &amp; Lanzetti, 2018##). In gastric cancer, CMTM3 promotes the degradation of EGFR to impede tumor metastasis (##REF##27121055##Yuan et al., 2016##), indicating that knocking out the CMTM3 gene can be a potential therapy for gastric cancer. In addition, CMTM3 accelerates the degradation of EGFR, inhibits the EGFR/STAT3/EMT signaling pathway, increases the expression of TP53, and enhances the TP53 signaling pathway in chordoma cells (##REF##34560882##Yuan et al., 2021##).</p>", "<p>Both CMTM3 and CMTM5 can regulate the endocytic trafficking of EGFR. CMTM3 reduces the expression of EGFR on the cell surface by promoting early endosome fusion by activating Rab5 (##REF##27867015##Yuan et al., 2017##). However, in HCC and prostate cancer, CMTM5 inhibits this signaling pathway by targeting its downstream pathway, namely, the PI3K/AKT signaling pathway (##REF##25387568##Xiao et al., 2015##). CMTM6, 7 and 8 are widely expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 expression is frequently suppressed or reduced in esophageal and nasopharyngeal cell lines, but this is not true for other cancer cell lines. Similar to CMTM3, CMTM7 also regulates the EGFR/AKT pathway in NSCLC (##REF##26528697##Liu et al., 2015##). A previous study revealed that CMTM7 can promote the internalization of EGFR and further inhibit the AKT signaling pathway (##REF##23893243##Li et al., 2014a##). CMTM8 accelerates the degradation of EGFR from the cell surface by relying on the MARVEL transmembrane domain protein, thereby influencing the EGFR signaling pathway by dampening ERK phosphorylation (##REF##22337876##Zhang et al., 2012##).</p>", "<title>Association with WNT signaling</title>", "<p>The WNT signaling pathway is vital for embryonic development, tissue regeneration, stem cell homeostasis, and cell proliferation. The WNT pathway is highly conserved throughout evolution, and its downstream effects were traditionally separated into canonical (β-catenin dependent) WNT signaling and non-canonical (β-catenin independent) signaling (##REF##34518209##Parsons, Tammela &amp; Dow, 2021##). Alteration of WNT signaling was involved in tumorigenesis of various cancer types, including colorectal cancer, liver cancer, and lung cancer (##REF##34518209##Parsons, Tammela &amp; Dow, 2021##).</p>", "<p>A previous study demonstrated that the interaction of CMTM6 with membrane-bound enolase-1 stabilized its expression, leading to activation of WNT signaling mediated by AKT–glycogen synthase kinase-3β (##REF##33434185##Mohapatra et al., 2021##). These findings indicate that CMTM6 can be a promising target for treating therapy-resistant oral squamous cell carcinoma by facilitating tumor cell immune evasion and reversing cisplatin resistance (##REF##33434185##Mohapatra et al., 2021##).</p>", "<p>Similarly, the WNT/β-catenin signaling pathway is involved in HNSCC development, including cell proliferation and differentiation with mesenchymal traits as early signs of disease (##REF##24871033##Lee et al., 2014##). CMTM6 expression is associated with WNT/β-catenin signaling. Depletion of CMTM6 affects the maintenance of stemness properties and inhibits TGFβ-induced EMT in HNSCC cells (##REF##31771985##Chen et al., 2020b##).</p>", "<p>Another study investigated the overexpression of CMTM genes on glioblastoma cell proliferation and invasion and revealed that CMTM1 and 3 promote tumor invasion, and their expression was significantly correlated with shorter overall survival. Consequently, the study showed that CMTM1 and CMTM3 can be targets for treating glioblastoma (##REF##25931111##Delic et al., 2015##). Additionally, using a human phosphokinase protein expression profiling assay, the authors demonstrated that CMTM1 and three downstream signaling may be correlated with the expression of growth factor receptor and Src kinases, as well as WNT activation.</p>", "<title>JAK2/STAT3 signaling pathway</title>", "<p>The JAK2/STAT3 pathway interacts non-covalently with various cytokine receptors and is activated upon receptors binding to hormones, growth factors, or cytokines. This pathway is pivotal in regulating cellular processes such as proliferation, differentiation, apoptosis, and immune modulation (##REF##35241923##Mengie Ayele et al., 2022##). It is implicated in the tumorigenesis and progression of both hematological malignancies and solid tumors due to its involvement in regulating cell growth-related gene expression, exerting adverse effects on prognosis (##REF##26151455##Thomas et al., 2015##).</p>", "<p><italic toggle=\"yes\">In vitro</italic>, CMTM3 overexpression inhibited cell proliferation and invasion, and the EMT process in HCC cells. The phosphorylation of JAK2 and STAT3 in HepG2 cells was also decreased. <italic toggle=\"yes\">In vivo</italic>, CMTM3 overexpression inhibited tumor growth in Bal-b/c nude mice. Thus, CMTM3 plays an important role in HCC metastasis by triggering EMT and suppressing the JAK2/STAT3 signaling pathway, indicating that CMTM3 is a potential target in the prevention and treatment of HCC (##REF##27629543##Li &amp; Zhang, 2017##).</p>", "<title>Potential of CMTM proteins as therapeutic targets</title>", "<p>CMTM1 consists of at least 23 alternatively spliced isoforms designated CMTM1_V1-V23, among which CMTM1_V17 is highly expressed in breast tumors. Moreover, a study revealed that CMTM1_V17 enhances cell proliferation and inhibits TNF-α-induced tumor cell apoptosis (##REF##25175386##Wang et al., 2014##). Survival analyses showed that CMTM1 could be an independent <italic toggle=\"yes\">post hoc</italic> factor in hepatocellular carcinoma (##REF##33585698##Song et al., 2021##). CMTM1 and CMTM3 are targets for treating glioblastoma (##REF##25931111##Delic et al., 2015##). CMTM2 expression is down-regulated and prognostically relevant in HCC and may serve as an independent prognostic factor (##REF##31990600##Guo et al., 2020##). Moreover, serum CMTM2 levels can be a valuable indicator of the pathogenesis of HBV-related diseases (##REF##33101541##Chen et al., 2020c##). Up-regulation of CMTM2 expression under the effect of Si-Jun-Zi decoction inhibits the tumor properties of gastric cancer (##REF##35873650##Li et al., 2022b##). CMTM3 affects EMT progression by inhibiting the EGFR/STAT3 signaling pathway, thereby inhibiting the development of tumorigenesis in chordoma and gastric cancer. CMTM3 could be used as its potential therapeutic target (##REF##34560882##Yuan et al., 2021##). Depletion of CMTM4 enhances the sensitivity of HCC tumors to anti-PD-L1 therapy, allowing more patients to benefit from immunotherapy (##REF##34558800##Chui et al., 2022##). A previous study reported that low expression of CMTM5 in hepatocellular carcinoma significantly correlated with poor overall survival (##REF##29213215##Xu &amp; Dang, 2017##). Another study suggested the prognostic value of CMTM5 expression and that it might prolong the survival of patients with TNBC (##REF##32305967##Chen et al., 2020a##). CMTM6 has been associated with the development of a variety of cancers. In hepatocellular carcinoma, CMTM6 interacts with Vimentin, thereby promoting invasive metastasis of HCC (##REF##33757532##Huang et al., 2021##); in breast cancer, CMTM6 stabilizes the HER2 protein and contributes to trastuzumab resistance (##REF##36627608##Xing et al., 2023##); and in gastric cancer, CMTM6 stabilizes the expression of PD-L1, and its high expression correlates with poor prognosis of gastric cancer, and the combined assay can be used as a prognostic indicator in gastric cancer (##REF##33509216##Zhang, Zhao &amp; Wang, 2021##). CMTM7 can interact with Catenin Alpha 1 to regulate Wnt/β-catenin signaling to inhibit breast cancer progression and be a novel target for breast cancer (##REF##36597146##Chen et al., 2023##). CMTM8 can interact with LPA1, activating oncogenic β-catenin signaling as a potential therapeutic target for pancreatic cancer (##REF##33553335##Shi et al., 2021##).</p>" ]
[ "<p>We thank all authors for their contributions to this review.</p>", "<title>Additional Information and Declarations</title>" ]
[ "<fig position=\"float\" id=\"fig-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16757/fig-1</object-id><label>Figure 1</label><caption><title>CMTM proteins have different regulatory network in various cancers.</title><p>The CMTM family is associated with pancreatic, breast, gastric, and liver cancer. (A) CMTM4 inhibits PI3K/AKT pathway by negatively regulating PAK4, thus inhibiting the proliferation of pancreatic cancer cells. CMTM8 as an LPA1-related chaperone mediates lysophosphatidic acid-induced metastasis of pancreatic cancer. CMTM5 mediates TNF-α to regulate pancreatic cancer cell apoptosis. (B) SOX10 can regulate the proliferation of gastric cancer by regulating the expression of CMTM7. CMTM3 can inhibit gastric cancer metastasis by regulating the STAT3/Twist1/EMT signaling pathway. (C) CMTM6 affects tumor immunity by regulating PD-L1 on the surface of breast cancer. (D) CMTM3 and CMTM7 inhibit the proliferation of hepatocellular carcinoma cells by inhibiting the JAK2/STAT3 and PI3K/AKT pathways, respectively. (E) CMTM4 is one of the regulators of PD-L1, which is involved in tumour immune regulation, and it is also involved in the EMT process, affecting the expression of some key molecules such as SNAIL.</p></caption></fig>", "<fig position=\"float\" id=\"fig-2\"><object-id pub-id-type=\"doi\">10.7717/peerj.16757/fig-2</object-id><label>Figure 2</label><caption><title>Roles of CMTM proteins in tumor growth and immunity.</title><p>(A) CMTM protein regulates cell cycle. CMTM3 and four express a high level of p21, leading to cell proliferation inhibition at G2/M phase. CMTM5 results in G0/G1 arrest and CMTM7 causes G1/S phase arrest by upregulating p27. (B) CMTM6 induces immune escape of tumor cells through tissue lysosomal mediated degradation of PD-L1. (C) The CMTM family mediates tumor-related signaling pathways and can be used as targets for cancer therapy.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"table-1\"><object-id pub-id-type=\"doi\">10.7717/peerj.16757/table-1</object-id><label>Table 1</label><caption><title>Characteristics of the CMTM family.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\" content-type=\"text\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">CMTM</th><th rowspan=\"1\" colspan=\"1\">Location</th><th rowspan=\"1\" colspan=\"1\">Mechanism</th><th colspan=\"2\" rowspan=\"1\">Main subcellular locations</th><th rowspan=\"1\" colspan=\"1\">Clinical significance</th><th rowspan=\"1\" colspan=\"1\">Reference</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">CMTM1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16q22.1</td><td rowspan=\"1\" colspan=\"1\">It causes tumorigenesis by affecting cell proliferation.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, peroxisome, nucleus, extracellular space</td><td colspan=\"2\" rowspan=\"1\">It is associated with chemo resistance and poor prognosis of patients.</td><td rowspan=\"1\" colspan=\"1\">##REF##28129775##Si et al. (2017)##, ##REF##25175386##Wang et al. (2014)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16q22.1</td><td rowspan=\"1\" colspan=\"1\">It affects HIV-1 transcription through AP-1 and CREB pathways.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, peroxisome, nucleus, extracellular space, Golgi apparatus, cytosol</td><td colspan=\"2\" rowspan=\"1\">It provides a new way to control HIV-1 transcription.</td><td rowspan=\"1\" colspan=\"1\">##REF##26986142##Zhang et al. (2016b##, ##REF##28816268##2017b)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16q22.1</td><td rowspan=\"1\" colspan=\"1\">Allele inactivation or methylation makes it lose its ability to negatively regulate cell proliferation.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, nucleus, extracellular space, endosome</td><td colspan=\"2\" rowspan=\"1\">It may be an independent prognostic indicator for gastric cancer.</td><td rowspan=\"1\" colspan=\"1\">##REF##29345297##Lu et al. (2018)##, ##REF##27121055##Yuan et al. (2016)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16q22.1</td><td rowspan=\"1\" colspan=\"1\">It regulates the cell cycle and affects tumor cell proliferation through synergistic protection of PD-L1.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, nucleus, extracellular space, Golgi apparatus</td><td colspan=\"2\" rowspan=\"1\">It is an effective target for clear cell renal cell carcinomas (ccRCCs) treatment and a factor of poor prognosis.</td><td rowspan=\"1\" colspan=\"1\">##REF##29180877##Bei et al. (2017)##, ##REF##28813410##Mezzadra et al. (2017)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14q11.2</td><td rowspan=\"1\" colspan=\"1\">It is involved in certain signaling pathways related to tumorigenesis and development.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, extracellular space</td><td colspan=\"2\" rowspan=\"1\">It may be a new target for tumor gene therapy.</td><td rowspan=\"1\" colspan=\"1\">##REF##28789377##Cai et al. (2017)##, ##REF##29691981##Guan et al. (2018a)##, ##REF##28457985##Zhang et al. (2017a)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3p22.3</td><td rowspan=\"1\" colspan=\"1\">It cooperates with PD-L1 to participate in immune escape.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, extracellular space, cytoskeleton, cytosol, lysosome, endosome</td><td colspan=\"2\" rowspan=\"1\">It may be a potential immunotherapy target, such as ovarian cancer.</td><td rowspan=\"1\" colspan=\"1\">##REF##28813417##Burr et al. (2017)##, ##REF##35174213##Yin et al. (2022)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3p22.3</td><td rowspan=\"1\" colspan=\"1\">Rab5 controls the EGFR-AKT signaling pathway and affects tumor development.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, extracellular space</td><td colspan=\"2\" rowspan=\"1\">It is related to the occurrence and development of gastric cancer.</td><td rowspan=\"1\" colspan=\"1\">##REF##30392914##Jin, Qin &amp; Jia (2018)##, ##REF##26528697##Liu et al. (2015)##</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CMTM8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3p22.3</td><td rowspan=\"1\" colspan=\"1\">It influences the EGFR signaling pathway through MARVEL to regulate cell proliferation, differentiation and apoptosis.</td><td rowspan=\"1\" colspan=\"1\">Plasma membrane, nucleus and extracellular space</td><td colspan=\"2\" rowspan=\"1\">It is involved in various tumors and is a new target for tumor gene therapy.</td><td rowspan=\"1\" colspan=\"1\">##REF##25551557##Both et al. (2014)##, ##REF##26503336##Gao et al. (2015)##</td></tr></tbody></table></alternatives></table-wrap>" ]
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[ "<fn-group content-type=\"competing-interests\"><title>Competing Interests</title><fn fn-type=\"COI-statement\" id=\"conflict-1\"><p>The authors declare that they have no competing interests.</p></fn></fn-group>", "<fn-group content-type=\"author-contributions\"><title>Author Contributions</title><fn fn-type=\"con\" id=\"contribution-1\"><p><xref rid=\"author-1\" ref-type=\"contrib\">Sai-Li Duan</xref> conceived and designed the experiments, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn fn-type=\"con\" id=\"contribution-2\"><p><xref rid=\"author-2\" ref-type=\"contrib\">Yingke Jiang</xref> conceived and designed the experiments, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn fn-type=\"con\" id=\"contribution-3\"><p><xref rid=\"author-3\" ref-type=\"contrib\">Guo-Qing Li</xref> performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn fn-type=\"con\" id=\"contribution-4\"><p><xref rid=\"author-4\" ref-type=\"contrib\">Weijie Fu</xref> performed the experiments, prepared figures and/or tables, and approved the final draft.</p></fn><fn fn-type=\"con\" id=\"contribution-5\"><p><xref rid=\"author-5\" ref-type=\"contrib\">Zewen Song</xref> analyzed the data, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn fn-type=\"con\" id=\"contribution-6\"><p><xref rid=\"author-6\" ref-type=\"contrib\">Li-Nan Li</xref> analyzed the data, authored or reviewed drafts of the article, and approved the final draft.</p></fn><fn fn-type=\"con\" id=\"contribution-7\"><p><xref rid=\"author-7\" ref-type=\"contrib\">Jia Li</xref> analyzed the data, authored or reviewed drafts of the article, and approved the final draft.</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Data Availability</title><fn id=\"addinfo-1\"><p>The following information was supplied regarding data availability:</p><p>This article is a literature review.</p></fn></fn-group>" ]
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[{"label": ["Bayraktar & Arun (2019)"], "person-group": ["\n"], "surname": ["Bayraktar", "Arun"], "given-names": ["S", "BK"], "article-title": ["Epidemiology, risk factors, and prevention"], "source": ["Breast Disease"], "year": ["2019"], "volume": ["1"], "fpage": ["39"], "lpage": ["61"], "pub-id": ["10.1007/978-3-030-04606-4_4"]}, {"label": ["Builder (2021)"], "person-group": ["\n", "\n"], "surname": ["Builder", "Salvo"], "given-names": ["V", "SG"], "article-title": ["Reproductive pathologies, conditions, and disorders"], "source": ["Mosby\u2019s Pathology for Massage Professionals"], "year": ["2021"], "publisher-loc": ["Amsterdam"], "publisher-name": ["Elsevier Health Sciences"], "fpage": ["356"], "lpage": ["399"]}, {"label": ["Choi et al. (2018)"], "person-group": ["\n"], "surname": ["Choi", "Kim", "Ahn", "Kim", "Bae", "Han", "Woo", "Lee"], "given-names": ["JH", "YB", "JM", "MJ", "WJ", "SU", "HG", "D"], "article-title": ["Identification of genomic aberrations associated with lymph node metastasis in diffuse-type gastric cancer"], "source": ["Experimental and Molecular Medicine"], "year": ["2018"], "volume": ["50"], "issue": ["12"], "fpage": ["1"], "lpage": ["11"], "pub-id": ["10.1038/s12276-018-0167-1"]}, {"label": ["Jin et al. (2019)"], "person-group": ["\n"], "surname": ["Jin", "Huang", "Liu", "Yu"], "given-names": ["K", "Q", "C", "X"], "article-title": ["New advances in diagnosis and treatment of pancreatic cancer"], "source": ["Chinese Journal of Digestive Surgery"], "year": ["2019"], "volume": ["12"], "fpage": ["657"], "lpage": ["661"], "pub-id": ["10.3760/cma.j.issn.1673-9752.2019.07.009"]}, {"label": ["Kanathezath et al. (2021)"], "person-group": ["\n"], "surname": ["Kanathezath", "Chembra", "Padingare Variyath", "Nair"], "given-names": ["A", "V", "SK", "GG"], "article-title": ["Identification of biomarkers and functional modules from genomic data in stage-wise breast cancer"], "source": ["Current Bioinformatics"], "year": ["2021"], "volume": ["16"], "issue": ["5"], "fpage": ["722"], "lpage": ["733"], "pub-id": ["10.2174/574893615999200922123104"]}, {"label": ["Li et al. (2023b)"], "person-group": ["\n"], "surname": ["Li", "Shu", "Geng", "Ying", "Guo", "Luo", "Xiang", "Wu", "Ma", "Chan"], "given-names": ["L", "X-S", "H", "J", "L", "J", "T", "L", "BB", "AT"], "article-title": ["A novel tumor suppressor encoded by a 1p36. 3 lncRNA functions as a phosphoinositide-binding protein repressing AKT phosphorylation/activation and promoting autophagy"], "year": ["2023b"], "fpage": ["1"], "lpage": ["18"], "pub-id": ["10.1038/s41418-023-01129-w"]}, {"label": ["Liu et al. (2023)"], "person-group": ["\n"], "surname": ["Liu", "Mroczek", "Mach", "St\u0119pie\u0144", "Aplas", "Pronobis-Szczylik", "Bukowski", "Mielczarek", "Gajewska", "Topolski"], "given-names": ["J", "M", "A", "M", "A", "B", "S", "M", "E", "PJC"], "article-title": ["Genetics, genomics and emerging molecular therapies of pancreatic cancer"], "source": ["Cancer"], "year": ["2023"], "volume": ["15"], "fpage": ["779"], "pub-id": ["10.3390/cancers15030779"]}, {"label": ["Ma et al. (2021)"], "person-group": ["\n"], "surname": ["Ma", "Wang", "Khatib", "Chang", "Heinrich", "Dominguez", "Forgues", "Candia", "Hernandez", "Kelly", "Zhao", "Tran", "Hernandez", "Davis", "Kleiner", "Wood", "Greten", "Wang"], "given-names": ["L", "L", "SA", "CW", "S", "DA", "M", "J", "MO", "M", "Y", "B", "JM", "JL", "DE", "BJ", "TF", "XW"], "article-title": ["Single-cell atlas of tumor cell evolution in response to therapy in hepatocellular carcinoma and intrahepatic cholangiocarcinoma"], "year": ["2021"], "volume": ["75"], "fpage": ["1397"], "lpage": ["1408"], "pub-id": ["10.1016/j.jhep.2021.06.028"]}, {"label": ["Moore (2022)"], "person-group": ["\n"], "surname": ["Moore"], "given-names": ["S"], "article-title": ["Cross-national variation in menopause symptoms\u2014a systematic review of recent evidence"], "year": ["2022"], "publisher-name": ["Master\u2019s Thesis, University of East Finland, Kuopio, Finland"]}, {"label": ["Pal (2022)"], "person-group": ["\n"], "surname": ["Pal"], "given-names": ["A"], "article-title": ["Epigenetics and DNA methylation"], "source": ["Protocols in Advanced Genomics and Allied Techniques"], "year": ["2022"], "publisher-loc": ["Cham"], "publisher-name": ["Springer"], "fpage": ["245"], "lpage": ["278"]}, {"label": ["Smyth et al. (2020)"], "person-group": ["\n"], "surname": ["Smyth", "Nilsson", "Grabsch", "van Grieken", "Lordick"], "given-names": ["EC", "M", "HI", "NC", "F"], "article-title": ["Gastric cancer"], "source": ["The Lancet"], "year": ["2020"], "volume": ["396"], "issue": ["10251"], "fpage": ["635"], "lpage": ["648"], "pub-id": ["10.1016/S0140-6736(20)31288-5"]}, {"label": ["Wang et al. (2023)"], "person-group": ["\n"], "surname": ["Wang", "Chu", "Wang", "Wang", "Liu", "Song", "Liu"], "given-names": ["J", "H", "Z", "X", "X", "Z", "F"], "article-title": ["In vivo study revealed pro-tumorigenic effect of CMTM3 in hepatocellular carcinoma involving the regulation of peroxisome proliferator-activated receptor gamma (PPAR\u03b3)"], "source": ["Cellular Oncology"], "year": ["2023"], "volume": ["46"], "issue": ["1"], "fpage": ["49"], "lpage": ["64"], "pub-id": ["10.1007/s13402-022-00733-1"]}, {"label": ["Yi (2021)"], "person-group": ["\n"], "surname": ["Yi"], "given-names": ["Y"], "source": ["Age at natural menopause and its determinants in women with type 1 diabetes"], "year": ["2021"], "publisher-loc": ["Pittsburgh"], "publisher-name": ["University of Pittsburgh"]}, {"label": ["Zhang et al. (2016a)"], "person-group": ["\n"], "surname": ["Zhang", "Dun", "Tang", "Yin", "Hu", "Zhao", "Xu", "Li"], "given-names": ["XW", "YJ", "X", "HQ", "ZP", "YP", "T", "Q"], "article-title": ["Expression of chemokine like factor-like myelin and lymphocyte and related proteins for vesicle trafficking and membrane link transmembrane domain-containing protein 2 in rats with varicocele"], "source": ["Beijing Da Xue Xue Bao Yi Xue Ban"], "year": ["2016a"], "volume": ["48"], "fpage": ["579"], "lpage": ["583"]}, {"label": ["Zhu et al. (2020)"], "person-group": ["\n"], "surname": ["Zhu", "Bei", "Li", "Zhang", "Zeng", "Guo", "Kong", "Fu", "Zhang", "Tan"], "given-names": ["X", "C", "D", "H", "W", "X", "J", "Y", "S", "S"], "article-title": ["Down-regulated CMTM7 promotes metastasis of hepatocellular carcinoma via its family member CMTM3"], "year": ["2020"], "pub-id": ["10.21203/rs.3.rs-30874/v1"]}]
{ "acronym": [], "definition": [] }
148
CC BY
no
2024-01-14 23:43:48
PeerJ. 2024 Jan 10; 12:e16757
oa_package/4c/05/PMC10787544.tar.gz
PMC10787573
0
[ "<title>Introduction</title>", "<p>The biomechanical properties of the cornea are an important biomarker for ocular health.##UREF##0##1## Evaluating corneal rigidity can help diagnose corneal disease and monitor therapeutic efficacy because tissue biomechanical properties and structure are inextricably linked.##UREF##1##2## This relationship has important implications for laser assisted <italic>in situ</italic> keratomileusis (LASIK), a surgical procedure for vision correction that involves structural modifications to the corneal stroma for refractive error correction.##UREF##2##3## Modern LASIK involves the use of a femtosecond laser to create a lamellar corneal flap. Then, the flap is lifted, and an excimer laser is used to directly ablate the stromal bed. After ablation, the flap is repositioned on the surface of the cornea. Specific refractive errors can be corrected based on the selection of the ablation region. For example, treatment for myopia involves ablation of the center of the cornea, and treatment for hyperopia involves ablation of the corneal mid-periphery. Such significant changes to the corneal structure have consequences for corneal biomechanical properties.##REF##16720023##4## In rare cases, patients who have undergone LASIK may experience ectasia as the procedure can compromise corneal biomechanical and structural integrity.##UREF##3##5##<sup>,</sup>##UREF##4##6## To minimize the risk of ectasia, the inclusion criteria for this refractive procedure are conservatively based on the structural properties of the cornea, including thickness and topography. However, these geometric parameters do not account for corneal stiffness, which plays an important role during early ectasia development.##REF##21383577##7##<sup>,</sup>##UREF##5##8## The next generation of customized refractive surgery may incorporate biomechanical mapping in addition to structural imaging, for a more personalized surgical treatment.##UREF##6##9##</p>", "<p>Despite the necessity of corneal biomechanical assessments, there are very few clinical tools that can measure these properties. Air-puff tonometry-based techniques, such as the Corvis ST and the ocular response analyzer, have been repeatedly used to measure corneal biomechanical properties after LASIK. However, these techniques can only measure corneal stiffness by inducing large, non-physiological displacements,##REF##17188041##10##<sup>,</sup>##UREF##7##11## which limits their ability to map corneal biomechanical properties and measure quantitative biomechanical parameters, e.g., Young’s modulus. Optical elastography techniques, including Brillouin microscopy and optical coherence elastography (OCE), have both been used to measure the biomechanical properties of the cornea after refractive surgery##UREF##8##12##<sup>,</sup>##REF##36616779##13## with significantly fewer limitations. While Brillouin microscopy has shown distinct changes in corneal stiffness due to refractive surgery, this technique cannot natively provide sample structural information,##REF##35290310##14## and understanding the relationship between the Brillouin modulus and Young’s modulus is still an area of investigation.##REF##35290310##14##<sup>,</sup>##REF##31548707##15##</p>", "<p>In this work, we examined the changes in mechanical properties in the cornea after LASIK in <italic>ex vivo</italic> porcine eyes. Three OCE modalities were used, including wave-based air-coupled ultrasound OCE (ACUS-OCE),##REF##33258863##16## compression OCE,##REF##37727230##17## and heartbeat OCE (Hb-OCE).##REF##32372574##18## ACUS-OCE provides an estimate of shear modulus across the corneal tissue, and both Hb-OCE and compression OCE provide a similar measurement in the axial direction. Combined, these techniques provide insight into the complex changes in the mechanical properties of the cornea before and after refractive surgery.</p>" ]
[ "<title>Methods</title>", "<p>Six pairs of whole porcine eye globes were shipped within 24 h of enucleation (Sioux-Preme Packing Co., Sioux Center, Iowa). One eye of each pair was kept as a control, and LASIK was performed on the fellow eye. The treated eyes were mounted in a custom-built eye holder, and intraocular pressure (IOP) was maintained using a closed-loop IOP controller.##REF##24877005##19## A LASIK flap was created with a diameter of 8.5 mm and thickness of (Intralase iFS150, Johnson &amp; Johnson Vision, Irvine, California). The flap was pulled back, and the stromal bed was ablated with the excimer laser (VISX Star S4, Johnson &amp; Johnson Vision, Irvine, California) with a maximum diameter of 6.5 mm. The ablation depth was set to to simulate a refractive treatment of approximately –10 diopters. Following the completion of the excimer ablation, the LASIK flap was replaced in the usual fashion. OCE imaging was performed immediately after LASIK on all eye globe pairs. Each eye globe was mounted in a custom-built eye holder, and OCE measurements were taken as IOP was controlled using the closed-loop IOP controller. Measurements were performed using an SD-OCT system with a 25 kHz A-line rate, axial resolution, and lateral resolution. The system had a displacement stability of in the common path configuration and in the dual-arm configuration. Three types of OCE techniques were performed, including ACUS-OCE, Hb-OCE, and compression OCE. Three pairs were measured using ACUS-OCE, and the remainder were measured using both the Hb-OCE and compression OCE techniques. Due to time constraints and tissue degradation, not all eyes could be measured by each method. ##FIG##0##Figure 1## shows a summary schematic for each technique. Sample hydration was maintained with phosphate-buffered saline.</p>", "<title>ACUS-OCE</title>", "<p>ACUS-OCE was performed as described in our previous works.##REF##35774330##20##<sup>,</sup>##UREF##9##21## Briefly, a spherically focused ACUS transducer with a central resonance frequency of 1 MHz, diameter, and diameter opening was co-focused with the OCT sample beam and mounted away from the surface of the cornea. Wave excitation was performed using five cycles of a 2 kHz square pulse (50% duty cycle) amplitude modulating a 1 MHz sinusoidal signal. This signal was then amplified using a 53 dB power amplifier (A150; Electronics &amp; Innovation, Rochester, New York) before driving the ACUS transducer.##REF##35774330##20##<sup>,</sup>##UREF##9##21##</p>", "<p>OCE measurements were taken in M-B-mode configuration at 25 kHz A-line scan rate with M-scan length of 1000 A-lines acquired over time at each imaging position. The ACUS-induced displacement produced at the corneal apex resulted in the propagation of a Lamb wave across the cornea. The wave propagation was sampled over 500 positions along an 8 mm line across the center of the cornea. Each measurement took to complete. The mechanical wave speed was estimated by tracking the peak of the axial particle velocity propagating across the scan region. Measurements were acquired at 10, 20, and 30 mmHg for both the untreated and LASIK-treated eyes.</p>", "<title>Hb-OCE</title>", "<p>Hb-OCE was performed on a separate set of three pairs of eyes. Imaging was performed in B-M-mode across the central 6 mm of the cornea with a B-scan width of 1000 A-lines and a frame rate of . Data acquisition was performed as the eye globe underwent a sinusoidal fluctuation of 1 mmHg peak to peak amplitude and 10 s period at baseline IOP of 10 and 20 mmHg, respectively. Each acquisition took 30 s for up to three IOP cycles. Fluctuations in IOP cause corneal compression and expansion, which can be captured by measuring the phase difference between consecutive OCT images. OCT phase images were denoised using the vector method,##UREF##10##22## and the resulting denoised phase data were converted to real displacement after two-dimensional unwrapping.##UREF##11##23## Displacement data were translated to strain using the weighted least squares method.##REF##22876350##24## The cumulative sum of inter-frame strains was calculated to determine the total strain change relative to the initial image with zero displacement. A detailed description of the Hb-OCE acquisition and processing methods can be found in our previous works.##REF##32372574##18##<sup>,</sup>##REF##33624461##25##</p>", "<title>Compression OCE</title>", "<p>The same set of eyes that was used for Hb-OCE was also used for compression OCE. A reference glass mounted to a piezoelectric ring actuator (HPSt 150/14-10/12 Piezomechanik GmbH, Munich, Germany) was aligned with the OCT beam in the common path configuration. A small amount of mineral oil was added to the surface of the cornea for lubrication, and the reference glass was gently lowered down onto the cornea until a 6 mm region of the tissue was applanated. OCT image acquisition followed the same method as in Hb-OCE. Images were acquired in B-M-mode across the center of the cornea at frame rate with a B-scan width of 1000 A-lines. The actuator was synchronized to the OCT frame trigger, such that one image was acquired while the cornea was compressed, and the consecutive image was acquired in the uncompressed state. Measurements were performed at 10 and 20 mmHg IOP. Data processing followed a similar methodology as the Hb-OCE technique; motion was detected between consecutive frames (compressed and uncompressed) using the OCT phase difference, the phase difference images were denoised using the vector method, the denoised data were converted to the displacements, and the displacements were translated to strain. Unlike the Hb-OCE method, total tissue displacement occurred between consecutive B-scans, so that inter-frame strain was equivalent to peak-to-peak strain in Hb-OCE. A thorough description of the compression OCE data acquisition and processing workflow can be found in our previous research.##REF##37727230##17##<sup>,</sup>##UREF##12##26##</p>" ]
[ "<title>Results</title>", "<p>##FIG##1##Figures 2(a)## and ##FIG##1##2(b)## illustrate representative OCT images of an untreated and LASIK-treated cornea, respectively. The corneal flap and the demarcation line showing the incision region are clearly visible. Laser ablation resulted in a visible lateral inhomogeneity and a thickness loss in the central corneal region. ##FIG##1##Figures 2(c)## and ##FIG##1##2(d)## show the wave propagation map (axial particle velocity) for the untreated and LASIK-treated corneas, respectively, at a given moment in time after the excitation. A guided Lamb wave is clearly seen propagating across the cornea. The increased magnitude of particle velocity in the LASIK-treated cornea suggests a decreased stiffness in the tissue, as the ACUS excitation energy was the same between measurements. ##FIG##1##Figures 2(e)##, ##FIG##1##2(g)##, ##FIG##1##2(i)## and ##FIG##1##2(f)##, ##FIG##1##2(h)##, ##FIG##1##2(j)## show the wave speed map at 10, 20, and 30 mmHg for the untreated and LASIK-treated corneas, respectively. The elastogram shows a marked increase in wave speed as a function of IOP, consistent with previous work.##UREF##9##21##<sup>,</sup>##REF##29275544##27##<sup>,</sup>##REF##34598188##28## However, the wave speed is notably lower in the LASIK-treated corneas compared to the untreated tissue at the same IOP.</p>", "<p>Mean wave speeds for the untreated corneas were , , and at 10, 20, and 30 mmHg IOP, respectively. Mean wave speeds for the LASIK-treated corneas were , , and at 10, 20, and 30 mmHg, respectively. Analysis of all samples showed a statistically significant increase in wave speed as a function of IOP for both the untreated (, , ) and the LASIK treated (, , ) corneas by one way ANOVA. Corneas treated with LASIK had a lower wave speed compared to the untreated samples, suggesting a decrease in stiffness due to the treatment, but this relationship was marginally not statistically significant ( by Wilcoxon signed ranks test) and may also be influenced to loss of corneal thickness during LASIK. ##FIG##2##Figure 3(a)## summarizes the ACUS-OCE results.</p>", "<p>Hb-OCE and compression OCE were performed on the same samples () since the data could be acquired with the same imaging setup. Measurements were performed only at 10 and 20 mmHg for both techniques due to experimental time constraints and sample degradation. Representative samples for untreated and LASIK are shown in ##FIG##3##Fig. 4##. Note that both techniques measure axial strain (i.e., along the cornea optical axis; millistrain: ) and an increase in strain is a decrease in stiffness. However, Hb-OCE induced larger amplitude displacements as compared to compression OCE, leading to larger strains. ##FIG##2##Figures 3(b)## and ##FIG##2##3(c)## illustrate that both Hb-OCE and compression OCE did not show a statistically significant difference in strain between 10 and 20 mmHg ( for untreated measured by Hb-OCE, for untreated measured by compression OCE, for LASIK measured by Hb-OCE, and for LASIK measured by compression OCE using Wilcoxon signed-rank test), likely due to small sample size and mild sample degradation. However, both methods showed a distinct difference in localized strain from the anterior stroma compared to the posterior. For both techniques, the cornea was segmented through the midline between the epithelium and endothelium. The absolute value of the difference between anterior and posterior strain was quantified for each sample. A box-and-whisker plot of the result is shown in ##FIG##4##Fig. 5##. Mean strain difference measured by Hb-OCE for the untreated and LASIK-treated corneas was and , respectively. The mean strain difference measured by compression OCE for the untreated and LASIK-treated corneas was and , respectively. While both techniques appeared to show a difference in stiffness, only compression OCE showed a statistically significant difference in regional strain after LASIK surgery ( for Hb-OCE, for compression OCE, Wilcoxon signed ranks test), likely due to limitations in sample size, delays between surgery and measurement, and changes in tissue stiffness and geometry due to hydration.</p>" ]
[ "<title>Discussion</title>", "<p>In this work, three OCE techniques were used to measure the stiffness of paired porcine corneas, where one eye was left untreated and the other underwent LASIK surgery. The Lamb wave speed measurement provided by ACUS-OCE can provide a reasonable estimate of the shear modulus of the tissue.##UREF##13##29## The compressive strain measured by Hb-OCE and compression OCE can both provide information regarding axial Young’s modulus.##REF##32749033##30## Both the quasistatic and the dynamic techniques measure distinct aspects of corneal elasticity, and all three techniques independently suggest that the LASIK procedure may cause a reduction in corneal stiffness. LASIK involves laser incision and ablation of the corneal stroma, directly compromising the component of the cornea with the greatest biomechanical contribution, i.e., the stroma. While some research suggests that the Bowman’s membrane contributes to corneal tensile strength,##REF##16720023##4## the OCE methods used here lack the resolution to distinguish the mechanical contribution of this thin layer. The measured reduction in stiffness supports what is expected based on the mechanism of LASIK. For example, the reduction in Lamb wave speed correlated with LASIK-induced damage to the collagen lamellae within the tissue, which compromised its shear modulus. That damage to the lamellae may have also caused a disruption to the axial Young’s modulus of the cornea, as suggested by the change in strain measured by Hb-OCE and compression OCE. In the untreated tissue, the anterior and posterior regions of the stroma responded uniformly to axial compression. However, after LASIK, the corneas showed a distinct increase in strain difference, suggesting that the procedure causes the corneal flap and residual stromal bed to respond separately to axial compression. While this behavior may be because the flap is not bound to the stromal bed in this <italic>ex vivo</italic> study, it does suggest that Hb-OCE and compression OCE may be able to measure regional differences in mechanical properties caused by refractive procedures. Furthermore, regional assessment of mechanical properties, as seen in this study, may provide additional insight regarding the specific mechanism of complications such as LASIK-induced ectasia.</p>", "<p>While these preliminary results are promising, there are some major limitations to this work. Primarily, this study was performed <italic>ex vivo</italic> and illustrated only the immediate biomechanical impact of the surgery on the cornea. However, the study sheds no light on the mechanical alterations in the tissue during and after the healing process. Nevertheless, OCE may be able to evaluate changes in mechanical properties throughout the wound healing process##REF##35460537##31## and is a direction of our future work. In this study, the corneal flap was laid flat against the residual stromal bed, and no external method for adherence was utilized. In a typical LASIK procedure, the flap adheres to the residual stromal bed naturally and begins to seal within a short period of time in part due to the pumping function of the corneal endothelium.##UREF##14##32## As the tissue begins to heal, its integrity may recover, but an overall softening is still expected.##REF##16720023##4##</p>", "<p>Another major limitation of this study is the limited sample size. While the results of this study may suggest that there is a difference between the two groups, the limited sample size that was used in this study prevents any thorough conclusions. Furthermore, in this study, contralateral symmetry between fellow eyes was assumed; the untreated fellow eye was thought to be biomechanically similar to the treated eye. However, future studies will directly examine corneal stiffness changes after LASIK by studying the same eye before and after treatment. Despite these limitations, here we lay the groundwork for future evaluations of corneal stiffness changes after refractive surgery procedures.</p>", "<p>In addition, there are several tested models for translating Lamb wave speed to viscoelastic properties, which have been implemented in OCE for the cornea. The complex boundary conditions of the LASIK-treated corneas that were present in this work make a thorough analysis of viscoelasticity more complex.##REF##27838594##33##<sup>,</sup>##REF##32737363##34## Han et al. described a Lamb wave model for estimating the Young’s modulus and viscosity of the cornea. However, this model does not account for the multilayer structure of the tissue, particularly in the case of the LASIK-treated samples, where the corneal stroma has been sliced and ablated; meaning that the tissue geometry does not meet the basic requirements of the model. Future work will focus on establishing a robust, multilayer model that more accurately represents LASIK-treated corneas. However, it should be noted that the exaggerated layered structure that we see in our results is limited to the <italic>ex vivo</italic> case. In the case of clinical LASIK, since the flap and residual stromal bed rapidly bond together, a multilayer model applicable for the <italic>ex vivo</italic> case may not reflect the corneal geometry and boundary conditions in clinical LASIK.</p>", "<p>In our previous work, we have shown that Hb-OCE and compression OCE had nonsignificant differences in strain <italic>in vivo</italic>.##UREF##12##26## Both techniques provide similar information despite the distinct differences between each technique. However, here, we did not note a similarity in strain measured by the two techniques, likely due to the distinct differences in displacement amplitude between the physical compression by the mechanical actuator in compression OCE and the 1 mmHg IOP peak-to-peak amplitude displacement in Hb-OCE. Hb-OCE measurements were performed with a dramatically higher strain compared to compression OCE, enabling us to estimate corneal stiffness at different regions along the stress–strain curve. Based on trends from our previous work, we expect that the differences between the techniques measured here would be much smaller had measurements been performed <italic>in vivo</italic>, since the corneal displacement measured <italic>in vivo</italic> was dramatically lower than the displacement induced here in the Hb-OCE measurements. In our previous work, we quantified the stiffness of the cornea using Hb-OCE and compression OCE by assessing the ratio of IOP change to corneal strain. However, the difference in the magnitude of IOP change between both methods (1 mm peak to peak for Hb-OCE and 0.2 peak to peak for compression OCE##REF##37727230##17##) ensures that the stiffness measured will not be comparable between both methods, and the lack of an appropriate model that accounts for ocular geometry and stress distribution in the tissue makes comparison to Young’s modulus estimation obtained from wave-based OCE difficult as well. Future work will focus on the development of appropriate models to estimate Young’s modulus given these parameters.</p>", "<p>The results of this work suggest that LASIK has an impact on the biomechanical properties of the cornea, potentially compromising cornea structural and mechanical integrity. Other refractive procedures likely cause similar changes to the cornea. However, procedures such as photorefractive keratectomy or small incision lenticule extraction, which have no or significantly smaller incisions in the corneal stroma, may maintain corneal biomechanical integrity without compromising refractive surgery outcomes.##REF##36616779##13## Our future work will further examine the biomechanical impact of various refractive surgery procedures using optical elastography procedures.</p>" ]
[ "<title>Conclusion</title>", "<p>In this work, three OCE techniques, ACUS-OCE, Hb-OCE, and compression OCE, were used to assess the mechanical properties of paired corneas, where one sample was left untreated and the other underwent LASIK. All three techniques showed that LASIK caused a reduction in corneal stiffness, though in the limited number of samples. In addition, Hb-OCE and compression OCE demonstrated the changes in elasticity distribution with depth after LASIK. Future work will examine changes in corneal biomechanical properties <italic>in vivo</italic> after LASIK and other refractive procedures.</p>" ]
[ "<title>Abstract.</title>", "<title>Significance</title>", "<p>The biomechanical impact of refractive surgery has long been an area of investigation. Changes to the cornea structure cause alterations to its mechanical integrity, but few studies have examined its specific mechanical impact.</p>", "<title>Aim</title>", "<p>To quantify how the biomechanical properties of the cornea are altered by laser assisted <italic>in situ</italic> keratomileusis (LASIK) using optical coherence elastography (OCE) in <italic>ex vivo</italic> porcine corneas.</p>", "<title>Approach</title>", "<p>Three OCE techniques, wave-based air-coupled ultrasound (ACUS) OCE, heartbeat (Hb) OCE, and compression OCE were used to measure the mechanical properties of paired porcine corneas, where one eye of the pair was left untreated, and the fellow eye underwent LASIK. Changes in stiffness as a function of intraocular pressure (IOP) before and after LASIK were measured using each technique.</p>", "<title>Results</title>", "<p>ACUS-OCE showed that corneal stiffness changed as a function of IOP for both the untreated and the treated groups. The elastic wave speed after LASIK was lower than before LASIK. Hb-OCE and compression OCE showed regional changes in corneal strain after LASIK, where the absolute strain difference between the cornea anterior and posterior increased after LASIK.</p>", "<title>Conclusions</title>", "<p>The results of this study suggest that LASIK may soften the cornea and that these changes are largely localized to the region where the surgery was performed.</p>", "<title>Keywords:</title>" ]
[]
[ "<title>Acknowledgments</title>", "<p>This work was funded by the National Institutes of Health (NIH), Grant Nos. R01EY033978, R01EY034114, R01EY022362, and Core Grant P30EY07551. Achuth Nair was supported by a fellowship from the Gulf Coast Consortia, on the National Library of Medicine Training Program in Biomedical Informatics and Data Science, T15LM007093. Fernando Zvietcovich acknowledges support from the Peruvian CONCYTEC PROCIENCIA program PE501082168-2023 and intramural funding from the Department of Engineering at Pontificia Universidad Católica del Perú.</p>", "<p><bold>Achuth Nair</bold>, PhD, is currently completing a postdoctoral fellowship at the University of Houston through the National Library of Medicine Training Program in Biomedical Informatics and Data Science. He received his PhD in biomedical engineering in 2022 from the University of Houston. His research interests include optical coherence tomography, optical coherence elastography, tissue biomechanics, and machine learning.</p>", "<p><bold>Fernando Zvietcovich</bold>, PhD, received his BS degree in electrical engineering and his MS degree in digital signal and image processing from Pontificia Universidad Católica del Perú, Lima, Peru, in 2012 and 2014, respectively. He received his PhD in electrical engineering from the University of Rochester, Rochester, New York, in 2020. He is currently working with the Biomedical Optics Laboratory, Department of Biomedical Engineering, University of Houston, Houston, Texas, USA. His research interest includes optical coherence tomography, medical elastography, medical image formation and processing, and computer vision and pattern recognition applied to medicine. His current research includes novel wavebased optical coherence elastography methods in soft tissues for advancing the diagnosis and monitoring of diseases and treatments in clinical environments. He was the past holder of the 2020 SPIE-Franz Hillenkamp Postdoctoral Fellowship in Problem-Driven Biomedical Optics and Analytics. He is currently an assistant professor at Pontificia Universidad Católica del Peru in the Engineering Department.</p>", "<p><bold>Manmohan Singh</bold>, PhD, is currently a research assistant professor in Dr. Kirill Larin’s Biomedical Optics Laboratory at the University of Houston. He received his PhD in biomedical engineering in 2018 from the University of Houston and completed a fellowship from the National Library of Medicine in Data Science and Biomedical Informatics in 2020. His research interests are focused on developing optical imaging modalities and image processing techniques to detect diseases noninvasively.</p>", "<p><bold>Mitchell P. Weikert</bold>, MD, MS, is a professor at the Cullen Eye Institute, Baylor College of Medicine. He received degrees in both electrical and biomedical engineering and worked as a design engineer in the petroleum industry. He obtained his medical degree from Baylor College of Medicine with honors. He served as the residency program director at Baylor from 2012 to 2023 and the medical director of the Lions Eye Bank of Texas from 2010 to 2016. His research interests include biometry, intraocular lens calculations, biomedical optics, and anterior segment imaging. He was recently recognized by Newsweek as one of America’s top 200 ophthalmologists.</p>", "<p><bold>Salavat R. Aglyamov</bold>, PhD, is a research assistant professor of mechanical engineering at the University of Houston. He received his BS and MS degrees in applied mathematics in 1991 and 1993, respectively, from Moscow State University, Russia. He received his PhD in biophysics in 1999 from the Institute of Theoretical and Experimental Biophysics, Russia. His research interests are in the areas of tissue biomechanics, elasticity imaging, medical imaging, ultrasound, and applied mathematics.</p>", "<p><bold>Kirill V. Larin</bold>, PhD, is a Cullen College of Engineering Distinguished Professor of biomedical engineering at the University of Houston. He received his MS degree in laser physics and mathematics from the Saratov State University in 1995 and his PhD in biomedical engineering from the University of Texas Medical Branch in Galveston in 2002. He has published more than 200 papers in the field of biomedical optics and biophotonics. He is a fellow of SPIE, OPTICA, and AIMBE.</p>", "<title>Disclosures</title>", "<p>AN, MS, FZ, and KVL all have a financial interest in ElastEye LLC. ElastEye LLC develops solutions for imaging corneal biomechanical properties. ElastEye LLC. may potentially benefit financially from the research findings on mechanical changes in the cornea presented here. This research was not supported by ElastEye LLC.</p>", "<title>Code and Data Availability</title>", "<p>The data presented in this work was available upon reasonable request.</p>" ]
[ "<fig position=\"float\" id=\"f1\"><label>Fig. 1</label><caption><p>(a) OCE system schematic. Hb-OCE is performed without any additional components. (b) ACUS-OCE sample arm configuration with ACUS transducer co-focused with objective lens. (c) Compression OCE sample arm in common path configuration. Note that for compression OCE, the reference arm shown in (a) is blocked.</p></caption></fig>", "<fig position=\"float\" id=\"f2\"><label>Fig. 2</label><caption><p>(a), (b) OCT structural B-mode of untreated and LASIK treated porcine cornea, respectively. (c), (d) Wave propagation map for each tissue type. (e), (g), (i)  Wave speed elastogram for untreated porcine cornea at 10, 20, and 30 mmHg, respectively. (f), (h), (j) Wave speed elastogram for LASIK treated porcine cornea at 10, 20, and 30 mmHg, respectively.</p></caption></fig>", "<fig position=\"float\" id=\"f3\"><label>Fig. 3</label><caption><p>(a) Lamb wave speed as a function of IOP, measured by ACUS-OCE. (b) Strain change as a function of IOP measured using Hb-OCE for untreated and LASIK corneas. (c) Strain change as a function of IOP measured using compression OCE for untreated and LASIK corneas.</p></caption></fig>", "<fig position=\"float\" id=\"f4\"><label>Fig. 4</label><caption><p>(a), (b) Hb-OCE and compression OCE strain maps for untreated porcine corneas. (c), (d) Hb-OCE and compression OCE strain maps for LASIK treated porcine corneas. The dashed white line represents the incision region for the LASIK treated corneas.</p></caption></fig>", "<fig position=\"float\" id=\"f5\"><label>Fig. 5</label><caption><p>(a) Absolute strain difference between the anterior and posterior cornea as measured by Hb-OCE. -value of 0.10 measured by Wilcoxon signed ranks test. (b) Absolute strain difference between the anterior and posterior cornea as measured by compression OCE. -value of 0.03 calculated by Wilcoxon signed ranks test.</p></caption></fig>" ]
[]
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[{"label": ["1."], "surname": ["Kling", "Hafezi"], "given-names": ["S.", "F."], "article-title": ["Corneal biomechanics: a review"], "source": ["Ophthal. Physiol. Opt."], "volume": ["37"], "issue": ["3"], "fpage": ["240"], "lpage": ["252"], "year": ["2017"], "pub-id": ["OPOPD5", "10.1111/opo.12345"], "issn": ["0275-5408"]}, {"label": ["2."], "surname": ["Roberts", "Dupps", "Downs"], "given-names": ["C. J.", "W. J.", "J. C."], "source": ["Biomechanics of the Eye"], "publisher-name": ["Kugler Publications"], "year": ["2018"]}, {"label": ["3."], "surname": ["Reinstein", "Archer", "Gobbe"], "given-names": ["D. Z.", "T. J.", "M."], "article-title": ["The history of LASIK"], "source": ["J. Refractive Surg."], "volume": ["28"], "issue": ["4"], "fpage": ["291"], "lpage": ["298"], "year": ["2012"], "pub-id": ["10.3928/1081597X-20120229-01"]}, {"label": ["5."], "surname": ["Comaish", "Lawless"], "given-names": ["I. F.", "M. A."], "article-title": ["Progressive post-LASIK keratectasia: biomechanical instability or chronic disease process?"], "source": ["J. Cataract Refractive Surg."], "volume": ["28"], "issue": ["12"], "fpage": ["2206"], "lpage": ["2213"], "year": ["2002"], "pub-id": ["10.1016/S0886-3350(02)01698-X"]}, {"label": ["6."], "surname": ["Binder"], "given-names": ["P. S."], "etal": ["et al."], "article-title": ["Keratoconus and corneal ectasia after LASIK"], "source": ["J. Refractive Surg."], "volume": ["21"], "issue": ["6"], "fpage": ["749"], "lpage": ["752"], "year": ["2005"], "pub-id": ["10.3928/1081-597X-20051101-15"]}, {"label": ["8."], "surname": ["Zvietcovich"], "given-names": ["F."], "etal": ["et al."], "article-title": ["Multi-meridian wave-based corneal optical coherence elastography in normal and keratoconic patients"], "source": ["ARVO Annual Meeting"], "fpage": ["2380"], "lpage": ["A0183"], "year": ["2022"], "pub-id": ["IOVSDA"], "issn": ["0146-0404"]}, {"label": ["9."], "surname": ["Roberts"], "given-names": ["C."], "article-title": ["Biomechanics of the cornea and wavefront-guided laser refractive surgery"], "source": ["J. Refractive Surg."], "volume": ["18"], "issue": ["5"], "fpage": ["S589"], "lpage": ["592"], "year": ["2002"], "pub-id": ["10.3928/1081-597X-20020901-18"]}, {"label": ["11."], "surname": ["Yang", "Roberts", "Mehta"], "given-names": ["E.", "C. J.", "J. S."], "article-title": ["A review of corneal biomechanics after LASIK and SMILE and the current methods of corneal biomechanical analysis"], "source": ["J. Clin. Exp. Ophthalmol."], "volume": ["6"], "issue": ["6"], "fpage": ["507"], "year": ["2015"], "pub-id": ["10.4172/2155-9570.1000507"]}, {"label": ["12."], "surname": ["Randleman", "Su", "Scarcelli"], "given-names": ["J. B.", "J. P.", "G."], "article-title": ["Biomechanical changes after LASIK flap creation combined with rapid cross-linking measured with Brillouin microscopy"], "source": ["J. Refractive Surg."], "volume": ["33"], "issue": ["6"], "fpage": ["408"], "lpage": ["414"], "year": ["2017"], "pub-id": ["10.3928/1081597X-20170421-01"]}, {"label": ["21."], "surname": ["Nair"], "given-names": ["A."], "etal": ["et al."], "article-title": ["Multiple optical elastography techniques reveal the regulation of corneal stiffness by collagen XII"], "source": ["Investig. Ophthalmol. Visual Sci."], "volume": ["63"], "issue": ["12"], "fpage": ["24"], "lpage": ["24"], "year": ["2022"], "pub-id": ["IOVSDA", "10.1167/iovs.63.12.24"], "issn": ["0146-0404"]}, {"label": ["22."], "surname": ["Matveyev"], "given-names": ["A."], "etal": ["et al."], "article-title": ["Vector method for strain estimation in phase-sensitive optical coherence elastography"], "source": ["Laser Phys. Lett."], "volume": ["15"], "issue": ["6"], "fpage": ["065603"], "year": ["2018"], "issn": ["1612-2011"], "pub-id": ["10.1088/1612-202X/aab5e9"]}, {"label": ["23."], "surname": ["Ghiglia", "Romero"], "given-names": ["D. C.", "L. A."], "article-title": ["Robust two-dimensional weighted and unweighted phase unwrapping that uses fast transforms and iterative methods"], "source": ["J. Opt. Soc. Am. A"], "volume": ["11"], "issue": ["1"], "fpage": ["107"], "lpage": ["117"], "year": ["1994"], "pub-id": ["JOAOD6", "10.1364/JOSAA.11.000107"], "issn": ["0740-3232"]}, {"label": ["26."], "surname": ["Nair"], "given-names": ["A."], "etal": ["et al."], "article-title": ["Multimodal heartbeat and compression optical coherence elastography for mapping corneal biomechanics"], "source": ["Front. Med."], "volume": ["9"], "fpage": ["833597"], "year": ["2022"], "pub-id": ["FMBEEQ", "10.3389/fmed.2022.833597"]}, {"label": ["29."], "surname": ["Zvietcovich", "Larin"], "given-names": ["F.", "K."], "article-title": ["Wave-based optical coherence elastography: the 10-year perspective"], "source": ["Progr. Biomed. Eng."], "volume": ["4"], "issue": ["1"], "fpage": ["012007"], "year": ["2021"], "pub-id": ["PRBEEZ", "10.1088/2516-1091/ac4512"], "issn": ["0920-5438"]}, {"label": ["32."], "surname": ["Bissen-Miyajima"], "given-names": ["H."], "etal": ["et al."], "article-title": ["Role of the endothelial pump in flap adhesion after laser in situ keratomileusis"], "source": ["J. Cataract Refractive Surg."], "volume": ["30"], "issue": ["9"], "fpage": ["1989"], "lpage": ["1992"], "year": ["2004"], "pub-id": ["10.1016/j.jcrs.2004.01.045"]}]
{ "acronym": [], "definition": [] }
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PMC10787576
0
[ "<title>Introduction</title>", "<p>Erectile dysfunction (ED) can occur at any age but is more likely after the age of 40, as it affects 10−15% of men aged 40−49 years, 20−40% of men between 60 and 69 years old, and 50−100% of men aged 70 and above [##REF##26953829##1##]. ED is defined as a persistent and recurrent inability to achieve and maintain a sufficiently firm penile erection for satisfactory sexual intercourse [##REF##8510302##2##]. As this is an age-related issue, given the rapid population aging, it is expected to affect 322 million individuals worldwide by 2025 [##REF##10444124##3##].</p>", "<p>However, extant research states that pohosphodiesterase type 5 inhibitor (PDE5I) treatment contributes to a remarkable recovery of erection. Still, in 20-30% of men, orally-administered PDE5I fails to produce satisfactory results [##REF##18046917##4##]. In 30-50% of such cases, according to some authors, intracavernosal injections of erectogenic drugs, intraurethral alprostadil, or the invasive insertion of a penile prosthesis can yield beneficial results, even though these treatment modalities are liable to more adverse side effects than PDE5I [##REF##29464656##5##].</p>", "<p>Guided by this evidence, the present study aimed to determine the most reliable predictors of PDE5I failure in patients affected by ED to improve treatment outcomes and patient compliance.</p>", "<p>This article was previously posted to the Research Square preprint on September 28, 2023.</p>" ]
[ "<title>Materials and methods</title>", "<p>After Institutional Review Board (IRB) approval (Approval number: E-2153), data for this retrospective study was gathered by reviewing our hospital records for the period spanning from January 2016 to January 2022, focusing on patients with ED in whom PDE5I treatment failed, due to which they subsequently received intracorporeal injections or were booked for penile prostheses. We also identified age-matched patients for whom PDE5I was beneficial to facilitate comparisons and allow the identification of factors that influence treatment outcomes. According to the management protocol in place at our hospital, patients experiencing ED should be given PDE5Is (Sildenafil 50 mg when needed, Tadalafil 20mg when needed) for at least eight weeks. In addition, they should be reevaluated at six-monthly intervals to assess their response and compliance with the treatment, based on which the PDE5I dose and type should be adjusted as required. The protocol was unified across all cases. Patients in whom treatment failed and those that required the adoption of the second and/or third line of treatment were of interest for the present investigation and defined as the PDE5I treatment failure group, and thus formed the study sample. Unmarried patients, patients with Peyronie’s disease, patients who have not completed the course of treatment or did not comply with dose modification, patients with contraindications for PDE5Is or at risk of adverse events (such as patients with a recent history of stroke or myocardial infarction, individuals with low or high blood pressure, as well as those with unstable angina, severe cardiac failure, severe liver impairment, or end-stage kidney dysfunction requiring dialysis), all patients received other oral supplement and/or hormonal treatment, all patients submitted to radical prostatectomy or other radical pelvic surgeries or radiation therapy, and those who refused PDE5I treatment or developed side-effects related to PDE5Is were excluded.</p>", "<p>When reviewing the medical records of patients that met the study inclusion criteria, demographic information (age, marriage duration, comorbidities, smoking status, and body mass index [BMI] obtained during the first clinic visit) was gathered and grade 0 was assigned to those without any comorbidities, whereas grades 1−3 indicated the presence of one, two and at least three chronic medical conditions, respectively. At this stage, we recorded the findings yielded by initial laboratory evaluation tests, including HbA1c, total testosterone (TT), free testosterone (FT), sex hormone-binding globulin (SHBG), estradiol, ollicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, vitamin D, cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride, prostate-specific antigen (PSA), thyroid-stimulating hormone (TSH), thyroxine (T4), and hemoglobin (HB) level. This group of patients was considered the treatment failure group and was compared to the control group, comprising age-matched patients in whom PDE5I treatment yielded satisfactory results.</p>", "<p>As per our protocol, venous blood samples were collected from each patient between 8 AM and 11 AM, following an overnight period of fasting. TT levels were measured using the electrochemiluminescence immunoassay (ECLIA), which is intended for use on Cobas E immunoassay analyzers. Although SHBG levels were determined, the ECLIA is intended for use on Cobas E immunoassay analyzers. FT levels were measured using the ETI-MAX 3000 instrument.</p>", "<p>All analyses were conducted using SPSS version 20 (IBM Corp., Armonk, NY) commercial software. When analyzing patient data, the mean and standard deviation were calculated for continuous variables while reporting relative frequencies for categorical variables. Student’s t-test and analysis of variance (ANOVA) were performed to compare means, while the chi-squared test was used for comparisons involving categorical data. The variables that emerged as statistically significant (at p &lt; 0.05) were used to design a logistic regression model. By comparing patients in whom PDE5I treatment was unsuccessful (the treatment group) to those in whom it was beneficial (controls), the aim was to identify the main risk factors that would contribute to treatment failure. Accordingly, the findings could be used to revise the treatment plan or to offer counselling to patients with ED.</p>" ]
[ "<title>Results</title>", "<p>In this study, the treatment group consisted of 288 patients who experienced treatment failure with PDE5Is, while the control group, matched for age, comprised 225 patients. The age distribution in the treatment failure group had a mean age of 65.3 years with a standard deviation of 12.4, whereas the control group had a mean age of 64.6 years with a standard deviation of 12.1.</p>", "<p>Table ##TAB##0##1## presents the results, revealing no statistically significant differences between the two groups in terms of marriage duration, comorbidities, smoking status, diabetes mellitus (DM), or BMI.</p>", "<p>However, statistically significant differences were observed between the treatment failure group and the control group in the levels of HbA1c (p &lt; 0.0001), free testosterone (p &lt; 0.0001), and SHBG (p &lt; 0.002), as indicated in Table ##TAB##1##2##.</p>", "<p>Notably, no statistically significant differences were found between the two groups in the following parameters: TT, estradiol, prolactin, FSH, LH, triglyceride levels, cholesterol levels, LDL levels, HDL levels, PSA levels, TSH levels, T4 levels, vitamin D levels, and Hb levels (Table ##TAB##1##2##).</p>" ]
[ "<title>Discussion</title>", "<p>Erectile dysfunction is the inability to achieve or sustain penile erection. This condition is influenced by various factors, including neural, psychological, vascular, and hormonal elements [##UREF##0##6##]. ED diagnosis involves a comprehensive clinical assessment, which consists of a detailed medical/physical examination and a review of the patient’s sexual and psychosocial history [##REF##31099420##7##].</p>", "<p>When this cycle is disrupted, men tend to seek medical help and are diagnosed with either ED as a primary condition or as a result of another sexual disorder. Consequently, it is vital to take a detailed sexual history and gather other relevant data (including age, sexual orientation, marital status, and past sexual experiences) to identify the cause of ED and to detect the possible comorbidities, toxic habits, or drugs that could result in ED [##REF##30057280##8##].</p>", "<p>In this context, certain risk factors that predispose patients to ED should also be considered, such as dyslipidemia, high blood level, smoking, diabetes mellitus (DM), cardiovascular diseases, excess weight, and a sedentary lifestyle [##REF##17026597##9##]. While ED is highly prevalent, especially in men aged 60 and above, certain risk factors and their role in treatment success are not sufficiently investigated. Given that PDE5Is are considered the first line of treatment for ED, the objective of the present study was to identify additional risk factors that may undermine PDE5I effectiveness. Therefore, following the guidelines for ED diagnosis and treatment, when gathering the data for this retrospective study, patient records were reviewed to obtain all information related to the physical examination of the genitourinary anatomy and the endocrine, as well as the vascular and neurological systems.</p>", "<p>We obtained the results of all laboratory tests performed at the patients’ first visit to our hospital, including HbA1c, lipid panel, testosterone level, thyroid function tests, prolactin level, and luteinizing hormone (LH) levels, as advocated by previous studies [##REF##34331033##10##,##UREF##1##11##]. In addition, given the high prevalence of vitamin D deficiency in our society (estimated at 60-100%) [##REF##29386955##12##], it was also recorded along with the PSA and CBC values.</p>", "<p>We focused on patients who had received PDE5I inhibitors and assigned those in whom this treatment was ineffective to the treatment group, while those who benefitted from this intervention served as controls. The decision to focus on this specific treatment mode was guided by an ample body of evidence indicating that PDE5Is are effective in treating ED in a majority of affected men [##REF##29884471##13##].</p>", "<p>Our study showed a high level of HbA1c associated with a higher risk of PDE5I treatment failure, as the mean HbA1c level in the control responder group was statistically significantly lower than in the treatment failure group (6.8 ± 1.2 vs. 8.5 ± 1.9, p &lt; 0.0001). In concurrence with what Cayetano-Alcaraz et al. stated, DM seems not only to be the risk factor for developing ED but has also been shown to compromise PDE5I effectiveness [##REF##35929992##14##]. Our study found no significant difference in the number of diabetic patients between the treatment failure group and the control responder group. However, there were statistically significant differences observed in poorly controlled diabetes mellitus, as indicated by HbA1c levels. This implies that higher HbA1c values are indicative of inadequately managed DM and a heightened susceptibility to vascular and neuropathic complications associated with diabetes.</p>", "<p>In our study, we meticulously defined inclusion and exclusion criteria, focusing on patients who had consistently taken PDE5Is for at least eight weeks to ensure a relevant and reliable analysis of the data, given that patient compliance is critical for the effectiveness of treatments, including PDE5Is, used in managing erectile dysfunction. Studies have shown wide variance in PDE5I discontinuation rates, from 14% to over 80%, attributed to various factors such as medical complications, inadequate results, severe side effects, and personal reasons like changes in libido, relationship issues, and dependence on medication for erectile function. Additional barriers like fear of side effects, medication accessibility, and financial burden also play a role. Moreover, some patients cease medication due to a lack of sexual spontaneity [##REF##17026597##9##,##REF##32104681##15##, ####REF##17761385##16##, ##REF##29667180##17##, ##REF##32231275##18####32231275##18##].</p>", "<p>Additionally, in our study, the exclusion criteria were rigorously applied to ensure the safety and applicability of the findings. Patients contraindicated for the use of PDE5Is due to recent cardiovascular events such as stroke or myocardial infarction, those with significant hypotension or uncontrolled hypertension, unstable angina, advanced cardiac failure, severe hepatic insufficiency, or end-stage renal disease necessitating dialysis [##REF##31512069##19##], were systematically excluded from the analysis. This was done to mitigate the risk of adverse events and to uphold the integrity of the study results.</p>", "<p>Based on our analyses, we identified low FT and high SHBG as factors that increased the likelihood of PDE5I treatment failure. As testosterone balance is important for erectile and sexual function, low FT was expected to correlate with ED severity, as confirmed in prior investigations. Also, our results show no statistical significance on the TT level, but in FT and SHBG, they were significant. Relying solely on TT levels is not sufficient for patients with ED, particularly in individuals aged over 60 who frequently exhibit increased SHBG levels. Morgado et al. estimated that one-fourth of men over 60 years old might be incorrectly diagnosed with normal gonadal function due to normal TT levels, while they actually have reduced calculated free testosterone (cFT). It is imperative to update the current diagnostic procedures for male hypogonadism to avoid these inaccuracies. It is recommended that clinical guidelines be expanded to include FT evaluations or cFT estimations to ensure low FT levels are identified even in cases where TT levels are within the normal range, particularly in the elderly population with ED [##REF##35347299##20##].</p>", "<p>Although these findings are highly informative, they need to be interpreted in light of the study's limitations. Specifically, this was a retrospective with an age-matching case-control study. In addition, there is no clear definition for PDE5I treatment failure in the literature; besides, it was not possible to ascertain the exact time of treatment failure after the PDE5I initiation due to infrequent follow-up visits (typically at six-monthly intervals, as is the standard in our hospital). Also, the daily dose of Tadalafil (5 mg) was not evaluated in that group of patients. Moreover, other risk factors (such as psychological factors, partner-related factors, and relationship quality) were not considered. Further research is required to obtain a more comprehensive picture of ED and the reasons behind treatment failure. On the other hand, as all our patients receive PDE5I for free from our hospital pharmacy, treatment cost and affordability were not included in the analysis.</p>" ]
[ "<title>Conclusions</title>", "<p>Men diagnosed with ED are more likely not to benefit from PDE5I treatment if they have high HbA1c levels and/or low free testosterone and/or high SHBG. Relying solely on TT levels is not sufficient for patients with ED, particularly in individuals aged over 60 who frequently exhibit increased SHBG levels.</p>", "<p>Therefore, management of these conditions should be a priority in this cohort, which may improve treatment effectiveness. Further prospective randomized studies are nonetheless required to assess the effects of other factors that were not incorporated into our analyses.</p>" ]
[ "<p>Introduction: Erectile dysfunction (ED) is a prevalent condition, especially in aging populations, with significant implications for quality of life. While phosphodiesterase type 5 inhibitors (PDE5Is) are the first-line treatment, a substantial percentage of patients do not respond satisfactorily. This study aimed to identify predictors of PDE5I treatment failure in ED patients.</p>", "<p>Methods: Data from January 2016 to January 2022 was reviewed for patients with ED who either failed PDE5I treatment or had a successful outcome. Demographic, medical, and laboratory data were collected and analyzed. Patients with contraindications or who did not complete the treatment were excluded.</p>", "<p>Results: The treatment failure group comprised 288 patients, while 225 age-matched patients formed the control responder group. There were no significant differences in marriage duration, comorbidities, smoking, or BMI. However, HbA1c levels, free testosterone, and SHBG were significantly different between the two groups.</p>", "<p>Conclusion: This study found that high HbA1c levels, low free testosterone, and high sex hormone-binding globulin (SHBG) were associated with PDE5I treatment failure. Managing high HbA1c levels and addressing hormonal imbalances may enhance PDE5I treatment effectiveness in ED patients. However, further research is needed to explore other factors contributing to treatment outcomes.</p>" ]
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[ "<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Comparing the demographic data of the patients with erectile dysfunction that benefitted (control responder group) and did not benefit (treatment failure group) from PDE5I treatment.</title><p>BMI: body mass index; DM: diabetes mellitus; P-value significant &lt;0.05</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nParameters\n</td><td rowspan=\"1\" colspan=\"1\">\nTreatment failure group (288)\n</td><td rowspan=\"1\" colspan=\"1\">\nControl responder group (225)\n</td><td rowspan=\"1\" colspan=\"1\">\nP-value\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nAge (years) mean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n65.3 ± 12.4\n</td><td rowspan=\"1\" colspan=\"1\">\n64.6 ± 12.1\n</td><td rowspan=\"1\" colspan=\"1\">\n0.52\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nMarriage duration (years) mean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n36.5 ± 8.2\n</td><td rowspan=\"1\" colspan=\"1\">\n35.7 ± 8.6\n</td><td rowspan=\"1\" colspan=\"1\">\n0.283\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nComorbidities grade 0 N (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n94 (32.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n83 (36.9%)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.417\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nComorbidities grade 1 N (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n104 (36.1%)\n</td><td rowspan=\"1\" colspan=\"1\">\n81 (36%)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.981\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nComorbidities grade 2 N (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n69 (24%)\n</td><td rowspan=\"1\" colspan=\"1\">\n49 (21.8%)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.609\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nComorbidities grade 3 N (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n21 (7.3%)\n</td><td rowspan=\"1\" colspan=\"1\">\n12 (5.3%)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.359\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nDM\n</td><td rowspan=\"1\" colspan=\"1\">\n182(63.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n140(62.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.816\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nSmoking N (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n124 (43%)\n</td><td rowspan=\"1\" colspan=\"1\">\n113 (50.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.236\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nBMI (kg/m<sup>2</sup>) mean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n29.7 ± 5.8\n</td><td rowspan=\"1\" colspan=\"1\">\n29.3 ± 4.5\n</td><td rowspan=\"1\" colspan=\"1\">\n0.39\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Comparing the laboratory results of the patients with erectile dysfunction that benefitted (control responder group) and did not benefit (treatment failure group) from PDE5I treatment.</title><p>HbA1c: hemoglobin A1c, SHBG: sex hormone-binding globulin, Estradiol, FSH: follicle-stimulating hormone, LH: luteinizing hormone, LDL: low-density lipoprotein, HDL: high-density lipoprotein, PSA: prostate-specific antigen, TSH: thyroid-stimulating hormone, T4: thyroxine, Hb: hemoglobin.</p><p>P-value significant &lt;0.05.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Parameters</td><td rowspan=\"1\" colspan=\"1\">Treatment failure group (288) mean ± SD</td><td rowspan=\"1\" colspan=\"1\">Control responder group (225) mean ± SD</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">HbA1c (%)</td><td rowspan=\"1\" colspan=\"1\">8.5 ± 1.9</td><td rowspan=\"1\" colspan=\"1\">6.8 ± 1.2</td><td rowspan=\"1\" colspan=\"1\">&lt;0.0001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Total testosterone (nmol/L)</td><td rowspan=\"1\" colspan=\"1\">17.3 ± 7.5</td><td rowspan=\"1\" colspan=\"1\">18.4 ± 8.7</td><td rowspan=\"1\" colspan=\"1\">0.125</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Free testosterone (pmol/L)</td><td rowspan=\"1\" colspan=\"1\">30.9 ± 13.3</td><td rowspan=\"1\" colspan=\"1\">39.3 ± 23.4</td><td rowspan=\"1\" colspan=\"1\">&lt;0.0001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">SHBG (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">52.6 ± 26.7</td><td rowspan=\"1\" colspan=\"1\">45.8 ± 22.9</td><td rowspan=\"1\" colspan=\"1\">0.002</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Estradiol (pmol/L)</td><td rowspan=\"1\" colspan=\"1\">127.9 ± 52.2</td><td rowspan=\"1\" colspan=\"1\">120.1 ± 51.1</td><td rowspan=\"1\" colspan=\"1\">0.09</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">FSH (IU/L)</td><td rowspan=\"1\" colspan=\"1\">6.9 ± 6.1</td><td rowspan=\"1\" colspan=\"1\">5.9 ± 6.3</td><td rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td rowspan=\"1\" colspan=\"1\">LH (IU/L)</td><td rowspan=\"1\" colspan=\"1\">8.4 ± 10.6</td><td rowspan=\"1\" colspan=\"1\">7.6 ± 8.8</td><td rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Prolactin (mIU/L)</td><td rowspan=\"1\" colspan=\"1\">198.1 ± 118.3</td><td rowspan=\"1\" colspan=\"1\">187.7 ± 119.7</td><td rowspan=\"1\" colspan=\"1\">0.33</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Vitamin D (nmol/L)</td><td rowspan=\"1\" colspan=\"1\">50.7± 28.1</td><td rowspan=\"1\" colspan=\"1\">53.3± 30.9</td><td rowspan=\"1\" colspan=\"1\">0.32</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Cholesterol (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">4.2 ± 2.7</td><td rowspan=\"1\" colspan=\"1\">4.3 ± 2.3</td><td rowspan=\"1\" colspan=\"1\">0.65</td></tr><tr><td rowspan=\"1\" colspan=\"1\">LDL (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">2.7 ± 0.8</td><td rowspan=\"1\" colspan=\"1\">2.6 ± 0.9</td><td rowspan=\"1\" colspan=\"1\">0.18</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">HDL (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">1.02 ± 0.30</td><td rowspan=\"1\" colspan=\"1\">1.07 ± 0.32</td><td rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Triglyceride (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">1.6 ± 0.84</td><td rowspan=\"1\" colspan=\"1\">1.7 ± 0.91</td><td rowspan=\"1\" colspan=\"1\">0.197</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">PSA (micg/L)</td><td rowspan=\"1\" colspan=\"1\">1.7 ± 1.9</td><td rowspan=\"1\" colspan=\"1\">1.6 ± 1.8</td><td rowspan=\"1\" colspan=\"1\">0.55</td></tr><tr><td rowspan=\"1\" colspan=\"1\">TSH (IU/L)</td><td rowspan=\"1\" colspan=\"1\">3.1 ± 4.7</td><td rowspan=\"1\" colspan=\"1\">3.5 ± 5.1</td><td rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">T4 (pmol/L)</td><td rowspan=\"1\" colspan=\"1\">15.2 ± 2.6</td><td rowspan=\"1\" colspan=\"1\">15.3 ±3.6</td><td rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr><td rowspan=\"1\" colspan=\"1\">HB (g/L)</td><td rowspan=\"1\" colspan=\"1\">14.3 ± 1.8</td><td rowspan=\"1\" colspan=\"1\">14.5 ± 1.5</td><td rowspan=\"1\" colspan=\"1\">0.19</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Muath Albarakati, Hossam S. El-Tholoth, Abdulaziz Alzahrani, Omar S. Alghamdi, Musa Alnuami, Abdalmagid Althobity, Khaled Bedaiwi </p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Muath Albarakati, Hossam S. El-Tholoth, Abdulaziz Alzahrani, Omar S. Alghamdi, Abdulrahman Alquliti, Abdulmalik Almardawi, Khaled Bedaiwi </p><p><bold>Drafting of the manuscript:</bold>  Muath Albarakati, Hossam S. El-Tholoth, Abdulaziz Alzahrani, Omar S. Alghamdi, Abdulrahman Alquliti, Musa Alnuami, Abdalmagid Althobity, Khaled Bedaiwi </p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Muath Albarakati, Hossam S. El-Tholoth, Abdulmalik Almardawi, Khaled Bedaiwi </p><p><bold>Supervision:</bold>  Musa Alnuami, Abdalmagid Althobity, Abdulmalik Almardawi, Khaled Bedaiwi </p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Prince Sultan Military Medical City (IRB) issued approval E-2153. We are pleased to announce that our research project, titled \"Predictors of \nPhosphodiesterase Type 5 Inhibitor (PDE5I) Treatment Failure in Patients Diagnosed with Erectile Dysfunction\" has been granted full approval by the Prince Sultan Military Medical City (IRB) Approval number: E-2153</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
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[{"label": ["6"], "article-title": ["Neuroanatomy and function of human sexual behavior: a neglected or unknown issue?"], "source": ["Brain Behav"], "person-group": ["\n"], "surname": ["Calabr\u00f2", "Cacciola", "Bruschetta"], "given-names": ["RS", "A", "D"], "fpage": ["0"], "volume": ["9"], "year": ["2019"]}, {"label": ["11"], "article-title": ["Is milder psychological stress responsible for more severe erectile dysfunction?"], "source": ["Andrologia"], "person-group": ["\n"], "surname": ["Liao", "Li", "Tang", "Li", "Tang"], "given-names": ["ZC", "XC", "YX", "DJ", "ZY"], "fpage": ["0"], "volume": ["52"], "year": ["2020"]}]
{ "acronym": [], "definition": [] }
20
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50515
oa_package/36/f9/PMC10787576.tar.gz
PMC10787577
0
[ "<title>Introduction</title>", "<p>The incidence of coronary artery disease (CAD) in women older than 65 years is similar to that in men and even surpasses that in men after 75 years [##UREF##0##1##]. Young women suffer less from heart diseases due to the vascular protective action of estrogen, which helps in preventing atherosclerosis. During menopause, a woman’s estrogen levels drop to approximately one-third of those during pre-menopausal years. The primary causes of morbidity and death for women in both developed and developing nations are ischemic heart disease and cerebrovascular events (i.e., strokes), which are exacerbated by changes in the synthesis of female hormones during menopause.</p>", "<p>Recognition of prodromal symptoms was reported to be critical for preemptive coronary heart disease screening, and effective diagnosis and treatment. At the time of presentation of acute coronary syndrome (ACS), 96% of women experience prodromal symptoms, with unusual fatigue (73%) and sleep disturbance (50%) being the most common symptoms [##REF##20045850##2##]. It is important to recognize the symptoms that are associated with acute subsequent cardiac events. However, symptoms among younger women are atypical, and patients with silent myocardial ischemia usually have more extensive and severe diseases.</p>", "<p>Coronary angiographic profiles reveal a distinct difference in the pattern of CAD between premenopausal and postmenopausal women, with a greater incidence of angiographically normal epicardial coronaries in the former, suggesting a non-atherosclerotic pathology. Even in those with significant coronary lesions, single-vessel disease is more common among premenopausal women, whereas among postmenopausal women, multi-vessel disease is the norm, with the majority having three-vessel disease [##REF##27471758##3##]. Gensini and Friesinger scores, which are used to assess the severity of CAD, are also significantly higher in postmenopausal women [##UREF##1##4##].</p>", "<p>Compared to premenopausal women with ACS, postmenopausal women with ACS tend to have more vulnerable culprit lesions. Moreover, characteristics of vulnerable plaques, such as thin-cap fibroatheroma, lipid-rich plaque, large lipid arc, longer lipid length, macrophage accumulation, microchannel, and cholesterol crystals, are more common in postmenopausal women [##REF##34673203##5##]. Postmenopausal and premenopausal women also differ in the symptoms, risk factors, disease characteristics, prognosis, and recurrence of CAD. However, due to the low incidence of CAD in premenopausal women, research on this particular population remains insufficient [##UREF##2##6##]. Therefore, this study aimed to investigate the complete sociodemographic characteristics, risk factors, clinical presentation, coronary angiographic profile, vessel score, and Gensini score of pre- and postmenopausal women with ACS.</p>" ]
[ "<title>Materials and methods</title>", "<p>This observational, cross-sectional study was performed at a medical teaching hospital in the central region of Bangladesh. Institutional ethical approval was obtained from the Ethics Review Committee of National Heart Foundation Hospital and Research Institute (Post Graduate Research Ref No. N.H.F.H.&amp; R.I 4-14/7/Ad/04, dated December 28, 2021) before enrolling subjects in the study. Written informed consent was obtained from all study participants. The inclusion criteria were pre- and postmenopausal women with ACS who underwent coronary angiogram while admitted to the Department of Cardiology of National Heart Foundation Hospital and Research Institute, Dhaka, Bangladesh, between December 2021 and November 2022. The exclusion criteria were those with associated valvular heart diseases, congenital heart diseases, cardiomyopathy, and extremely severe concomitant diseases (e.g., severe dementia and advanced malignancy), as well as those who were unwilling to participate in the study.</p>", "<p>Postmenopause was defined as a lack of menstrual bleeding for 12 months or a history of hysterectomy [##REF##18313505##7##]. Premenopause was defined as not having experienced menopause or oophorectomy [##REF##6823874##8##].</p>", "<p>Patients were categorized into two groups according to menopausal status. Group I comprised premenopausal women with ACS, and Group II compromised postmenopausal women with ACS. ACS was diagnosed in patients consistent with a compatible clinical presentation and was further confirmed by electrocardiogram. Cardiac biomarker testing was done to diagnose the subtype of ACS. Complete sociodemographic characteristics, risk factors, clinical presentation, coronary angiographic profile, vessel score, and Gensini score were recorded in this study. Gensini score, which was first described in 1975 by Goffredo G. Gensini, is a widely used angiographic scoring system for quantifying the severity of CAD; it considers the geometrical severity of the lesion, cumulative effects of multiple obstructions, and the significance of the affected myocardium. Gensini score considers three main parameters for each coronary lesion: severity score, region multiplying factor, and collateral adjustment factor. First, a non-linear score is assigned to each lesion according to the reduction of the lumen diameter. A multiplier is then applied to the lesions depending on the functional significance of the area supplied by that segment. The final score is the sum of the lesion scores. Thus, Gensini Score = severity score × segment location multiplying factor × collateral adjustment factor. In particular, a lesion is defined as significant when it causes a ≥1% reduction in luminal diameter by visual assessment. The relative severity of the lesion is indicated using a score of 1 for 1%-25% obstruction and doubling that number as the severity of the obstruction progresses. For example, reductions of 25%, 50%, 75%, 90%, 99%, and complete occlusion are given Gensini scores of 1, 2, 4, 8, 16, and 32, respectively. In addition, the Gensini score considers the typical blood flow to the left ventricle in each conduit or section of a vessel, taking into account the differences between the left and right dominant coronary systems. Depending on where a lesion is located in the coronary tree and the functional significance of the area it supplies, a multiplier is given to each lesion score. A collateral adjustment factor is utilized-and the adjustment is decreased by the degree of illness in the vessel that is the source of collaterals if a segment is fully occluded or 99% stenosed and receiving collaterals. The total of all the lesion scores determines the final Gensini score [##REF##6823874##8##].</p>", "<p>Statistical analysis</p>", "<p>The sample size was calculated using Gpower version 3.1.9.4, where the mean difference between two independent means (two groups) was applied, taking effect size as 0.50, alpha error as 0.05, power as 80%, and location ratio as 1. The total sample size was 140. Data entry and all statistical analyses were performed using SPSS version 22 (IBM Corp., Armonk, NY). Categorical data were reported in terms of percentage and frequency, and continuous variables were reported in terms of mean±standard deviation (SD).</p>", "<p>Means of continuous variables were compared using Student’s t-test. Associations between categorical variables were evaluated using the chi-squared test. Correlation studies of continuous variables were conducted using Pearson’s correlation coefficient. A p-value of &lt;0.05 (with 95% confidence interval) indicated statistical significance.</p>" ]
[ "<title>Results</title>", "<p>A total of 140 cases of ACS diagnosed in the Department of Cardiology from December 2021 to November 2022 were included in this study. The mean ages of Group I and Group II were 41.53 ± 5.45 years and 57.23 ± 7.45 years, respectively. The largest age group in Group I was 41-45 years (45.7%, n=32), whereas that in Group II was 56-60 years. Among the respondents, hypertension, dyslipidemia, family H/O premature CAD, and diabetes mellitus (DM) were the most common risk factors in both groups. No statistically significant differences in hypertension and dyslipidemia incidence were observed between the two groups (p&gt;0.05). However, DM and smokeless tobacco were more prevalent in Group II (p&lt;0.05). Family H/O premature CAD was significantly more common in Group I, as was oral contraceptive pill (OCP) intake. Most of the patients in Group I were obese (58.6%, n=41), followed by normal (25.7%, n=18), and overweight (15.7%, n=11). Similarly, in Group II, most patients were obese (50.0%, n=35), followed by overweight (28.6%, n=20), normal (20%, n=14), and underweight (1.4%, n=1). Analysis revealed that there were no statistically significant differences between the two groups (p&gt;0.05) (Table ##TAB##0##1##).</p>", "<p>Among the study population, clinical presentation other than angina pain revealed that atypical presentation (e.g., palpitation, epigastric pain, tingling and numbness sensation in the limbs, and feelings of uneasiness) was common in Group I, whereas shortness of breath was common in Group II. There was a statistically significant difference in atypical presentation between the two groups (p&lt;0.05). Clinically, it was evident that in Group I the majority of patients presented with unstable angina (58.6%), followed by non-ST-elevation myocardial infarction (NSTEMI) (30%) and ST-elevation myocardial infarction (STEMI) (11.4%), whereas in Group II, these rates were 42.9%, 37.1%, and 20.0%, respectively. Analysis revealed that there was no statistically significant difference in subsets of ACS between the two groups (p&gt;0.05) (Table ##TAB##1##2##).</p>", "<p>The mean values of fasting blood sugar (FBS), HbA1C, and serum creatinine were higher in Group II than in Group I. There were statistically significant differences in the mean values of HbA1c and serum creatinine between the two groups (p&lt;0.05), whereas there was no statistically significant difference in that of FBS (p&gt;0.05). The mean values of troponin-I and creatine kinase myocardial band (CKMB) were also higher in Group II than in Group I, but these differences were not statistically significant (p&gt;0.05). The mean value of HDL-C was lower in Group II than in Group I, and this difference was significant (p&lt;0.05) (Table ##TAB##2##3##).</p>", "<p>The severity of CAD in the study patients was assessed in terms of the number of coronary artery involvement. In Group I, 31.4% (n=22) of patients had single-vessel disease, 17.1% (n=12) had double-vessel disease, and 20% (n=14) had triple vessel disease, whereas in Group II, these rates were 15.7% (n=11), 18.6% (n=13), and 48.6% (n=34), respectively. Single-vessel disease was more prevalent in premenopausal women (p&lt;0.002), and triple-vessel disease was more prevalent in postmenopausal women (p&lt;0.001). The normal vessel was also significant regarding the site of coronary artery lesions of RCA, LCX, and LAD vessels were normal more in Group I, and proximal involvement was more common in Group II, with these findings being statistically significant (p&lt;0.05). LMCA was involved in 18.6% (13) of patients in Group I and 15.7% (11) of patients in Group II, but this finding was not statistically significant and more prevalent in premenopausal women (Table ##TAB##3##4##).</p>", "<p>Among the respondents, mean Gensini score was 33.5±36.9 in Group I and 56.1±43.4 in Group II, which was a statistically significant difference (p&lt;0.05) (Table ##TAB##4##5##, Figure ##FIG##0##1##).</p>" ]
[ "<title>Discussion</title>", "<p>In this study, the mean ages of patients in Group I and Group II were 41.53 ± 5.45 years and 57.23 ± 7.45 years, respectively. Most of the patients in Group I belonged to the 41-45 years age group, whereas most in Group II belonged to the 56-60 years age group; these results agreed with those of a study done by Ahmed et al., who found mean ages of 41.6 ± 3.8 years and 56.0 ± 7.2 years in their study, respectively [##UREF##1##4##].</p>", "<p>Regarding risk factors, hypertension, DM, dyslipidemia, family H/O premature CAD, and obesity were the most common in both groups. Smoking was not identified in either group, as smoking is a rare occurrence in female patients in our country. However, DM and smokeless tobacco were significantly more common in postmenopausal women. Prevalence of DM also increased with age and also long-standing uncontrolled DM was an important risk factor for multi-vessel coronary artery involvement and diffuse disease. Smokeless tobacco intake is more common in our country, especially in rural areas. Family history of premature CAD and OCP intake were significantly more common in premenopausal women. Ahmed et al., who performed a study on pre- and postmenopausal women with ACS in Bangladesh [##UREF##1##4##], found similar risk factor types in their work, namely dyslipidemia, obesity, smoking, DM, hypertension, and a family history of premature CAD. Hypertension was also a common risk factor in 76% of patients. A different study done by Islam et al. [##REF##30174432##9##] showed an association between smokeless tobacco intake and CAD, which agreed with our results.</p>", "<p>Diabetes carries a greater risk in female patients, as it is associated with platelet abnormalities, endothelial dysfunction, and negating the protective effects of estrogen [##REF##27471758##3##]. Many recent studies have also shown that hypertension and DM are the two major risk factors for CAD in women [##REF##27471758##3##,##UREF##3##10##, ####REF##28321018##11##, ##REF##29922097##12####29922097##12##]. The proportion of patients with none of the above conventional risk factors and still having CAD was significantly higher in the premenopausal group, signifying that there are factors other than conventional ones contributing to CAD in young women [##REF##27471758##3##].</p>", "<p>Our results showed that shortness of breath and atypical presentation (e.g., palpitation, epigastric pain, tingling and numbness sensation in the limbs, and feelings of uneasiness) were significantly more common in premenopausal patients, which matched the results of a previous study by Yihu et al. [##REF##28321018##11##]. Unstable angina was the most common diagnosis in both groups, followed by NSTEMI and STEMI. This finding may be the result of coronary vasospasm being common in premenopausal women due to hormonal protection at reproductive age, as seen in previous studies [##REF##27471758##3##]. Mean values of HbA1c and serum creatinine were significantly higher in the postmenopausal group, and HDL-C was lower, which may be due to the multiple comorbidities and lack of hormonal protection with progressively increasing age. In menopause, lipid metabolism is significantly affected, which is mainly manifested by an increase in LDL-C and a decrease in HDL-C [##REF##28321018##11##].</p>", "<p>The mean left ventricular ejection fraction (LVEF) of Group II patients was lower than that of Group I patients. LVEF decreased with advancing age in those with ACS. Postmenopausal patients suffer more from STEMI and NSTEMI than premenopausal patients, which may affect the reduction of LVEF in postmenopausal patients. Premenopausal women have significantly more normal coronary angiogram and SVD results, which may be due to the higher incidence of microvascular dysfunction in premenopausal women. TVD was significantly more common in postmenopausal women due to DM, increased age, and more AMI, as reported in other studies [##UREF##1##4##].</p>", "<p>The number of coronary artery involvement in premenopausal women demonstrated more frequent single-vessel involvement (24% vs. 52%), whereas triple-vessel involvement was more common in postmenopausal women (12% vs. 40%, p&lt;0.05). The coronary artery lesion site distribution in Group I and Group II revealed that the left anterior descending artery was most commonly involved, followed by the right coronary artery and the left circumflex artery. Left main coronary artery involvement was more common in Group I than in Group II. Differences in the involvement of the right coronary artery and the left anterior descending artery were highly statistically significant between the two groups (p&lt;0.001).</p>", "<p>In Group I, proximal LAD involvement was more common. In Group II, in addition to proximal LAD involvement, proximal LCX, and proximal and mid-RCA involvement were also common. At present, there is no authoritative statement about why LAD disease occurs more often in premenopausal women. Taking the anatomical structure of the left anterior descending artery into account, it is more active, and the left ventricle must consume more oxygen and nutrients. The anterior descending artery is more easily involved, as it is the main blood supply of the left ventricle, and it supports large areas of the ventricle. In their study, Ahmed et al. showed that proximal left anterior descending artery (LAD) lesions were the most common in the premenopausal group (56%), followed by RCA (36%) and LCX (30%). Our study found similar results, as the most common lesion in premenopausal women was LAD, followed by LCX and RCA. Similar findings were found by Ke-fei et al. [##UREF##4##13##], with the involvement of LAD, LCX, and RCA at 50.4%, 39.1%, and 40.7%, respectively. Vessel score indicated that single-vessel involvement was most common in Group I, whereas triple-vessel involvement was most common in Group II. Statistically significant differences in SVD and TVD were found between the two groups (p&lt;0.05). In terms of severity assessment, the average Gensini score was 33.5± 36.9 in Group I and 56.1±43.4 in Group II, and this difference was highly statistically significant (p&lt;0.001).</p>", "<p>The Gensini and vessel scores of patients were much lower, which meant the lesions were confined mostly to one or two blood vessels. This result was most likely because young women are more likely to have inflammation, coronary spasm, plaque erosion, or rupture, and spontaneous coronary artery dissection. On the other hand, the clinical manifestations of postmenopausal women were more complex, in which collateral circulation was more easily formed, as they had a longer disease duration and long-term progression of the disease course. Many studies have also shown that postmenopausal women have higher CAD severity compared to premenopausal women, as evidenced by vessel score and Gensini score [##UREF##1##4##,##UREF##4##13##].</p>", "<p>There are several limitations of our study. The study was conducted at a public hospital in Dhaka city, in which the respondents were self-selected purposively; therefore, it cannot be assumed that this sample is representative of the entire population of Bangladesh, as results may differ in different socio-demographic or cultural situations. Furthermore, in this study, the in-hospital outcome was not considered. Women with ACS who underwent CAG were included in the study, which may inadequately reflect the angiographic profile of women in the region. Finally, patient grouping was done on the basis of menstrual history, which may have been inadequate.</p>" ]
[ "<title>Conclusions</title>", "<p>Our results showed that the risk of ACS in women is low but not uncommon during the premenopausal period. Hypertension, DM, family history of premature CAD, and dyslipidemia were the most common risk factors for ACS in both groups, and OCP intake was also common in the premenopausal group. Smokeless tobacco intake was common in the postmenopausal group.</p>", "<p>Coronary angiographic findings revealed less severe lesions and higher single-vessel involvement (most commonly LAD involvement) in premenopausal women compared to postmenopausal women. However, LM involvement and multiple-vessel involvement were found in the case of a high-risk group. A large-scale community-based study should be carried out to obtain more information. Social awareness regarding primary and secondary prevention, early diagnosis, and treatment are the keys to reducing the morbidity, mortality, and burden of CAD.</p>" ]
[ "<p>Background: Postmenopausal women present with more severe coronary artery disease (CAD) in addition to multiple comorbidities. However, there are limited data available to compare the risk factors, clinical characteristics, and angiographic severity of CAD between pre- and postmenopausal women with the acute coronary syndrome (ACS).</p>", "<p>Aim: This study aimed to assess and compare the severity of CAD in pre- and postmenopausal women with ACS.</p>", "<p>Methods: This cross-sectional observational study was conducted at the Department of Cardiology of NHFH RI. A total of 140 female patients with ACS were enrolled and then divided into Group I (premenopausal) and Group II (postmenopausal) on the basis of menopause history. Clinical data and coronary angiographic severity were compared between both groups.</p>", "<p>Results: The mean age of the premenopausal group was 41.53 ± 5.45 years, and that of the postmenopausal group was 57.23 ± 7.45 years. Family history of premature CAD was significantly more common in the premenopausal group than in the postmenopausal group (35(50%) vs. 23(32.9%); p=0.017)). DM and smokeless tobacco were more prevalent in the postmenopausal group (48(68.6%) vs. 28(40%); p=0.001 and 14(20%) vs. 2(2.9%); p=0.002). Atypical presentation was more common in the premenopausal group (21(30%) vs. 9(12.9%); p=0.013). Most of the patients in both groups presented with unstable angina followed by NSTEMI and STEMI. Mean left ventricular ejection fraction was lower in the postmenopausal group than in the premenopausal group (50.71 ± 8.38% vs. 53.74 ± 7.46%; p=0.026). Normal coronary angiogram and single-vessel disease were more prevalent in the premenopausal group (22(31.4%) vs. 12(17.1%); p=0.04) and (22(31.4%) vs. 11(15.7%); p=0.002), whereas triple-vessel disease was more prevalent in the postmenopausal group (34(48.6% vs. 14(20%); p=0.001). The left anterior descending artery was the most commonly involved vessel in the postmenopausal group (67(95.7%) vs. 60(85.7%); p=0.04). Finally, the mean Gensini score was higher in the postmenopausal group than in the premenopausal group (56.1 ± 43.4 vs. 33.5 ± 36.9; p=0.001).</p>", "<p>Conclusion: Family history of premature CAD and atypical presentation were common in premenopausal ACS patients. DM and smokeless tobacco use were more prevalent in the postmenopausal group than in the premenopausal group. Normal coronary angiogram and single-vessel disease were more prevalent in the premenopausal group, and triple-vessel disease was more common in the postmenopausal group. CAD was more severe in the postmenopausal group.</p>" ]
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[]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Comparison of Gensini score between the two groups with atypical presentation.</title></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Baseline demographic characteristics of the study population (n=140).</title><p>HTN=hypertension, DM=diabetes mellitus, SD=standard deviation, OCP=oral contraceptive pill, BMI=body mass index, CAD=coronary artery disease</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDemographic characteristics\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nGroup I n=70\nn(%)\n</td><td rowspan=\"1\" colspan=\"1\">\nGroup II n=70\nn(%)\n</td><td rowspan=\"1\" colspan=\"1\">\np-value\n</td></tr><tr><td rowspan=\"7\" colspan=\"1\">\n \n \n \n \n \n \nAge (year)\n</td><td rowspan=\"1\" colspan=\"1\">\n≤35\n</td><td rowspan=\"1\" colspan=\"1\">\n12 (17.1)\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n36–40\n</td><td rowspan=\"1\" colspan=\"1\">\n16 (22.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n41–45\n</td><td rowspan=\"1\" colspan=\"1\">\n32 (45.7)\n</td><td rowspan=\"1\" colspan=\"1\">\n3 (4.3)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n46–50\n</td><td rowspan=\"1\" colspan=\"1\">\n10 (14.3)\n</td><td rowspan=\"1\" colspan=\"1\">\n10 (14.3)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.805\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n51–55\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n20 (28.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n56–60\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n21 (30.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n&gt;60\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n16 (22.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nMean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n41.53 ± 5.45\n</td><td rowspan=\"1\" colspan=\"1\">\n57.23 ± 7.45\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr><td rowspan=\"7\" colspan=\"1\">\n \n \n \n \n \n \nRisk factors\n</td><td rowspan=\"1\" colspan=\"1\">\nHTN\n</td><td rowspan=\"1\" colspan=\"1\">\n52 (74.3)\n</td><td rowspan=\"1\" colspan=\"1\">\n58 (82.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.217\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDM\n</td><td rowspan=\"1\" colspan=\"1\">\n28 (40.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n48 (68.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nDyslipidemia\n</td><td rowspan=\"1\" colspan=\"1\">\n30 (42.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n36 (51.4)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.310\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nFamily H/O premature CAD\n</td><td rowspan=\"1\" colspan=\"1\">\n35 (50.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n23 (32.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.017\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nOCP\n</td><td rowspan=\"1\" colspan=\"1\">\n38 (54.3)\n</td><td rowspan=\"1\" colspan=\"1\">\n0(0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nSmokeless tobacco\n</td><td rowspan=\"1\" colspan=\"1\">\n2 (2.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n14 (20.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.002\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSmoking\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nUnderweight\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n1 (1.4)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.316\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nNormal\n</td><td rowspan=\"1\" colspan=\"1\">\n18 (25.7)\n</td><td rowspan=\"1\" colspan=\"1\">\n14 (20)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.421\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nBMI (kg/m<sup>2</sup>)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nOverweight\n</td><td rowspan=\"1\" colspan=\"1\">\n11 (15.7)\n</td><td rowspan=\"1\" colspan=\"1\">\n20 (28.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.067\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nObese\n</td><td rowspan=\"1\" colspan=\"1\">\n41 (58.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n35 (50.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.309\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nTotal\n</td><td rowspan=\"1\" colspan=\"1\">\n70 (100.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n70 (100.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nMean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n26.43 ± 4.28\n</td><td rowspan=\"1\" colspan=\"1\">\n25.70 ± 3.78\n</td><td rowspan=\"1\" colspan=\"1\">\n0.282\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Clinical characteristics of the study patients (n=140).</title><p>STEMI=ST-segment elevation myocardial infarction, NSTEMI=non-ST-segment elevation myocardial infarction, UA=unstable angina</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nClinical characteristics\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nGroup I (n=70)\nn (%)\n</td><td rowspan=\"1\" colspan=\"1\">\nGroup II (n=70)\nn (%)\n</td><td rowspan=\"1\" colspan=\"1\">\np value\n</td></tr><tr><td rowspan=\"2\" colspan=\"1\">\n \nClinical presentation\n</td><td rowspan=\"1\" colspan=\"1\">\nShortness of breath\n</td><td rowspan=\"1\" colspan=\"1\">\n12 (17.1)\n</td><td rowspan=\"1\" colspan=\"1\">\n21 (30.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.073\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nAtypical presentation\n</td><td rowspan=\"1\" colspan=\"1\">\n21 (30.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n9 (12.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.013\n</td></tr><tr><td rowspan=\"3\" colspan=\"1\">\n \n \nClinical diagnosis\n</td><td rowspan=\"1\" colspan=\"1\">\nSTEMI\n</td><td rowspan=\"1\" colspan=\"1\">\n8 (11.4)\n</td><td rowspan=\"1\" colspan=\"1\">\n14 (20.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.164\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nNSTEMI\n</td><td rowspan=\"1\" colspan=\"1\">\n21 (30.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n26 (37.1)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.371\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nUA\n</td><td rowspan=\"1\" colspan=\"1\">\n41 (58.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n30 (42.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.063\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nTotal\n</td><td rowspan=\"1\" colspan=\"1\">\n70 (100.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n70 (100.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Comparison of biochemical and echocardiographic variables between the groups (n=140).</title><p>FBS=fasting blood sugar, HbA1C=Glycated hemoglobin, CKMB=creatine kinase myocardial band, TG=triglycerides, HDL=high-density lipoprotein, LDL=low-density lipoprotein, TC=total cholesterol, LVEF=left ventricular ejection fraction.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nVariables\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nGroup I (n=70)\n</td><td rowspan=\"1\" colspan=\"1\">\nGroup II (n=70)\n</td><td rowspan=\"1\" colspan=\"1\">\np-value*\n</td></tr><tr><td rowspan=\"9\" colspan=\"1\">\n \n \n \n \n \n \n \n \nLab investigation\n</td><td rowspan=\"1\" colspan=\"1\">\nFBS (mmol/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n6.8 ± 2.4\n</td><td rowspan=\"1\" colspan=\"1\">\n7.3 ± 2.2\n</td><td rowspan=\"1\" colspan=\"1\">\n0.232\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nHbA1C (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n6.8 ± 1.7\n</td><td rowspan=\"1\" colspan=\"1\">\n7.7 ± 2.0\n</td><td rowspan=\"1\" colspan=\"1\">\n0.008\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSerum creatinine (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.9 ± 0.1\n</td><td rowspan=\"1\" colspan=\"1\">\n1.1 ± 0.4\n</td><td rowspan=\"1\" colspan=\"1\">\n0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nCKMB (U/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n38.0 ± 39.6\n</td><td rowspan=\"1\" colspan=\"1\">\n42.3 ± 23.8\n</td><td rowspan=\"1\" colspan=\"1\">\n0.470\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nTroponin-I (ng/mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n7.7 ± 22.6\n</td><td rowspan=\"1\" colspan=\"1\">\n17.2 ± 59.7\n</td><td rowspan=\"1\" colspan=\"1\">\n0.225\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nTG (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n127 ± 57.2\n</td><td rowspan=\"1\" colspan=\"1\">\n144.7 ± 68.9\n</td><td rowspan=\"1\" colspan=\"1\">\n0.118\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nHDL(mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n40.5 ± 10.2\n</td><td rowspan=\"1\" colspan=\"1\">\n35.3 ± 6.0\n</td><td rowspan=\"1\" colspan=\"1\">\n&lt;0.001\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nLDL (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n105.1 ± 26.8\n</td><td rowspan=\"1\" colspan=\"1\">\n115 ± 38.4\n</td><td rowspan=\"1\" colspan=\"1\">\n0.066\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nTC (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n156.4 ± 44.1\n</td><td rowspan=\"1\" colspan=\"1\">\n166.2 ± 57.3\n</td><td rowspan=\"1\" colspan=\"1\">\n0.258\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n≤30\n</td><td rowspan=\"1\" colspan=\"1\">\n1 (1.4)\n</td><td rowspan=\"1\" colspan=\"1\">\n0 (0.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n31–44\n</td><td rowspan=\"1\" colspan=\"1\">\n7 (10.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n16 (22.9)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nLVEF (%)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n45–54\n</td><td rowspan=\"1\" colspan=\"1\">\n27 (38.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n27 (38.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n&gt;55\n</td><td rowspan=\"1\" colspan=\"1\">\n35 (50.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n27 (38.6)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nTotal\n</td><td rowspan=\"1\" colspan=\"1\">\n70 (100.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n70 (100.0)\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nMean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n53.74 ± 7.46%\n</td><td rowspan=\"1\" colspan=\"1\">\n50.71 ± 8.38%\n</td><td rowspan=\"1\" colspan=\"1\">\n0.026\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Severity of coronary artery disease in the study population by number of coronary artery involvement (n=140).</title><p>Single vessel disease, double vessel disease, triple vessel disease, right coronary artery, left circumflex artery, left anterior descending artery, left main coronary artery.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Characteristics</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Group I (n=70) n (%)</td><td rowspan=\"1\" colspan=\"1\">Group II (n=70) n (%)</td><td rowspan=\"1\" colspan=\"1\">p-value</td></tr><tr><td rowspan=\"4\" colspan=\"1\">      Number of involved vessels</td><td rowspan=\"1\" colspan=\"1\">None</td><td rowspan=\"1\" colspan=\"1\">22 (31.4)</td><td rowspan=\"1\" colspan=\"1\">12 (17.1)</td><td rowspan=\"1\" colspan=\"1\">0.049</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">SVD</td><td rowspan=\"1\" colspan=\"1\">22 (31.4)</td><td rowspan=\"1\" colspan=\"1\">11 (15.7)</td><td rowspan=\"1\" colspan=\"1\">0.002</td></tr><tr><td rowspan=\"1\" colspan=\"1\">DVD</td><td rowspan=\"1\" colspan=\"1\">12 (17.1)</td><td rowspan=\"1\" colspan=\"1\">13 (18.6)</td><td rowspan=\"1\" colspan=\"1\">0.825</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">TVD</td><td rowspan=\"1\" colspan=\"1\">14 (20.0)</td><td rowspan=\"1\" colspan=\"1\">34 (48.6)</td><td rowspan=\"1\" colspan=\"1\">0.001</td></tr><tr><td rowspan=\"4\" colspan=\"1\">      Number of coronary artery involvement</td><td rowspan=\"1\" colspan=\"1\">RCA</td><td rowspan=\"1\" colspan=\"1\">55 (78.6)</td><td rowspan=\"1\" colspan=\"1\">63 (90.0)</td><td rowspan=\"1\" colspan=\"1\">0.063</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">LCX</td><td rowspan=\"1\" colspan=\"1\">55 (78.6)</td><td rowspan=\"1\" colspan=\"1\">55 (78.6)</td><td rowspan=\"1\" colspan=\"1\">1.00</td></tr><tr><td rowspan=\"1\" colspan=\"1\">LAD</td><td rowspan=\"1\" colspan=\"1\">60 (85.7)</td><td rowspan=\"1\" colspan=\"1\">67 (95.7)</td><td rowspan=\"1\" colspan=\"1\">0.042</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">LMCA</td><td rowspan=\"1\" colspan=\"1\">13 (18.6)</td><td rowspan=\"1\" colspan=\"1\">11 (15.7)</td><td rowspan=\"1\" colspan=\"1\">0.564</td></tr><tr><td rowspan=\"5\" colspan=\"1\">        Site of coronary artery lesion</td><td colspan=\"4\" rowspan=\"1\">RCA[A1] </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Normal</td><td rowspan=\"1\" colspan=\"1\">24 (43.7)</td><td rowspan=\"1\" colspan=\"1\">7 (11.0)</td><td rowspan=\"1\" colspan=\"1\">0.001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Proximal</td><td rowspan=\"1\" colspan=\"1\">19 (34.5)</td><td rowspan=\"1\" colspan=\"1\">43(68.3)</td><td rowspan=\"1\" colspan=\"1\">0.001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Mid</td><td rowspan=\"1\" colspan=\"1\">8 (14.5)</td><td rowspan=\"1\" colspan=\"1\">10 (15.9)</td><td rowspan=\"1\" colspan=\"1\">0.841</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Distal</td><td rowspan=\"1\" colspan=\"1\">4 (7.3)</td><td rowspan=\"1\" colspan=\"1\">3 (4.8)</td><td rowspan=\"1\" colspan=\"1\">0.565</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"5\" rowspan=\"1\">LCX</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Normal</td><td rowspan=\"1\" colspan=\"1\">25 (47.2)</td><td rowspan=\"1\" colspan=\"1\">11 (21.0)</td><td rowspan=\"1\" colspan=\"1\">0.006</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Proximal</td><td rowspan=\"1\" colspan=\"1\">22 (41.5)</td><td rowspan=\"1\" colspan=\"1\">33 (64.7)</td><td rowspan=\"1\" colspan=\"1\">0.018</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Mid</td><td rowspan=\"1\" colspan=\"1\">1 (1.9)</td><td rowspan=\"1\" colspan=\"1\">2 (3.9)</td><td rowspan=\"1\" colspan=\"1\">0.535</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Distal</td><td rowspan=\"1\" colspan=\"1\">5 (9.4)</td><td rowspan=\"1\" colspan=\"1\">5 (9.8)</td><td rowspan=\"1\" colspan=\"1\">0.949</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">OM</td><td rowspan=\"1\" colspan=\"1\">2 (2.9)</td><td rowspan=\"1\" colspan=\"1\">4 (5.7)</td><td rowspan=\"1\" colspan=\"1\">0.404</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"5\" rowspan=\"1\">LAD</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Normal</td><td rowspan=\"1\" colspan=\"1\">23 (39.7)</td><td rowspan=\"1\" colspan=\"1\">9 (15.5)</td><td rowspan=\"1\" colspan=\"1\">0.004</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Proximal</td><td rowspan=\"1\" colspan=\"1\">35 (58.3)</td><td rowspan=\"1\" colspan=\"1\">49 (76.6)</td><td rowspan=\"1\" colspan=\"1\">0.003</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Mid</td><td rowspan=\"1\" colspan=\"1\">1 (1.7)</td><td rowspan=\"1\" colspan=\"1\">6 (9.4)</td><td rowspan=\"1\" colspan=\"1\">0.063</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Distal</td><td rowspan=\"1\" colspan=\"1\">1 (1.7)</td><td rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td rowspan=\"1\" colspan=\"1\">0.300</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Diagonal</td><td rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td rowspan=\"1\" colspan=\"1\">3 (4.3)</td><td rowspan=\"1\" colspan=\"1\">0.800</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"5\" rowspan=\"1\">LMCA</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Normal</td><td rowspan=\"1\" colspan=\"1\">57 (81.4)</td><td rowspan=\"1\" colspan=\"1\">59 (84.3)</td><td rowspan=\"1\" colspan=\"1\">0.654</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Disease</td><td rowspan=\"1\" colspan=\"1\">13 (18.6)</td><td rowspan=\"1\" colspan=\"1\">11 (15.7)</td><td rowspan=\"1\" colspan=\"1\">0.654</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB5\"><label>Table 5</label><caption><title>Severity of coronary artery disease in the study patients in terms of Gensini score (n=140).</title><p>SD=standard deviation.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nVariable\n</td><td rowspan=\"1\" colspan=\"1\">\nGroup I (n=70)\n</td><td rowspan=\"1\" colspan=\"1\">\nGroup II (n=70)\n</td><td rowspan=\"1\" colspan=\"1\">\np-value\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nGensini score\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nMean ± SD\n</td><td rowspan=\"1\" colspan=\"1\">\n33.5 ± 36.9\n</td><td rowspan=\"1\" colspan=\"1\">\n56.1 ± 43.4\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nMedian\n</td><td rowspan=\"1\" colspan=\"1\">\n21\n</td><td rowspan=\"1\" colspan=\"1\">\n56\n</td><td rowspan=\"1\" colspan=\"1\">\n \n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"4\" rowspan=\"1\">\nMean Rank                        58.6                                                   82.4                                                                                 0.001\n</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Ayesha Siddika, Fazila-Tun-Nesa Malik, Md Kalimuddin, Nahidul Hasan, Nazir Ahmed, Mohammad Badiuzzaman, Mir Nesaruddin Ahmed, Ashok Dutta, Mir Ishraquzzaman, Md. Shamim Chowdhury</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Ayesha Siddika, Fazila-Tun-Nesa Malik, Md Kalimuddin, Nahidul Hasan, Nazir Ahmed, Mohammad Badiuzzaman, Mir Nesaruddin Ahmed, Ashok Dutta, Mir Ishraquzzaman, Md. Shamim Chowdhury</p><p><bold>Drafting of the manuscript:</bold>  Ayesha Siddika, Fazila-Tun-Nesa Malik, Md Kalimuddin, Nahidul Hasan, Nazir Ahmed, Mohammad Badiuzzaman, Mir Nesaruddin Ahmed, Ashok Dutta, Mir Ishraquzzaman, Md. Shamim Chowdhury</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Ayesha Siddika, Fazila-Tun-Nesa Malik, Md Kalimuddin, Nahidul Hasan, Nazir Ahmed, Mohammad Badiuzzaman, Mir Nesaruddin Ahmed, Ashok Dutta, Mir Ishraquzzaman, Md. Shamim Chowdhury</p><p><bold>Supervision:</bold>  Ayesha Siddika, Fazila-Tun-Nesa Malik, Md Kalimuddin, Nahidul Hasan, Nazir Ahmed, Mohammad Badiuzzaman, Mir Nesaruddin Ahmed, Ashok Dutta, Mir Ishraquzzaman, Md. Shamim Chowdhury</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050514-i01\" position=\"float\"/>" ]
[]
[{"label": ["1"], "article-title": ["Heart disease and stroke statistics--2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee"], "source": ["Circulation"], "person-group": ["\n"], "surname": ["Thom", "Haase", "Rosamond"], "given-names": ["T", "N", "W"], "fpage": ["0"], "lpage": ["151"], "volume": ["113"], "year": ["2006"]}, {"label": ["4"], "article-title": ["Comparison of angiographic severity of coronary artery diseases between pre- and postmenopausal women with acute coronary syndrome"], "source": ["Bangladesh Heart J"], "person-group": ["\n"], "surname": ["Ahmed", "Das", "Tushar"], "given-names": ["R", "PR", "AZ"], "fpage": ["113"], "lpage": ["123"], "volume": ["36"], "year": ["2021"]}, {"label": ["6"], "article-title": ["Coronary artery disease in premenopausal versus postmenopausal women- risk factors, clinical profile and angiographic characteristics"], "source": ["IOSR J Dent Med Sci"], "person-group": ["\n"], "surname": ["Nambirajan", "Kumar", "Chakkravarthi"], "given-names": ["J", "PP", "D"], "fpage": ["36"], "lpage": ["41"], "volume": ["19"], "year": ["2020"]}, {"label": ["10"], "article-title": ["Comparison of risk factor profile and angiographic pattern among pre-menopausal and post-menopausal women presenting with angina: results from a prospective single center observational study"], "source": ["J Cardiovasc Dis Diagn"], "person-group": ["\n"], "surname": ["Kandoria", "Bhardwaj", "Mahajan"], "given-names": ["A", "R", "K"], "fpage": ["266"], "volume": ["5"], "year": ["2017"]}, {"label": ["13"], "article-title": ["Symptom recognition and healthcare experiences of young women with acute myocardial infarction"], "source": ["Circ Cardiovasc Qual Outcomes"], "person-group": ["\n"], "surname": ["Lichtman", "Leifheit-Limson", "Watanabe"], "given-names": ["JH", "EC", "E"], "fpage": ["0"], "lpage": ["8"], "volume": ["8"], "year": ["2015"]}]
{ "acronym": [], "definition": [] }
13
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50514
oa_package/63/5b/PMC10787577.tar.gz
PMC10787583
0
[]
[ "<title>METHODS</title>", "<p>The study included 47 patients with mastocytosis (24 women, 24 men) with mean age in the 50 range (26–72) years. Systemic mastocytosis with skin lesions was diagnosed in 33 patients. Median tryptase was 43.6 ng/mL (range 7.71–288). The mean BMI was 28 range (17.78–41.02). The control group consisted of 18 healthy volunteers (10 women, 8 men; aged 32–70; the mean age was <italic toggle=\"yes\">M</italic> = 50.89). BMI ranged from 19.16 to 32.03, and its mean value was 26.14. All participants gave their informed consent to participate in the study.</p>", "<p>No statistically significant differences were found between patients with mastocytosis and the control group in terms of age, sex, BMI, and the occurrence of food allergies and hypersensitivity. The exclusion criteria were pregnancy, neoplastic diseases, heart, kidney and liver failure, confirmed infections of the digestive system, chronic inflammation of the intestines, probiotic intake and antibiotic therapy in the last month before collecting a stool sample. The study consisted of three parts. The first part consisted of the clinical assessment based on completed questionnaires on the most frequently reported symptoms, the FFQ questionnaire, and an interview in 24 h of nutritional memory. Secondly, the examination of the intestinal microflora with the method of traditional culture. Stool samples were collected from 45 people diagnosed with mastocytosis. In the third stage, 16 people with mastocytosis who participated in the previous stages of the study, were randomly selected for 16S rRNA sequencing using next generation sequencing (NGS). The control group consisted of 18 volunteers. The consent was given for the examination by the independent Bioethical Committee for Scientific Research of the MUG (approval no. NKBBN/374/2016.</p>", "<p>The stool samples were collected by the patients into sterile containers in accordance with the instructions described in detail in Supplementary files. Briefly, the procedure consisted in collecting material from 8 different sites with a spatula to fill a 150 mL container to about ¾ of its height. The stool was cultured with the quantitative method at the Clinical Microbiology Laboratory of the University Clinical Hospital, where the material was immediately sent from the MUG Allergology Clinic or collected by the patient at home (stored in a refrigerator at 2–8°C) and delivered no later than 12 h before the planned medical visit. The obtained stool samples were marked with a special number and stored at ‐ 80°C until further determination.</p>", "<title>Examination of the intestinal microflora</title>", "<p>The method of serial dilutions was used to determine the number of cells of individual bacterial species in the tested stool samples. Assuming that the bacteria present in the titer of at least 10<sup>5</sup> viable cells in 1 g of stool can modulate the properties of the intestinal microbiota and shape its influence on the functioning of the entire human body, bacteria were isolated from the stool suspension in dilutions from 10<sup>5</sup> to 10<sup>10</sup>.</p>", "<title>Examination of the intestinal microbiota using the NGS technique</title>", "<p>DNA isolation from the collected samples was carried out at A&amp;A Biotechnology in Gdynia. Genomic DNA was isolated by a modified method based on the Genomic Mini AX Bacteria + kit (A&amp;A Biotechnology). Prior to the preparation of the V3‐V4 amplicon library, DNA eluates were checked for quantity and quality. Libraries for sequencing were prepared by random DNA fragmentation followed by ligation of the 5' and 3′ adapters. The adapter ligated fragments were then PCR amplified and gel purified. Libraries were prepared according to the 16S Metagenomic Sequencing Library Preparation Guidelines##UREF##5##\n11\n## using Herculase II Fusion DNA Plymerase Nextera XT Index Kit V2. The quality of the library was checked according to the Illumina qPCR Quantification Protocol Guide.##UREF##6##\n12\n## Libraries have been prepared by Macrogen. For sequencing, the material was transferred to the Macrogen Laboratory, IT and Business Headquarter &amp; Support Center located in the Republic of Korea. Sequencing was performed using the Illumina MiSeq 2x300 bp platform.</p>", "<p>Bioinformatics analysis was performed using CLC Genomic Workbench v. 12 (Qiagen) + Microbial Genomics Modul Plugin v. 4.1 (Qiagen). Data import for analysis and then their format was based on the read pairing (forward and reverse) and the removal of incorrect reads. The first stage of bioinformatics analysis was to remove duplicates and low‐quality readings (quality limit = 0.05). Then, the obtained sequences were assigned to appropriate taxonomic levels (i.e. genus, species) creating operational taxonomic units (Operational Taxonomic Units‐ OTU). The OTU alignment with the corresponding sequences was made using the Greengenes reference database.</p>", "<p>Statistical analyzes were performed using the Statistica 13.3 TIBC software, IBM SPSS Statistics 25 and MS Excel spreadsheet. The level of statistical significance was <italic toggle=\"yes\">p</italic> &lt; 0.05. Statistical tests used in the analyzes are one‐way analysis of variance (ANOVA), Levene's test, post‐hoc NIR tests, Spearman and/or Pearson correlation, chi‐square test, Mann‐Whitney <italic toggle=\"yes\">U</italic> test. Additional analysis of the gut microbiota was based on the determination of alpha and beta diversity using CLC Genomics Workbench version 22.0.</p>", "<p>Alpha diversity describes species diversity and richness in a single sample. The Sh annon and Simpson index as well as Chao1 and Phylogenetic diversity metrics were used to assess alpha diversity, while the non‐parametric Kruskal‐Wallis test was used in the statistical analysis.</p>", "<p>Beta diversity describes the differences in the taxonomic composition between samples. Principal coordinate analysis (PCoA) based on the matrix was used to assess beta diversity in Jaccard, Bray‐Curtis and Unifrac. Multivariate PERMANOVA analysis of variance was used to show differences in beta diversity, and threshold values ​​&lt;0.05 were defined as statistically significant differences.</p>" ]
[ "<title>RESULTS</title>", "<p>Results of the clinical assessment. The examined respiratory and digestive system symptoms, skin symptoms of mastocytosis were related to the frequency of consumption of the studied groups of products (containing glutamate, amines, salicylates, gluten, lactose). Skin symptoms include pruritus, erythroderma, flush, urticaria, angioedema, itchy lips, swelling of the eyelids, recurrent aphthous stomatitis, dry skin, and folliculitis. Gastrointestinal symptoms include abdominal cramps, nausea, vomiting immediately after eating, diarrhea, loose stools, constipation, flatulence, dyspepsia, and heartburn/burning in the esophagus.</p>", "<p>The strongest correlations concerned gastrointestinal symptoms. A fairly strong correlation between the average consumption of the above‐mentioned product classes and the average total of reported symptoms was obtained (rho = 0.60; <italic toggle=\"yes\">p</italic> = 0.000; <italic toggle=\"yes\">N</italic> = 43). Such a correlation between the average consumption of these product classes and the reported symptoms was not statistically significant in the control group (rho = 0.44; <italic toggle=\"yes\">p</italic> = 0.070; <italic toggle=\"yes\">N</italic> = 18).</p>", "<p>In order to determine the nutritional habits, data collected with the food frequency of consumption questionnaire (FFQ‐6) was used. FFQ‐6 questionnaire is a semi‐quantitative questionnaire validated for the Polish population concerning the frequency of consumption of 165 food products (food and beverages) at a specific time in the last month or year. When assessing the reproducibility of the results obtained using FFQ‐6, it was considered a valid measurement tool for assessing the frequency and amount of food consumed. The analysis showed no statistically significant differences in the studied groups (<italic toggle=\"yes\">p</italic> &lt; 0.05). The nutritional habits in patients with mastocytosis and in the control group were similar.</p>", "<p>The results of the 24‐h diet diary were compared with the nutritional standards for the Polish population.##UREF##7##\n13\n## It has been shown that the majority of patients with mastocytosis have low dietary vitamin and mineral content. All patients had an iodine and vitamin D content below the lower limit of normal, and most had insufficient dietary potassium, iron, calcium, magnesium, vitamin E, vitamin C and folate (Table ##TAB##0##1##). Particularly noteworthy is the fact that as many as 94.5% of patients had too little fiber.</p>", "<title>Results of the examination of the intestinal microflora by the inoculation method</title>", "<p>Fecal culture was performed using the quantitative method in 45 patients with mastocytosis. Abnormal stool test results were obtained in 35 patients with mastocytosis. Based on literature data,##UREF##8##\n14\n## the abnormal stool test result was defined as <italic toggle=\"yes\">Escherichia coli</italic> &lt;10<sup>6,</sup> undetectable probiotic flora (i.e. below the adopted cut‐off point of bacterial detection) and the presence of pathogenic microorganisms. The most common cause of abnormal stool results was <italic toggle=\"yes\">E</italic>. <italic toggle=\"yes\">coli</italic> titer &lt;10<sup>6</sup>. Among the microorganisms with high pathogenic potential in patients with mastocytosis, <italic toggle=\"yes\">Klebsiella pneumoniae</italic>, <italic toggle=\"yes\">Clostridium perfringens</italic>, <italic toggle=\"yes\">Clostridium baratii</italic>, <italic toggle=\"yes\">Candida glabrata</italic>, <italic toggle=\"yes\">and Candida albicans</italic> were found.</p>", "<title>The examination of the intestinal microbiota using the NGS technique</title>", "<p>The highest alfa diversity expressed by the Simpson index was observed in the control group and it was statistically significantly greater than in the group of patients with mastocytosis (<italic toggle=\"yes\">p</italic> = 0.04, Kruskal‐Wallis). When analyzing species biodiversity expressed by the Shannon and Chao1 indexes, no statistically significant differences were found in the studied groups (Figure ##FIG##0##1##).</p>", "<p>Beta diversity was assessed using the matrix Jaccard, Bray‐Curtis and Unifrac. Based on the analysis PCoA showed a visible shift to the right of the control group (green points) relative to the group of patients with mastocytosis (red points) (Figure ##FIG##1##2##). Statistically significant differences in the composition of microbial communities were demonstrated by comparing the control group with the group of patients with mastocytosis (PERMAVOVA, Bray‐Curtis, <italic toggle=\"yes\">p</italic> = 0.00011). Subsequent PERMANOVA analyzes using Jaccard, UniFrac weighted, Euclidean matrices also showed significant differences between the control group and mastocytosis (<italic toggle=\"yes\">p</italic> = 0.00011 Jaccard, <italic toggle=\"yes\">p</italic> = 0.00038 UniFrac weighted, <italic toggle=\"yes\">p</italic> = 0.0001 Euclidean).</p>", "<p>Firmicutes were the most numerous types of bacteria (phylum) in all studied groups, the relative abundance of which was expressed as a percentage, respectively: 81% in the control group and 82% in the group of patients with mastocytosis (Figure ##FIG##2##3##). <italic toggle=\"yes\">Bacteroidetes</italic> constituted 8% in the control group and 7% in patients with mastocytosis, respectively. Another dominant type of bacteria was Actinobacteria, which accounted for 4.8% in patients with mastocytosis and 5.5% in the control group. There were statistically significant differences between mastocytosis and the control group (<italic toggle=\"yes\">p</italic> = 0.04852) in the relative abundance of an unidentified genus belonging to the <italic toggle=\"yes\">Enterobacteriacea</italic> family (<italic toggle=\"yes\">Gammaproteobacteria</italic> group).</p>", "<p>When analyzing the composition of the intestinal microbiota depending on the form of mastositis (ISM, BMM, SSM, MIS), no statistically significant differences were found in general bacterial profiles and in the biodiversity of microbial communities expressed by alpha and beta diversity.</p>", "<p>In the group of patients with mastocytosis, a strong positive correlation between the concentration of mast cell tryptase and the bacteria of the genus Suterella, belonging to the type of Proteobacteria, was demonstrated, Figure ##FIG##3##4##. (Pearson <italic toggle=\"yes\">r</italic> = 0.88, <italic toggle=\"yes\">p</italic> = 0.0017) and the bacteria shown in the Table ##TAB##1##2##.</p>" ]
[ "<title>DISCUSSION</title>", "<p>This study showed that the incidence of food intolerance and allergy was similar in patients with mastocytosis and in the control group. The above observations are consistent with the reports of Jarkvist et al., who showed that the incidence of food intolerance in patients with mastocytosis is similar to that in the general population.##REF##32534147##\n15\n## The dietary interview conducted on the basis of the 24‐h nutritional memory clearly showed that 94.5% of patients with mastocytosis have insufficient dietary fiber content. While there is evidence in the available literature that the host genotype affects microbiota differentiation, diet is believed to be a key factor in shaping the gut microflora.##REF##16278440##\n16\n##, ##UREF##9##\n17\n## Experimental studies in mice showed that dietary changes explained as much as 57% of the total variation in the structure of the gut microbiota, while the genetic basis was responsible for only 12% of the differences.##REF##19865183##\n18\n## Based on previous researches, it has been established that a plant‐based diet rich in fats or fiber can promote specific bacterial profiles.##REF##23607879##\n19\n##, ##UREF##10##\n20\n## For example, a high‐fat, low‐fiber diet leads to an increase in the ratio of Firmicutes to Bacteroidetes.##UREF##3##\n7\n## Indeed, this study showed a higher percentage of Firmicutes and a lower percentage of Bacteroidetes in patients with mastocytosis. Folkerts et al. suggested that a low‐fiber Western European diet may contribute to a greater manifestation of allergic diseases.##UREF##3##\n7\n##, ##REF##24390308##\n21\n## The arguments of the researchers are confirmed by the parallel increase in the incidence of allergic diseases to the decreased consumption of fiber in developed countries. Regulating the mast cell activity with a high‐fiber diet is therefore a promising direction for new therapeutic strategies in patients with mastocytosis that we believe should be considered.</p>", "<p>As expected, the highest alpha‐diversity expressed by the Simpson index was observed in the control group and it was statistically significantly greater than in the group of patients with mastocytosis. Greater richness and diversity in the gut microbiome is believed to be more desirable and associated with health.##UREF##11##\n22\n## On the other hand, when analyzing beta diversity on the basis of PCoA, a visible shift to the right on the horizontal axis of the control group in relation to patients with mastocytosis was shown. The above results indicate differences in beta diversity defined as differences in the taxonomic composition between samples (mastocytosis vs. control group). The above observations may provide evidence of differences in the general structure of the intestinal microbiome in patients with mastocytosis and in the control group, resulting from the mechanism of allergy. This observation requires further research.</p>", "<p>When analyzing the quantitative composition of individual types of bacteria, 5 types of bacteria dominated in the control group: Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and <italic toggle=\"yes\">Verrrucomicrobia</italic>, which is consistent with the previous findings on the intestinal microbiome of a healthy human.##UREF##10##\n20\n##, ##UREF##12##\n23\n## In the sequencing process, patients with mastocytosis showed lower abundance of bacteria with beneficial effects for the host: <italic toggle=\"yes\">Faecalibacterium prausnitzii</italic>, <italic toggle=\"yes\">Lachnospira and Roseburia</italic>.<italic toggle=\"yes\">Faecalibacterium prausnitzii</italic> are one of the most important producers of butyrate, the main source of energy for colonocytes. The anti‐inflammatory effect of butyric acid in the intestines consists of inhibiting the activity of the transcription factor NF‐kB, reducing interferon gamma IFN‐γ and increased PPARγ activity. In addition to its anti‐inflammatory effect, <italic toggle=\"yes\">Faecalibacterium prausnitzii</italic> has a positive effect on the permeability of the intestines, strengthening the intestinal barrier.##REF##28045459##\n24\n## Earlier reports showed reduced abundance of <italic toggle=\"yes\">Faecalibacterium prausnitzii</italic> in asthma, Crohn's disease, obesity and depressive disorders.##REF##28045459##\n24\n##, ##REF##25882912##\n25\n##, ##REF##30208875##\n26\n##\n<italic toggle=\"yes\">Roseburia</italic> strengthens the intestinal barrier function and is an important producer of SCFA short‐chain fatty acids.##UREF##13##\n27\n##\n<italic toggle=\"yes\">Lachnospira</italic>, in addition to its ability to produce butyric acid, can also produce acetic acid, which, by affecting Treg cells, inhibited the allergic inflammatory reaction of the airways in a mouse model.##REF##34069415##\n28\n##\n</p>", "<p>The analysis of the commensal microflora of mastocytosis patients showed that <italic toggle=\"yes\">Escherichia coli</italic> titer was below &lt;10<sup>6</sup>. Furthermore, the studied <italic toggle=\"yes\">Escherichia coli</italic> belongs to an extremely diverse species, most of which are non‐pathogenic. Many beneficial functions of E.coli living in the large intestine have been described; apart from participation in the synthesis of vitamins, its immunomodulatory properties in the host organism are also mentioned. According to some researchers, the lower abundance of non‐pathogenic <italic toggle=\"yes\">E</italic>. <italic toggle=\"yes\">coli</italic> strains is associated with allergic diseases.##UREF##14##\n29\n##, ##REF##23536867##\n30\n## Pang et al. It has been demonstrated that oral administration to mice of a non‐pathogenic strain of <italic toggle=\"yes\">E</italic>. <italic toggle=\"yes\">coli</italic> resulted in a statistically significant reduction of allergic symptoms in both the upper and lower respiratory tract.##REF##23536867##\n30\n## The mechanism of suppressing the allergic reaction consisted of reducing the production of pro‐inflammatory Th‐2 lymphocytes cytokines and increasing the activity of Treg lymphocytes secreting the anti‐inflammatory IL‐10.##REF##23536867##\n30\n## The researchers suggest that the above observations are consistent with the current “hygiene theory” and that the reduction of the commensal flora is disadvantageous in terms of achieving immune tolerance.</p>", "<p>Commensal and probiotic bacteria that make up the intestinal microbiome are involved in the fermentation of fiber, the products of which are SCFA: acetate, propionate, butyrate.##REF##32278320##\n31\n##, ##REF##27328006##\n32\n##, ##REF##29522844##\n33\n## Diakos et al. were the first to provide evidence that butyrate can inhibit mast cell degranulation and reduce the production of tumor necrosis factor TNF‐alpha.##REF##16949031##\n9\n## Wang et al. confirmed the beneficial effect of butyrate on the degranulation of mast cells and the reduction of the production of pro‐inflammatory cytokines.##REF##22772452##\n10\n## Butyrate may regulate the function of mast cells through secondary pathways mediated by group 2 innate lymphoid cells (ILC2) 70.71. When subsequent observations bring hope for the possibility of using probiotic strains for specific prophylactic or therapeutic purposes in diseases related to the activation of mast cells, a question arises regarding the composition of probiotic microflora in this group of patients. To our knowledge, this is the first study to characterize the gut microbiome of people with mastocytosis using the NGS method, supported using traditional culture methods.</p>", "<p>This work is one of the first studies characterizing the intestinal microbes in patients with mastocytosis. It has been shown that patients with systemic mastocytosis differ significantly not only in the overall structure of the gut microbiome expressed in alpha and beta diversity from healthy individuals, but also have a unique gut microbial profile. This is confirmed by previous reports of other researchers.##UREF##15##\n34\n## The dominant types of bacteria were <italic toggle=\"yes\">Firmicutes</italic>, <italic toggle=\"yes\">Bacteroidetes</italic>, <italic toggle=\"yes\">Actinobacteria</italic>, <italic toggle=\"yes\">Verrucomicrobia</italic> and <italic toggle=\"yes\">Proteobacteria</italic>. 373 operational taxonomic units unique for mastocytosis were found, which differed significantly from the healthy control group. In patients with mastocytosis, statistically significant differences in the abundance of bacteria belonging to the family <italic toggle=\"yes\">Erysipelotrichaceae</italic> have been described. Although these microorganisms have been described relatively recently by Verbarg et al., there is a growing number of reports documenting the role of <italic toggle=\"yes\">Erysipelotrichaceae</italic> in inflammatory diseases of the gastrointestinal tract.##REF##14742484##\n35\n## A greater number of this family of bacteria was found in patients with colorectal cancer.##REF##22761885##\n36\n## Changes in the abundance of <italic toggle=\"yes\">Erysipelotrichacea</italic> were also observed in patients with inflammatory bowel disease.##REF##23280118##\n37\n## In contrast, Spencer et al provided strong evidence for the association of <italic toggle=\"yes\">Erysipelotrichacea</italic> with host lipid disorders. The researchers showed that <italic toggle=\"yes\">Erysipelotrichi</italic> and <italic toggle=\"yes\">Gammaproteobacteria</italic> were directly related to choline deficiency‐induced steatosis. In this study, it was observed that the percentage of <italic toggle=\"yes\">Erysipelotrichi</italic>it was even greater in patients with mastocytosis who reported abdominal pain. In subsequent studies, it would be interesting to establish the effect <italic toggle=\"yes\">Erysipelotrichacea</italic>on lipid metabolism and inflammation of the gastrointestinal tract in this group of patients. The present study showed no statistically significant differences between the results of microbial stool culture and the concentration of mast cell tryptase. However, in the correlation analysis, the results obtained from bacterial sequencing of 16S rRNA showed a strong positive correlation of tryptase concentration with bacteria at the level of the genus: <italic toggle=\"yes\">Suterella</italic>, <italic toggle=\"yes\">Eubacterium</italic>, <italic toggle=\"yes\">Odoribacter</italic>, and <italic toggle=\"yes\">Anaerostipes</italic>. There is evidence that tryptase affects the differentiation of fibroblasts, causing fibrotic changes in the intestines.##REF##32507895##\n38\n## The above observations clearly show that in future studies of the intestinal microbiota one should look for bacteria characteristic of mastocytosis at the level of the genus, and not only refer to general changes.</p>" ]
[ "<title>CONCLUSION</title>", "<p>The nutrition habits and BMI of mastocytosis patients are similar to the general population, except for too little fiber intake and mineral content. The gastrointestinal symptoms of mastocytosis patients may be related to the low richness of microbiota species and the amount of Suterella, Barnesiellaceae, Eubacterium, Odoribacter, and Anaerostipes, which correlated with tryptase levels.</p>" ]
[ "<title>Abstract</title>", "<title>Background</title>", "<p>Mastocytosis is a rare neoplastic disease of the bone marrow associated with the proliferation and accumulation of mast cells in various internal organs, including the gastrointestinal tract. There are few studies describing the gut microbiome of patients with mastocytosis using next generation sequencing supported using traditional culture methods. The aims of the study were, firstly, the determination of nutrition habits, composition of the intestinal microflora and BMI in mastocytosis, and secondly, analysis of mastocytosis severity and symptoms depending on the composition of the intestinal microflora.</p>", "<title>Methods</title>", "<p>The study included 47 patients with indolent systemic mastocytosis and 18 healthy controls. All participants gave their informed consent to participate in the study. The study consisted of 3 parts: I‐clinical assessment, II ‐ examination of the intestinal microflora using the biochemical method, III ‐ 16S rRNA sequencing.</p>", "<title>Results</title>", "<p>The nutrition habits and BMI of mastocytosis patients were similar to controls; however, most patients with mastocytosis had a low dietary vitamin and mineral content. As many as 94.5% of patients had too little fiber intake and mineral content. The most common cause of the abnormal stool test result with traditional culture was a titer of <italic toggle=\"yes\">E</italic>. <italic toggle=\"yes\">coli</italic> &lt;10<sup>6</sup>. The low richness of microbiota species indicated by the Simpson index was observed in mastocytosis, <italic toggle=\"yes\">p</italic> = 0.04. There were no significant differences in the composition of the intestinal microflora depending on the type of mastocytosis; however, the tryptase level correlated with the amount of Suterella, Barnesiellaceae, Eubacterium, Odoribacter, and Anaerostipes.</p>", "<title>Conclusions</title>", "<p>The nutritional habits and BMI of mastocytosis patients are similar to the general population, except for too little fiber intake and mineral content. The gastrointestinal symptoms of mastocytosis patients may be related to the low richness of microbiota species and the amount of Suterella, Barnesiellaceae, Eubacterium, Odoribacter, Anaerostipes, which correlated with tryptase levels.</p>", "<p content-type=\"self-citation\">\n<mixed-citation publication-type=\"self-citation\" id=\"clt212310-cit-0039\">\n<string-name>\n<surname>Harcęko‐Zielińska</surname>\n<given-names>E</given-names>\n</string-name>, <string-name>\n<surname>Niedoszytko</surname>\n<given-names>M</given-names>\n</string-name>, <string-name>\n<surname>Górska</surname>\n<given-names>A</given-names>\n</string-name>, et al. <article-title>The influence of nutritional habits, body mass index and intestinal microbiota in mastocytosis on clinical symptoms using conventional culture and next generation sequencing</article-title>. <source>Clin Transl Allergy</source>. <year>2024</year>;<elocation-id>e12310</elocation-id>. <pub-id pub-id-type=\"doi\">10.1002/clt2.12310</pub-id>\n</mixed-citation>\n</p>" ]
[ "<title>KEY MESSAGE</title>", "<p>Mastocytosis is a rare (accounting for less than 0.01% of the general population) group of neoplastic diseases of the bone marrow associated with the growth of mast cells and their accumulation in one or more organs, such as the skin, mucous membranes, liver, spleen, or bone marrow.##REF##15941072##\n1\n## About 80% of patients report gastrointestinal complaints, mainly disturbances in the rhythm of bowel movements.##UREF##0##\n2\n## Histamine stimulates the secretion of stomach acid, which increases the risk of developing gastric ulcer and intestinal ulceration. The etiology of frequent diarrhea in patients with mastocytosis remains unknown. Studies suggest an impact of mast cell infiltration in the intestinal wall and the abnormality of the small intestine mucosa itself, which may lead to malabsorption and weight loss.##UREF##1##\n3\n##\n</p>", "<p>The human microbiome consists of 500–1000 different microorganisms with a total biomass of approximately 1.5 kg<sup>4</sup>. Live cells of a fully developed intestinal microflora contain genetic information, which, according to preliminary findings, exceeds the number of host cells by up to a hundred times.##UREF##2##\n4\n##, ##REF##15505215##\n5\n## According to updated data from 2016, the ratio of bacterial cells to human host cells is closer to 1: 1.##REF##27541692##\n6\n##\n</p>", "<p>The diet is an important factor in modifying the intestinal microbiome. A high‐fiber diet plays a key role in the manifestation of diseases related to excessive mast cell proliferation. Short‐chain fatty acids—SCFAs produced by individual bacteria can inhibit mast cell degranulation at the level of the MAPK signaling pathway.##UREF##3##\n7\n## Little is known about the role of the gut microflora in the pathogenesis of this disease.##UREF##4##\n8\n## Due to the rapid technological progress and the constantly expanding knowledge in this subject, this is an exciting time in the study of the human microbiome, which may become an important step in the development of personalized therapy focused on prevention, health maintenance or treatment with a relatively low risk of adverse effects.##REF##16949031##\n9\n##, ##REF##22772452##\n10\n## We found no clinical studies on mastocystosis and microbiome in the PubMed database. The observations made so far have focused mainly on the role of dietary fiber and its metabolites in the pathophysiology of mast cell diseases.##REF##22772452##\n10\n##\n</p>", "<p>The aims of the study were the determination of nutrition habits, composition of the intestinal microflora, and BMI in mastocytosis; secondly, the analysis of the mastocytosis severity and symptoms depending on the composition of the intestinal microflora.</p>", "<title>AUTHOR CONTRIBUTIONS</title>", "<p>\n<bold>Ewelina Harcęko‐Zielińska</bold>: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); project administration (equal); resources (equal); software (equal); supervision (equal); validation (equal); writing – original draft (equal); writing – review &amp; editing (equal). <bold>Marek Niedoszytko</bold>: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); investigation (equal); methodology (equal); project administration (equal); resources (equal); supervision (equal); validation (equal); writing – original draft (equal); writing – review &amp; editing (equal). <bold>Aleksandra Górska</bold>: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); project administration (equal); resources (equal); supervision (equal); writing – review &amp; editing (equal). <bold>Sylwia Małgorzewicz</bold>: Conceptualization (equal); data curation (equal); methodology (equal); resources (equal); supervision (equal); writing – review &amp; editing (equal). <bold>Marta Gruchała‐Niedoszytko</bold>: Conceptualization (equal); data curation (equal); formal analysis (equal); resources (equal); writing – original draft (equal); writing – review &amp; editing (equal). <bold>Marek Bronk</bold>: Data Curation (equal); investigation (equal); methodology (equal); resources (equal); software (equal); supervision (equal); validation (equal); writing – review &amp; editing (equal). <bold>Sławomir Dąbrowski</bold>: Data Curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); supervision (equal); validation (equal). <bold>Marta Chełmińska</bold>: Project Administration (equal); supervision (equal); writing – review &amp; editing (equal). <bold>Ewa Jassem</bold>: Funding acquisition (equal); project administration (equal); supervision (equal); writing – review &amp; editing (equal).</p>", "<title>CONFLICT OF INTEREST STATEMENT</title>", "<p>The authors declare no conflicts of interest.</p>", "<title>Supporting information</title>" ]
[ "<title>ACKNOWLEDGMENTS</title>", "<p>This study was funded by the Medical University of Gdansk ST. Medical University of Gdansk ST number, (ST 01‐10022/0000679/01/231/231/0/2022 &amp; ST number 01‐10023/0004956/01/231/231/0/2023).</p>", "<title>DATA AVAILABILITY STATEMENT</title>", "<p>Data available on request from the authors.</p>" ]
[ "<fig position=\"float\" fig-type=\"FIGURE\" id=\"clt212310-fig-0001\"><label>FIGURE 1</label><caption><p>Alpha diversity expressed by the Simpson index in the control group (green), patients with mastocytosis (red).</p></caption></fig>", "<fig position=\"float\" fig-type=\"FIGURE\" id=\"clt212310-fig-0002\"><label>FIGURE 2</label><caption><p>Beta diversity analysis in the study groups. Green points refer to the control group and red points to mastocytosis. Percentages in the axis descriptions represent the percentage of variation explained by the principal Principal coordinate analysis (PCoA) coordinates as measured using the Bray‐Curtis matrix. Figure (A) shows the 2D view, while figure (B) shows the beta diversity of the studied groups in 3D visualization. The results shown in the figure were calculated and visualized using the CLC Genomics Workbench 22.0 software.</p></caption></fig>", "<fig position=\"float\" fig-type=\"FIGURE\" id=\"clt212310-fig-0003\"><label>FIGURE 3</label><caption><p>Relative microbial abundance at the class level in the study groups.</p></caption></fig>", "<fig position=\"float\" fig-type=\"FIGURE\" id=\"clt212310-fig-0004\"><label>FIGURE 4</label><caption><p>An analysis of the correlation between tryptase concentration and intestinal microorganisms in patients with mastocytosis.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"clt212310-tbl-0001\" content-type=\"TABLE\"><label>TABLE 1</label><caption><p>The content of vitamins and minerals in the diet of patients with mastocytosis the results of the dietary interview were related to the nutritional standards for the Polish population based on, Normy żywienia dla populacji Polski”, Mirosław Jarosz, Instytut Żywności I Żywienia, 2017.</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Norm</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Mean</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">% Abnormal results</th></tr></thead><tbody valign=\"top\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Carbohydrate [g]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">&gt;130</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">232.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Protein [g]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">M</italic> &gt; 50, <italic toggle=\"yes\">F</italic> &gt; 41</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐64.2 F‐ 65.2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐28.6, F‐0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cholesterol [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">&lt;300</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">327.7</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">66.67</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Fiber [g]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17.2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">94.5</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Sodium [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1500</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1854.2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Potassium [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3500</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2786.6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">99.9</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iron [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐10, F‐18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐10.2, F‐14.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐71.4, F‐72.7</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Calcium [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1000</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">562</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88.9</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Phosphorus [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">700</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">924.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">33.3</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iodine [ug]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">150</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20.8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Magnesium [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐420, F‐320</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐199, F‐299</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐100, F‐63.6</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Vitamin A [ug]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐620, F‐500</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐769, F‐2553</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐29, F‐90.9</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Vitamin D [ug]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Vitamin E [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐10, F‐8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐5.7, F‐6.9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Vitamin C [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐90, F‐75</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐63.2, F‐134.1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M‐62.5, F‐57.1</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Vitamin B6 [mg]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11.1</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Vitamin B12 [ug]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51.1</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Folates [ug]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">400</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">299</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">77.8</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"clt212310-tbl-0002\" content-type=\"TABLE\"><label>TABLE 2</label><caption><p>Correlation between tryptase concentration and bacteria at the genus level in patients with mastocytosis.</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Type</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Genus</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Pearson</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">p</italic>‐value</th></tr></thead><tbody valign=\"top\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Bacteroidetes</italic>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Barnesiellaceae</italic>\n<xref rid=\"clt212310-note-0002\" ref-type=\"table-fn\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.0046</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Firmicutes</italic>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Eubacterium</italic>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.74</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.0037</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Bacteroidetes</italic>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Odoribacter</italic>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.71</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.0094</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Firmicutes</italic>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">Anaerostipes</italic>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.0343</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"clt212310-sup-0001\" position=\"float\" content-type=\"local-data\"><caption><p>Supporting Information S1</p></caption></supplementary-material>" ]
[ "<table-wrap-foot id=\"clt212310-ntgp-0001\"><fn id=\"clt212310-note-0001\"><p>Abbreviations: F, female; M, man.</p></fn></table-wrap-foot>", "<table-wrap-foot id=\"clt212310-ntgp-0002\"><fn id=\"clt212310-note-0002\"><label>\n<sup>a</sup>\n</label><p>Unidentified genus belonging to the family Barnesiellaceae.</p></fn></table-wrap-foot>" ]
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[ "<media xlink:href=\"CLT2-14-e12310-s001.docx\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["2"], "mixed-citation": ["\n"], "string-name": ["\n"], "surname": ["Jensen"], "given-names": ["RT"], "article-title": ["Gastrointestinal abnormalities and involvement in systemic mastocytosis"], "source": ["Hematol Oncol Clin N Am"], "year": ["2000"], "volume": ["14"], "issue": ["3"], "fpage": ["579"], "lpage": ["623"], "pub-id": ["10.1016/S0889-8588(05)70298-7"]}, {"label": ["3"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "surname": ["Kruszewski", "Niedoszytko", "Jassem", "Anna"], "given-names": ["J", "M", "E", "B"], "source": ["Mastocytoza: Rozpoznanie i Leczenie"], "volume": ["174"], "publisher-name": ["Agencja \u201cBenkowski\u201d"], "year": ["2007"], "ext-link": ["https://books.google.com/books/about/Mastocytoza.html?hl=pl&id=aaaLtgAACAAJ"]}, {"label": ["4"], "mixed-citation": ["\n"], "string-name": ["\n", "\n"], "surname": ["Xu", "Gordon"], "given-names": ["J", "JI"], "source": ["Honor Thy Symbionts"], "publisher-name": ["Proc Natl Acad Sci U S A."], "year": ["2003"], "pub-id": ["10.1073/pnas.1734063100"]}, {"label": ["7"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "surname": ["Folkerts", "Stadhouders", "Redegeld"], "given-names": ["J", "R", "FA"], "article-title": ["Effect of dietary fiber and metabolites on mast cell activation and mast cell\u2010associated diseases"], "source": ["Front Immunol"], "year": ["2018"], "volume": ["9"], "pub-id": ["10.3389/fimmu.2018.01067"]}, {"label": ["8"], "mixed-citation": ["\n"], "string-name": ["\n", "\n"], "surname": ["Afrin", "Khoruts"], "given-names": ["LB", "A"], "article-title": ["Mast cell activation disease and microbiotic interactions"], "source": ["Clin Therapeut"], "year": ["2015"], "volume": ["37"], "issue": ["5"], "fpage": ["941"], "lpage": ["953"], "pub-id": ["10.1016/j.clinthera.2015.02.008"]}, {"label": ["11"], "mixed-citation": ["\n"], "collab": ["Illumina"], "article-title": ["16S Metagenomic Sequencing Library Preparation"]}, {"label": ["12"], "mixed-citation": ["\n"], "collab": ["Illumina"], "article-title": ["Sequencing Library QPCR Quantification Guide"], "year": ["2011"]}, {"label": ["13"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "surname": ["Szponar", "Wolnicka", "Rychlik"], "given-names": ["L", "K", "E"], "article-title": ["Album Fotografii Produkt\u00f3w i Potraw"], "year": ["2000"], "ext-link": ["www.castudio.com.pl"]}, {"label": ["14"], "mixed-citation": ["\n"], "string-name": ["\n"], "surname": ["Blount"], "given-names": ["ZD"], "article-title": ["The unexhausted potential of E. coli"], "source": ["Elife"], "year": ["2015"], "volume": ["4"], "pub-id": ["10.7554/ELIFE.05826"]}, {"label": ["17"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n"], "surname": ["Zoetendal", "Akkermans", "van Akkermans Vliet", "de Visser", "de Vos"], "given-names": ["EG", "ADL", "WM", "JAGM", "WM"], "article-title": ["The host genotype affects the bacterial community in the human gastrointestinal tract"], "source": ["Microb Ecol Health Dis"], "year": ["2001"], "volume": ["13"], "issue": ["3"], "fpage": ["129"], "lpage": ["134"], "pub-id": ["10.1080/089106001750462669"]}, {"label": ["20"], "mixed-citation": ["\n"], "string-name": ["\n", "\n"], "surname": ["Stachowicz", "Kiersztan"], "given-names": ["N", "A"], "article-title": ["Rola mikroflory jelitowej w patogenezie oty\u0142o\u015bci i cukrzycy"], "source": ["Postepy Hig Med Dosw"], "year": ["2013"], "volume": ["67"], "fpage": ["288"], "lpage": ["303"], "pub-id": ["10.5604/17322693.1044746"]}, {"label": ["22"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "surname": ["Liu", "Ma", "Xu"], "given-names": ["A", "T", "N"], "article-title": ["Adjunctive probiotics alleviates asthmatic symptoms via modulating the gut microbiome and serum metabolome"], "source": ["Microbiol Spectr"], "year": ["2021"], "volume": ["9"], "issue": ["2"], "pub-id": ["10.1128/SPECTRUM.00859-21"]}, {"label": ["23"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "surname": ["Harris", "Kassis", "Major", "Chou"], "given-names": ["K", "A", "G", "CJ"], "article-title": ["Is the gut microbiota a new factor contributing to obesity and its metabolic disorders?"], "source": ["Journal of Obesity"], "year": ["2012"], "volume": ["2012"], "fpage": ["1"], "lpage": ["14"], "pub-id": ["10.1155/2012/879151"]}, {"label": ["27"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "surname": ["Nie", "Ma", "Luo"], "given-names": ["K", "K", "W"], "article-title": ["Roseburia intestinalis: a beneficial gut organism from the discoveries in genus and species"], "source": ["Front Cell Infect Microbiol"], "year": ["2021"], "volume": ["11"], "pub-id": ["10.3389/FCIMB.2021.757718"]}, {"label": ["29"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n"], "surname": ["Boudeau", "Glasser", "Julien", "Colombel", "Darfeuille\u2010Michaud"], "given-names": ["J", "AL", "S", "JF", "A"], "article-title": ["Inhibitory effect of probiotic Escherichia coli strain Nissle 1917 on adhesion to and invasion of intestinal epithelial cells by adherent\u2010invasive E. coli strains isolated from patients with Crohn\u2019s disease"], "source": ["Aliment Pharmacol Ther"], "year": ["2003"], "volume": ["18"], "issue": ["1"], "fpage": ["45"], "lpage": ["56"], "pub-id": ["10.1046/J.1365-2036.2003.01638.X"]}, {"label": ["34"], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n"], "surname": ["Komarow", "Makovoz", "Rodrigues", "Eisch", "Scott", "Metcalfe"], "given-names": ["H", "A", "R", "R", "L", "D"], "article-title": ["Survey of the oral and intestinal microbiome in patients with systemic mastocytosis"], "source": ["J Allergy Clin Immunol"], "year": ["2022"], "volume": ["149"], "issue": ["2"], "fpage": ["AB131"], "pub-id": ["10.1016/j.jaci.2021.12.446"]}]
{ "acronym": [], "definition": [] }
38
CC BY
no
2024-01-14 23:43:49
Clin Transl Allergy. 2024 Jan 13; 14(1):e12310
oa_package/cf/3b/PMC10787583.tar.gz
PMC10787586
0
[ "<title>INTRODUCTION</title>", "<p>Biomaterial discovery and engineering are one of the largest and most active topics in biomedical research, with a large and direct impact on the development of implantable prostheses and engineering of artificial tissues.##UREF##0##1,2## However, clinical and regulatory norms strictly regulate the development and use of implantable biomaterials, whose costs and validation <italic>iter</italic> are one of the major limiting factors for a further progress in the field. As an example, the estimated cost for the validation of new biomaterials can span the range €60 000–€120 000 per product, depending on the final device invasiveness.##UREF##0##1,3## Therefore, there is currently a large interest in new techniques and methods aimed at the validation of biomaterials in a less expensive way. This goal can be reached by reducing the working time of highly specialized personnel and by reducing the number of employed laboratory animals, which has a large ethical impact.</p>", "<p>Procedures for the biomaterial validation are defined by the ISO10993 norm##UREF##3##4## that relies on the visual inspection of tissue sections to score any evidence of an undergoing foreign body reaction (FBR), like the recruitment of immune cells.##REF##18162407##5## This preclinical research step requires the sacrifice of a huge number of laboratory animals, contradicting the basic 3R's principles##REF##25836957##6## with an unsustainable ethical burden. Other characteristic signs of FBR, such as angiogenesis, collagen I–III deposition, and fat infiltration, are also analyzed on tissue sections from laboratory animals with protocols (primarily immune-histochemistry), which are over 40 years old.</p>", "<p>Therefore, it is highly relevant to develop inspection methods that allow for a more complete and real time analysis of the vascular capillaries,##UREF##4##7## providing invaluable information on the biocompatibility of a medical device, well beyond the current ISO 10993-6 norms.</p>", "<p>Recent scientific advances have consistently and effectively demonstrated the possibility to directly visualize in long-lasting observations the recruitment of the immune cells##REF##35046959##8## and the related fibrotic reaction <italic>in vivo</italic> by means of non-linear excitation microscopy.##UREF##5##9–12## Similar intravital inspections would be extremely valuable to test biomaterials,##UREF##5##9,10## greatly improving the quality and reliability of the assessment of the reaction to implants.##REF##30717232##13## To achieve this aim, significant advances in deep tissue optical microscopy are necessary. Although invasive skull intravital microscopy techniques have been largely demonstrated,##REF##28243510##14,15## through-skin optical imaging would have high potential to reduce observation invasiveness, but it is still challenging. A part from the possibility of optical clearance of the skin,##REF##37288448##16## one can try to overcome this limitation by implanting an optical window in the animal, also known as window chamber (for example, cranial or dorsal), to avoid passing through the dense skin <italic>stratum corneum</italic>.##REF##28243510##14## Among the variety of existing imaging windows developed so far,##REF##28243510##14,17## one of the most used ones is the dorsal skin-fold chamber.##UREF##7##18,19## As an example, Dondossola <italic>et al.</italic>##UREF##5##9## presented a detailed high-resolution visualization of the reaction to samples of electrospun fibers subcutaneously implanted in mice for up to 14 days. More recently, the cytotoxic effect of the topical release of anticancer drugs by tiny micro-dispensers (volume ) implanted in mice was visualized <italic>ex vivo</italic> and <italic>in vivo</italic>.##UREF##9##20## In this case, Jonas and colleagues used either endoscopic confocal##REF##33562152##21## or two-photon excitation fluorescence (TPEF) microscopy##REF##34769180##22## to image the response of cancer cells to drugs released locally at the tumor site.</p>", "<p>The application of the window chamber concept to immune reaction of implants using high resolution optical microscopy would require a thorough analysis of the possibility of identifying immune cells and microvessels at the implant site <italic>in vivo</italic> or on fresh biopsies, to replace the time-consuming procedure of histological analysis on thin tissue sections. The relevant questions are basically two. Is it possible to perform label-free high-resolution microscopy of tissue <italic>in vivo</italic> replacing histology analysis? Is it possible to follow the histological changes in longitudinal studies <italic>in vivo</italic>? To address these issues, we should prove that non-linear excitation microscopy provides high resolution cellular details <italic>in vivo</italic> without the need of tissue staining. We should also be able to prove that highly porous microstructures implanted in lab animals offer a reliable frame of reference for repeated, longitudinal observation of the tissue dynamics.</p>", "<p>Here, we performed a direct comparison of H&amp;E histopathology to non-linear optical imaging <italic>in vivo</italic> of a microstructure, called Microatlas,##UREF##10##23## implanted in the chorioallantoic membrane (CAM) of chicken embryos and observed label-free. The Microatlas is a photopolymerized microstructure composed of a highly regular microscaffold, consisting of a rectangular lattice, with the dimension of , which is infiltrated by CAM cells when implanted. The micro-structure includes several features, which act as beacons for its 3D orientation and for the optical allignment of the device under a microscope for longitudinal studies. It is fabricated in a biocompatible resin, typically used for dental applications,##REF##25797444##24## mildly doped with a photosensitizer. The polymerized resin is fluorescent under non-linear excitation, and it can be, therefore, visualized in microscopy images without hindering the visualization of the growing tissue. As we will show in this contribution, the choice of the geometrical structure allows a good infiltration of tissue and vessels while keeping a good resistance to the bio-mechanical stresses induced by the growing tissue, at least in the chicken embryo animal model employed here.</p>", "<p>We further show that cell type recognition and quantification of their density and shape can be obtained by non-linear excitation optical imaging <italic>in vivo</italic>, perfectly matching the standard H&amp;E histopathology analysis performed on tissue fixed sections <italic>ex vivo</italic>. Second harmonic generation (SHG) microscopy on fresh tissue CAMs also allows us to ascertain the presence and geometrical features of the micro-vessels in the Microatlas.</p>" ]
[ "<title>METHODS</title>", "<title>Samples preparation</title>", "<title>Sample preparation for two-photon laser polymerization</title>", "<p>The photoresist employed for 2PP was the SZ2080,##REF##25797444##24## a negative organic–inorganic biocompatible resin, extensively validated for cell culture. It is made of two components: methacryloxypropil trimethoxysylane (MAPTMS, 97%, Sigma-Aldrich) and zirconium propoxide (ZPO, 70% in propanol, Sigma-Aldrich). ZPO enhances the material's mechanical stability. 1% wt. Irgacure 369 (IRG, Sigma-Aldrich) is added as a photo-initiator. The mass density as measured on a bulk polymerized specimen##UREF##28##82## is 1200 kg/m<sup>3</sup>. The molar concentration of Irg369 is about 33 .</p>", "<p>About 35 <italic>μ</italic>l of SZ2080 photoresist were deposited by drop casting on a 12 mm diameter circular glass coverslip (#1.5, Bio-Optica, Italy). The operation was always performed leaving a free external annulus on the glass substrate. This annulus has the role of assuring the correct holding inside the support. The solvent was removed through an evaporation phase (i.e., baking procedure) which occurred under chemical hood for at least 48 h at room temperature. Then, the resist reached a sol-gel state and allowed creating the initial chemical bonds between monomers and oligomers of the photoresist, preparing a starting substrate for the following laser-induced cross-linking.</p>", "<title>The chick-embryo implantation and preparation for microscopy</title>", "<title>Implantation</title>", "<p>Groups of 12–24 fertilized eggs were collected per week from a local farm (L'orto in casa ss., Correzzana, Italy) and stored in a dark cold room at the temperature of 10–15 °C. The eggs were dry washed with a brush and then stored in a clean egg-cup, blunt-end down. Fertilized chicken eggs can be stored at ∼13 °C up to 5 days before incubation without initiating development with a negligible degradation.##REF##27101515##83## Twelve eggs were incubated per experiment in a programmed egg incubator MG50 JR (FIEM, Italy), with serial automatic turning of eggs by an adjustable grid. The turning was activated from the beginning of the process with a constant rate and the temperature was maintained at 37.7 °C. A suitable air exchange was assured through proper ventilation holes. An Arduino-UNO based (Arduino, Italy) humidity system was connected to the incubator to maintain the humidity at 45% ± 5% during the <italic>in ovo</italic> cultivation. The <italic>in ovo</italic> incubation lasts 96 h. The Microatlas implantation <italic>in ovo</italic> occurred at the EID 7 <italic>ex ovo</italic> and was performed under a sterile cabinet. Control specimens (i.e., non-treated embryos) were implanted with a sterile circular glass coverslip, free from any microfabricated structures. For <italic>ex vivo</italic> measurements, at the end of the experiment (respectively, EID, 10–11–14) all the albumen content was removed and quickly substituted by formalin 4% (∼40 ml) assuring to completely cover the embryo. Then, the tissue portion was placed in a fridge at 4 °C to complete the fixing procedure, which lasted 72 h. Once formalin fixed, the embryo was washed three times in saline solution and stocked at 4 °C in Phosphate Buffered Saline (PBS). Both implanted and control regions were extracted after the washing procedure.</p>", "<title>Ex vivo and in vivo imaging</title>", "<p><italic>Ex vivo</italic> samples analyzed by confocal fluorescence microscopy were processed to assure selective nuclei staining. Cell nuclei staining was performed following a previously defined protocol herein briefly summarized. The cellular membrane of dissected CAM portions was permeabilized in 0.25% v/v of nonionic surfactant Triton-X-100 (Sigma Aldrich, USA) for 15 min. Then, each sample was gently washed three times in PBS and stained with far-red nucleus fluorescent probe: DRAQ5 (AB1084104, Abcam, Italy) (having excitation peak of 647 nm and emission spectrum between 665 and 681 nm) at a concentration 0.2% v/v for 10 min. Finally, the sample was mounted with 30 <italic>μ</italic>l of the embedding solution Mowiol 4–88 on rectangular microscopy glass (30 × 22 mm<sup>2</sup>, 1#, ThermoFisher, USA). The samples were then stored for 24 h at room temperature or 72 h at 4 °C.</p>", "<p>For histopathological analysis, the membrane CAM was fixed in 4% PFA, stained with Hematoxylin and Eosin, laid on a thin paper sheet and embedded in paraffin before being sliced along the cross section and along the plane of the membrane (4  section thickness).</p>", "<p>The non-linear excitation microscopy experiments on chicken embryos <italic>in vivo</italic> were performed after the Microatlas implantation at the EID 7 on embryos <italic>ex ovo</italic>. A drop of PBS buffer was added on top of the embryo implant site to allow the image acquisition with a water immersion microscope objective. The embryo was kept in the thermostated box of the microscope that kept the sample stage at for the whole experiment.</p>", "<title>Optical setups</title>", "<title>Two-photon laser polymerization setup</title>", "<p>2PP fabrication was performed by a laboratory-made femtosecond Ytterbium (Yb) - doped laser system, based on a cavity dumped mode-locked oscillator. The lasing wavelength was λ = 1042 nm, the pulse duration is ≅340 fs, the repetition rate 1 MHz, and the average maximum output power ≅8 W. The laser beam passed through a software controlled mechanical shutter (Uniblitz Electronics, LS Series, USA; maximum operating frequency ≅1 kHz) and was tightly focused by a plan-apochromat 100× oil immersion objective with numerical aperture (NA) 1.4 (Carl Zeiss, Germany) onto the photosensitive material, passing through the sample glass substrate.</p>", "<p>A spatial light modulator (SLM) was introduced alongside the laser path and consisted of a driver unit with standard digital video interface (DVI or HDMI) and a phase only liquid crystal on silicon full HD micro-display. A Galilean beam expander, with a magnification of 5×, was used, before the SLM, to enlarge the beam (3.6 × 3.2 mm<sup>2</sup>) to completely illuminate all the surface of the SLM micro display (15.36 × 8.64 mm<sup>2</sup>). Finally, a Keplerian telescope, made as a series of two lenses (750 and 200 mm) (Thorlabs, USA), decreased the image size to fit the objective back projection.</p>", "<p>In the Microatlas fabrication, only the first diffraction order was used while the others were blocked with an anodized aluminum slab.</p>", "<p>SLM phase masks (400 × 400 pixels in size) were computed by the SLM Pattern Generator software, HOLOEYE. That software started from a binary image processed with the Gerchberg–Saxton iterative Fourier transform algorithm. By feeding computer generated holograms to the SLM display, we split the laser beam in multiple parallel ones with dynamically changeable positions and powers. The pixel pixel-micron conversion factor was experimentally determined along both X and Y directions observing an anisotropy due to the rectangular shape of the SLM screen,\n</p>", "<p>Therefore, aiming to a theoretical distance of 50 <italic>μ</italic>m, the corresponding pixel distance obtained was 31 pixels along the X axis and 29 pixels along the Y axis implying a mean line length along each direction approximately of 49.6 <italic>μ</italic>m.</p>", "<p>The sample was mounted on an aluminum circular support connected to a gimbal mechanical system (Gimbal Mounts 100, Thorlabs, USA). The gimbal system presents an inner threaded hole, which allowed fabrication with various types of sample-holder, just fitting the cavity, giving the sample-holder a wide versatility. The sample holder-gimbal complex was mounted onto a planar (X, Y) brushless motion stage (ANT130XY Series, Aerotech, USA). The Z-direction, instead, was controlled by a motorized stage, balanced by two air compressed pneumatic pistons (ANT130LZS Series, Aerotech, USA), which counterbalance the gravity and avoid vibrations. These three stages were controlled via software (Automation 3200 CNC Operator Interface, Aerotech, USA) and equipped with a feedback position and velocity control system having a resolution on the order of nm. A red-light LED illumination was positioned under the sample-holder, in the central cavity of the gimbal, allowing the visualization during the writing process of the working area, as well as of the polymerized structures, through a CMOS camera (DCC1545M, Thorlabs, Germany). The three-axes stages and all the other set-up components were placed on a granite arch (ZALI, Precision granite technology, Italy), in turn placed above a pneumatic vibration isolator workbench (Newport, Stabilizer, High Performance Lamina Flow Isolator, I-2000 Series, USA).</p>", "<p>After the laser fabrication process, the samples were developed to remove all the un-polymerized photoresist. Briefly, the sample glass surface was soaked for 25 min in a glass beaker filled with a 50% (v/v) 2-pentanone, 50% (v/v) isopropyl alcohol solution (Sigma-Aldrich, USA). Then, the samples were washed with abundant isopropyl alcohol and then gently dried by room temperature Nitrogen.</p>", "<title>Confocal microscopy</title>", "<p>The Microatlas confocal fluorescence acquisitions were performed by a NIKON A1R or on a LEICA SP5 confocal microscope acquiring at 512 × 512 and 1024 × 1024 pixel<sup>2</sup> resolution (spacing along the optical axis = 1 <italic>μ</italic>m per cell counting and 0.3 <italic>μ</italic>m per nuclei segmentation) using the 488 and 633 nm laser lines. On the Nikon microscope either a 20× dry NA = 0.8 or a 40× water immersion NA = 1.15 objective was used. On the Leica microscope, either a 20× dry NA = 0.5 or a 40× oil immersion NA = 1.3 were used. DRAQ5 was used for staining nuclei with an excitation wavelength of 633 nm and collecting light through an emission bandpass filter in the range 645–720 nm.</p>", "<title>Non-linear excitation microscopy</title>", "<p>The Microatlas TPEF and SHG acquisitions were performed on custom setup based on a Ti:sapphire femtosecond laser (MaiTai Deepsee, Newport, USA) with pulse duration of  250 fs at the sample plane, having a tunable emission wavelength (690 nm &lt; λ &lt; 1020 nm). The beam passes through a commercial scan-head (FV300, Olympus, Japan) and reaches the BX51 Olympus upright optical microscope. The employed objective was a 25× water-matched (working distance 2 mm and NA 1.0, Olympus, Germany). Spectral separation of the emitted light was achieved by dichroic mirrors and bandpass filters in front of each photomultiplier tube (Hamamatsu H7422-40 for the 400/40 nm and 535/50 nm, 590/50 nm channels).##REF##27406819##11## The entire microscope was surrounded by a custom-made thermostatic cabinet in which the temperature was kept at 37 °C during <italic>in vivo</italic> inspections (Air thermostating by “The Cube,” Life Imaging Services, Basel, Switzerland). Multistacks were collected at 512 × 512 or 1024 × 1024 pixel resolution with an axial spacing along the optical axis of 1 <italic>μ</italic>m.</p>", "<title>Image processing and data analysis</title>", "<title>Noise reduction on microscopy images</title>", "<p>Confocal and TPEF images have been processed with Noise2Void (N2V), a recently published noise reduction deep learning method.##UREF##29##84## This technique is able to mitigate all forms of non-structured noise, such as Gaussian and Poisson noise (signal fluctuations, shot noise, readout noise and quantization errors). The processing was performed on a desktop tower (Alienware Aurora R10, Dell, USA) equipped with a dedicated GPU (GeForce RTX 3090 &amp; 24GB GDDR6X, NVIDIA, USA). All the acquired z-stacks were divided into groups characterized by the same imaging modality and experimental conditions (i.e., z-step and number of channels) in order to train each sub-set independently, while minimizing the number of required learning sessions. We employed a standard 3D-N2V configuration with U-Net depth = 2, kernel size = 3, and a mean square error (MSE) loss. The largest patch shape able to fit into memory was 64 × 64 × 8 × 1 pixels (XYZC). For each sub-set of images, a model was trained for 200 epochs (1–2 h) with batch normalization active. See also SI5, “Example of N2V Denoising on linear and non-linear images” for additional details.</p>", "<title>Nuclei segmentation, cell density and anisotropy measurement</title>", "<p>Cell nuclei visible in the processed fluorescence Z-stacks (as a positive signal on confocal images and as a negative signal on the non-linear excitation images) were 3D segmented by means of Imaris software (Oxford Instruments, UK) through the “magic wand” tool of the Surface View Mode. After segmentation of each nucleus, quantitative parameters were extracted such as surface area, volume, and ellipsoid axes. For the reported analysis, we mainly focused on the estimated ellipsoid axis since they provide a more unbiased representation when compared to volume or surface estimations, that are limited by the efficacy of the 3D reconstruction.</p>", "<p>About 80 cells were manually segmented from fluorescence images taken on three implanted and three control areas. For cell density quantification, three different time points (day 3, day 4, and day 7 after implantation) were considered. The cell density of each sample (Microatlas and control) was calculated assuming a homogeneous distribution of the cells along the vertical coordinate as follows:\n</p>", "<p>At least six random regions of interests (ROIs), 100 × 100 <italic>μ</italic>m<sup>2</sup> in size were inspected per Microatlas and in the control samples. To assure the full 3D reconstruction of the cells in the volume, the height of the inspected volume varied in the range 20–45 <italic>μ</italic>m. The cells were quantified by using the ImageJ Multipoint tool, manually selecting each cell throughout the volume, and summing up the number of counts (cells) for each ROI.</p>", "<title>Nuclei segmentation of cells in H&amp;E histology images</title>", "<p>The H&amp;E-stained CAM sections were digitally scanned using a NanoZoomer-SQ (C13140) whole-slide scanner (Hamamatsu). The scanner captured images with a 40× dry objective (NA 0.75) and a spatial resolution of 220 nm/pixel (115454 dpi). Data were stored in NanoZoomer Digital Pathology Images (ndpi file format) using JPEG (Q = 80) compression scheme. Images have been manually annotated on QuPath.##REF##29203879##85## For the evaluation of the cell density on H&amp;E images of the tissue sections, we ascribed to the tissue section the volume of the section (4 ) plus the average cell size (.</p>", "<title>Vessels' segmentation</title>", "<p>The blood vessels were segmented and analyzed along the whole acquired thickness: 80–100 <italic>μ</italic>m per Microatlas, while the control samples were analyzed along the total CAM thickness (30–50 <italic>μ</italic>m). By using ImageJ ROI tool, vessels were manually segmented by following the capillary tube midline while computing the tube thickness (as the distance between the tube walls) every 100 <italic>μ</italic>m along the planar dimension and averaging all the data per vessel. Vessels whose length were lower than 100 <italic>μ</italic>m, comprised a thickness value obtained as the average of measures between the two extremities. Capillaries were inspected alongside different acquired stacks, assuring the uniqueness of each vessel between adjacent axial planes.</p>", "<title>Second harmonic generation microscopy image analysis</title>", "<p><italic>In vivo</italic> non-linear excitation microscopy images were processed to quantify collagen I fibers directionality (by means of SHG) and blood vessels density (by means of autofluorescence signal). SHG multistacks were processed by the ImageJ Directionality plugin. Through this tool, the distribution of the orientation degree of collagen fibers present in the Region of interest was assessed per Microatlas pore. Signal from three sequential axial acquisitions were averaged and then the whole Microatlas height, ∼30% each of the total multistack, was arbitrary divided in three macro groups (LOW–MEDIUM–HIGH), ∼30 <italic>μ</italic>m each, starting from the glass interface, and the directionality averaged in those groups.</p>", "<title>Collagen segmentation and quantification</title>", "<p>TPEF and SHG acquisitions were performed on the same H&amp;E-stained slides used for assessing the immunological response. Multistacks were collected using the non-linear excitation Olympus microscopy setup. The imaging was conducted at a resolution of 1024 × 1024  with an axial spacing of 2  along the optical axis. Tile-scanning was employed to create a mosaic image of both EID14 and control samples.</p>", "<p>The collected stacks were analyzed using ImageJ software. Within each tile, we identified the most in-focus z-slices and summed their signals. A median filter with a radius of 2 pixels was applied to enhance contrast and reduce noise. The two-photon primed fluorescence of the H&amp;E stain was used to estimate the total tissue area in terms of pixel count. Simultaneously, the SHG signal was manually thresholded to highlight the collagen-covered areas.</p>", "<p>To compare the EID14 implanted sample with the control, we calculated the fraction of collagen relative to the total tissue area. This involved computing the ratio between collagen-positive pixels and total tissue pixels across 25 different tiles for each CAM sample. After conducting the analysis, no significant differences (P &gt; 0.05) were observed between the implanted and control samples based on the results of the two-tailed unpaired t-test.</p>", "<title>Statistical analysis</title>", "<p>In all the presented analyses, Kolmogorov test was used to inspect normality distribution inside each macro group, one-way ANOVA to test significant differences between experimental groups, and once assessed the homogenous distribution, Mann–Whitney test was used to compare implanted and untreated groups. All the collected data were fit in Origin (Originlab, USA).</p>" ]
[ "<title>RESULTS</title>", "<title>Geometry of the Microatlas device</title>", "<p>The whole micro-structured device carries four identical Microatlas grids [M in ##FIG##0##Fig. 1(a)##] each being realized as a rectangular lattice [##FIG##0##Fig. 1(b)##] fabricated by two-photon polymerization (2PP) in the SZ2080 biocompatible resin, on the surface of an optically transparent substrate, a borosilicate circular glass [##FIG##0##Fig. 1(c)##]. The porous microstructure, obtained by means of tightly focused ( 1  beams, can be chosen arbitrarily in the fabrication process: the overall regular structure of the Microatlas can extend for hundreds of micrometers in the fabrication plane and for about 100 <italic>μ</italic>m along the optical axis [perpendicular to the plane of the substrate, ##FIG##0##Fig. 1(b)##]. Here, we adopted a pore size of about for the lattice unit of the Microatlas to allow angiogenesis: each pore of the Microatlas has an inner rectangular size of 49.6 × 49.6 × 20 <italic>μ</italic>m<sup>3</sup> (SI1. “Microatlas design and parallel two-photon polymerization protocols”). At the same time, the structure rigidity should not be decreased by the enhanced porosity (larger pore size) in order to stand the stresses induced by the tissue growing inside. This can be achieved by inserting sparse thicker pillars in the structure that increases its structural stability. With the aim of future massive exploitation in <italic>in vivo</italic> implants, we first focused our efforts on keeping the fabrication time of this highly porous implantable structure low, and on testing what was the reaction of the CAM to its implant.</p>", "<title>Fast prototyping of rigid microstructures with high porosity</title>", "<p>Our strategy was to insert square-shaped pillars [P in ##FIG##0##Fig. 1(b)##] 3–4 times thicker than the rest of the Microatlas grid elements interleaved every 100 <italic>μ</italic>m (i.e., every two pores) and with an X-shape cross section [##FIG##0##Fig. 1(d)##]. In this way, we were able to obtain stable scaffolds for up to 4 days after the implantation and to mitigate the increase in the fabrication time due to the larger volume to be polymerized. Moreover, the X-shaped cross section of the pillars helps in minimizing their fluorescence signal contribution to the images. The fluorescence of (the photosensitizer within) the polymerized resist can be induced by two-photon excitation at 800 nm (Ref. ##UREF##10##23##) with a two-photon action ()##UREF##11##25## that is ten time larger than that of redox plasmatic enzymes like NAD (). However, the typical concentration of the enzymes is about ten times larger () than that of the photosensitizer in the resist (), resulting in a very similar level of fluorescence emission. The X-shaped cross section of the pillars helps also in further reducing the fabrication time, while, at the same time, providing high rigidity to the pillars. The pillars X-shape cross section had a 5  side and a 1.5 <italic>μ</italic>m arms' thickness. These structures [##FIG##0##Fig. 1(d)##] were fabricated with laser writing speed of 2.5 mm/s, and voxel size <sup>3</sup>. The resulting 36 pillars, interleaved in the Microatlas grid, were then realized four at a time thanks to a four “square” mask entailing a parallel fabrication with overall process time of 1.50 min (see Table SI1.1).</p>", "<p>In order to minimize the fabrication time per device, the laser 2PP writing of the device was implemented in parallel mode, by coupling the optical scanning setup with a spatial light modulator (SLM) to generate multiple laser foci.##REF##31409835##26## This process (see SI2, “Two-photon laser polymerization setup and parallel two-photon polymerization protocol”), assisted by a smart choice of the beam spots array for the fabrication of the various features of the device, considerably increased the process throughput,##REF##31409835##26## with a total fabrication time per Microatlas of 2.5 min (see SI2, “Microatlas design” and in particular, Table SI1.1). This was achieved with a X–Y inter-voxel spacing R = 0.35 <italic>μ</italic>m as the optimal choice leading to stable and perfectly reproducible lattices, with X–Y writing speed of 3 mm/s. The writing speed along the Z direction was lowered to 1 mm/s to reduce vibrations during the polymerization process. The final device for implant is constituted by four porous Microatlas grids (M), four dense spacers (S), and several reference objects [F, C indicated in ##FIG##0##Figs. 1(a)## and ##FIG##0##1(d)##]. Since the fabrication time scales with the third power of the object size,##UREF##12##27## also the geometrical parameters of these supporting additional elements were carefully chosen. The most demanding elements in terms of fabrication time were the dense spacers [##FIG##0##Fig. 1(e)## and S in ##FIG##0##Fig. 1(a)##], with a total side of 500 <italic>μ</italic>m and horn length of 150 <italic>μ</italic>m. These dense microstructures (tight woodpile polymerization pattern with lattice unit = ) needed a fabrication time of about 30 mins with a speed of 3 mm/s (see Table SI1.1). Finally, the entire device fabrication was based on a total of six phase masks with a total fabrication time of about 47 min and a time saving of 70% with respect to the single-focus fabrication with the same laser spot geometry. Notably, the fabrication time increases only by 6% per each additional scaffold (Table SI1.1).</p>", "<title>Visualization of Microatlas implanted in chicken embryos allows cell density evaluation</title>", "<p>The quantification of the number density of cells is essential for any assessment of the immune reaction to an implant. The Microatlas implanted in living chicken embryos can provide this information. To prove this, our main aim is to show that optical microscopy imaging of the CAM provides enough information also to assess the immune reaction to the implant in terms of recruitment of cells. This is achieved by a thorough comparison of the histological analysis (<italic>ex vivo</italic> imaging on H&amp;E-stained sections) to non-linear excitation microscopy, a very well-established technique for <italic>in vivo</italic> imaging. Confocal fluorescence microscopy on <italic>ex vivo</italic> samples (in which the cell nuclei were stained with the DRAQ5 dye) was also used as a cross-check of the shape and internal structure of the nuclei. <italic>Ex ovo</italic> implantations of the devices [##FIG##1##Fig. 2(a)##] were performed at Embryonic Incubation day 7 (EID7) following a modified protocol of the well-established CAM assay.##UREF##10##23,28## We pursued a multimodal study of different aspects of the immune reaction according to the general Scheme ##FIG##2##1##.</p>", "<p>In our <italic>in vivo</italic> experiments, non-linear excitation microscopy was applied without the need of a specific labeling. The SHG signal was used to monitor the formation of collagen type I [##FIG##1##Figs. 2(b)–2(d)##, blue channel] and to quantify the fibrotic reaction. TPEF of endogenous metabolic cofactors was used to single out cells, in particular granulocytes [##FIG##1##Figs. 2(h)## and ##FIG##1##2(j)##, green channel and white arrows]. In all experiments, we acquired and analyzed images taken on at least three implanted chicken embryos, at 3, 4, and 7 days after the implantation (corresponding to EID10, EID11, and EID14, respectively) and compared these results to those obtained from untreated embryos. The cell density was evaluated <italic>in vivo</italic> on the implanted Microatlas and in control CAMs by TPEF label-free imaging and cross-checked against the analysis of images taken on H&amp;E stained tissue sections and against confocal fluorescence microscopy images taken on <italic>ex vivo</italic> samples stained on the nuclei with the DRAQ5 dye. At first, the cells' nuclei were counted on randomly selected 1  thick non-linear microscopy optical sections, finding a substantial stability of the tissue density in the control regions in the time range EID10–EID14 [##FIG##1##Fig. 2(e1)##]. In contrast, the cell infiltration in the Microatlas implanted in embryos showed a significant increase with the implant duration. The cell density in the Microatlas increases steadily from EID11 with a doubling interval of 1.6   days in the first 7 days of implant, which can be taken as a clear sign of cell infiltration in the Microatlas. The increase in cell infiltration at EID14 could suggest a more pronounced cell infiltration by B-/T-cells because of the physiological evolution of the reaction to the implant.</p>", "<title><italic>Ex vivo</italic> histopathology of the implanted CAM</title>", "<p>Direct histopathological analysis with H&amp;E staining is the gold standard of tissue analysis##UREF##13##29## and provides detailed information on the tissue state and possible inflammatory reaction to the Microatlas implant. Therefore, to validate the results of the fluorescence microscopy analysis of the cell density in the implants, we performed H&amp;E analysis on 4  sections of the CAM membrane [##FIG##1##Fig. 2(f)## and Figs. SI3.1 and SI3.2], both for controls and implanted embryos in the time windows EID10–EID14. On the H&amp;E-stained cross sections of the control CAM samples, we can clearly identify the main tissue components:##UREF##13##29## the fibrous chorionic ectoderm, the central mesenchymal layer rich in vessels, and the allantoic endoderm (Figs. SI3.1 and SI3.2). The mesodermal layer of the allantois becomes fused with the adjacent mesodermal layer of the chorion after a few days from fertilization. The vascular network that develops in the mesenchymal double layer and that is connected with the embryonic circulation has a respiratory function and allows the uptake of calcium from the egg shell.##UREF##14##30–32## Upon implant, the vessels are growing in size [##FIG##1##Fig. 2(f)## at EID14 and Figs. SI3.1 and SI3.2, at EID11–EID15], indicating the recruitment of cells in response to the implanted Microatlas, initially recognized as a foreign body. Cells were counted on the H&amp;E-stained tissue sections and the cellular volumetric density was estimated close to implant site and in control samples [##FIG##1##Fig. 2(e2)##] by considering the average nuclear size and the section thickness. The general trend observed on the fluorescence microscopy images taken <italic>in vivo</italic> is replicated here, with a steady increase in the cellular density found close to the implant site corresponding to a doubling interval of 4   days in the first 7 days of the implant. In the control samples, as observed on the fluorescence images, the cellular density reaches a plateau level at least from EID10. To estimate the agreement between the H&amp;E and the fluorescence imaging analysis, we have computed the relative increase in the cellular density in implants, with respect to the control tissues, as a function of the incubation time. This relative cellular density increases with time [##FIG##1##Fig. 2(e3)##], with a doubling interval of 3.5   days and 2.0   days for the H&amp;E and the fluorescence imaging analysis, respectively. It is relevant for our purpose to notice that the trend is similar in the two cases and the only significant difference (p is found for EID11 [##FIG##1##Fig. 2(e3)##]. On the contrary, the cell density in the control tissues is found to agree between the two methods within 10% [##FIG##1##Fig. 2(e4)##]. These observations support our hypothesis that the cellular density can be evaluated on the fluorescence images as well as on the histological sections.</p>", "<title><italic>In vivo</italic> histopathology of the CAM</title>", "<p>We can push the equivalence of H&amp;E <italic>ex vivo</italic> histology and <italic>in vivo</italic> fluorescence imaging even further, by proving that we can also identify and recognize the cells from their morphology on fluorescence images. To this purpose, we applied both confocal (on stained samples) and non-linear excitation imaging, label-free. On the confocal images, we visualized the cytoplasm through its auto-fluorescence [green channel, ##FIG##1##Fig. 2(h)##] and the nuclei through the DRAQ5 staining [red channel, ##FIG##1##Fig. 2(h)##]. Confocal images display very similar details to those found on the H&amp;E images of the tissue sections [##FIG##1##Figs. 2(g)## and ##FIG##1##2(h)##] both in terms of the vessel bed structure [##FIG##1##Fig. 2(h)##] and of the cells [##FIG##1##Fig. 2(h)## insets], identified from the autofluorescence of metabolic enzymes.##REF##11964266##33## By means of non-linear excitation fluorescence microscopy, we can pursue a completely label-free approach <italic>in vivo</italic>, exploiting the cell cytoplasm auto-fluorescence and the SHG signal arising from collagen. The major difference with confocal fluorescence imaging is that in non-linear excitation cell nuclei appear dark, having very low autofluorescence, while the autofluorescence of the Microatlas is stronger [##FIG##1##Fig. 2(i)##]. This signal, however, is not hindering the observation of the cells within the Microatlas [##FIG##1##Fig. 2(j)##]. Therefore, both in confocal and non-linear fluorescence imaging, we can easily identify the granulocytes from their cell nuclei in the Microatlas pores and outside it [see arrows in ##FIG##1##Fig. 2(j)##, zoom inserts]. In the case of non-linear excitation microscopy, we can also easily segment vessels (from autofluorescence) and collagen (from the SHG signal).</p>", "<p>Identification of the different types of cells can be done on H&amp;E images [##FIG##3##Fig. 3(a)##] at high resolution [##FIG##3##Fig. 3(a3)##], based on the details of the cell and nuclei morphology. Chicken embryo has a number of histological peculiarities with respect to rodents, one of the most widely used animal model for immunological tests. Avian erythrocytes have small nuclei. Among the macrophages polymorphonucleated system (MPS) cells, granulocytes comprise heterophils more than neutrophils. Heterophil granulocytes typically possess nuclei with three lobes, while most eosinophil and basophil granulocytes possess nuclei with two lobes. All these features can be taken into account for immune cell segmentation on fluorescence images. Moreover, fibroblasts, endothelial and red blood cells [see, for example, ##FIG##3##Fig. 3(a3)##] could be also identified from their shape. However, no information on the tissue reconstituted within the Microatlas can be retrieved on the histological images since the Microatlas is ex-planted to cut the tissue sections. Moreover, in histology we are limited to a 2D reconstruction of the cell morphology and longitudinal studies are lengthy. Instead, fluorescence images allow us to distinguish cells equally well [##FIG##0##Figs. 3(b1)## and ##FIG##3##3(b2)##]. Cells in the collagenous mesenchymal layer could be identified as fibroblasts due to their characteristic elongated nuclei [##FIG##3##Figs. 3(b3)## and ##FIG##3##3(b4)##]. On the vessel endothelium [##FIG##3##Figs. 3(c1)## and ##FIG##3##3(c3)##], we could identify endothelial cells from their shallow cytoplasm [##FIG##3##Figs. 3(c2)## and ##FIG##3##3(c4)##]. The nucleated red blood cells with their characteristic small nucleus can also be clearly discerned inside the vessels both in cross sections [##FIG##3##Figs. 3(c2)## and ##FIG##3##3(c4)##] and in longitudinal sections [##FIG##3##Figs. 3(c1)## and ##FIG##3##3(c3)##]. Notably, this analysis can be pursued both on confocal images [on <italic>ex vivo</italic> samples, see ##FIG##3##Figs. 3(b1)##, ##FIG##3##3(b3)##, ##FIG##3##3(c1)##, and ##FIG##3##3(c2)##] and on non-linear excitation images [on <italic>in vivo</italic> samples, ##FIG##3##Figs. 3(b3)##, ##FIG##3##3(b4)##, ##FIG##3##3(c3)##, and ##FIG##3##3(c4)##], both in and around the Microatlas.</p>", "<p>Certain types of immune cells can also be identified label-free on linear and non-linear excitation images. As an example, polymorphonucleated cells (heterophil, eosinophil and basophil granulocytes) can be recognized among the other cells from their characteristic shape and their multi-lobed nucleus [##FIG##3##Fig. 3(d)##, bottom row, and ##FIG##4##Fig. 4##]. Nuclear lobes are not present in lymphocytes (T/B cells), nor in monocytes and macrophages. Lymphocytes have highly regular round-to-oval nuclei. Monocytes have kidney-shaped nuclei, without lobes, and macrophages have irregular oval nuclei. Mast cells have round-to-oval nuclei, without lobes. Since polymorphonucleated cells (hereafter identified simply as granulocytes) are more easily discernable and they are the first line of reaction to an implant, we have focus on them in this work. The capability to identify this type of cells, with such a high degree of accuracy, is particularly relevant to ascertain the degree of the immune response. It is particularly noteworthy that similar results can be obtained with confocal fluorescence (nuclei stained with DRAQ5 in <italic>ex vivo</italic> samples) and with two-photon excitation microscopy images <italic>in vivo</italic>, in which case no staining was necessary at all. It is also important to notice that the presence of the Microatlas, with its dim fluorescence signal, does not prevent our morphological analysis but offers a frame of reference for repeated longitudinal studies. These findings open the way to the use of the Microatlas as an implantable platform for the quantification of the immune reaction to a biomaterial, once tight coupling between the two is ensured.</p>", "<p>Based on our results, we were able to build a morphological atlas of cells exploiting the direct comparison of H&amp;E, confocal fluorescence and non-linear excitation images, as reported in ##FIG##3##Fig. 3(d)## for fibroblasts, endothelial and red blood cells, and for granulocytes.</p>", "<title>Indications of a mild immune reaction to the implant</title>", "<p>We can now pass to a more detailed analysis of the FBR as a function of the implant duration. Starting at EID11 we observe hyperemia [##FIG##3##Figs. 3(a1)## and SI3.2], concomitant with growth of the thickness of the CAM, due to an augmented blood flow carrying granulocytes for a prompt immune reaction to the implant. At EID14, we can recognize fibroblasts and cells from the mononuclear phagocyte system (MPS, comprising granulocytes, and likely also monocytes and macrophages) [##FIG##3##Fig. 3(a3)##]. We can distinguish them both in confocal and non-linear excitation fluorescence images [##FIG##3##Fig. 3(b)##] and in the H&amp;E stained sections [##FIG##3##Fig. 3(d)##]. In fact, fibroblasts can be singled out in the mesoderm [##FIG##3##Figs. 3(a3)## and ##FIG##3##3(d)##, second row from top], with their characteristic elongated shape of the nuclei. Among the MPS cells, the granulocytes (comprising in chicken embryo, heterophils, eosinophils and basophils) can be highlighted by their smaller and bilobed (heterophils) and trilobed (eosinophils and basophils) nuclei [##FIG##3##Figs. 3(a3)## and ##FIG##3##3(d)##, second row from bottom]. The marked presence of this kind of cells is indeed an indication that an inflammatory reaction is ongoing at the implant site. It is possible that concomitant to the recruitment of MPS cells at EID14, T- and B-cells are also converging to the implant site, complementing the reaction due to MPS cells. However, the limited deposition of collagen I [##FIG##3##Figs. 3(b2)##, ##FIG##3##3(b4)##, ##FIG##4##3(c3)##, and ##FIG##3##3(c4)##] that we observe through the SHG signal (blue channel) in correspondence of the implant region is a clear indication that the reaction is not massive. Moreover, no indication of necrosis and calcifications is visible at all.</p>", "<title>Shape and size of cells' nuclei in the Microatlas</title>", "<p>High resolution fluorescence microscopy allows us also to fully characterize the geometry of cell nuclei in the capillaries [##FIG##4##Fig. 4(a)##], in the tissue around [##FIG##4##Fig. 4(b)##, gray box] and inside the Microatlas [##FIG##4##Fig. 4(b)##, inner dashed box]. In this way, we can gain additional information for the identification of the type of cells directly on the fluorescence images. To this purpose, infiltrated cells, and more specifically cell nuclei, can be randomly isolated and segmented and the morphological features of their nuclei can be characterized by force fitting their surface to a 3D ellipsoid with axes,##REF##29884887##34##\n [##FIG##4##Fig. 4(c)##]. This simple description can capture the major differences in the nuclear shape, like the one of the granulocytes for which the nuclei appear bilobate [##FIG##4##Fig. 4(c)##] and can be approximated by a prolate ellipsoid. The recognition of the nuclear shape anisotropy can be performed on images of histological sections stained with hematoxylin an eosin [##FIG##4##Figs. 4(d)## and ##FIG##4##4(e)##] and on non-linear excitation fluorescence microscopy images, in a label-free approach [##FIG##4##Fig. 4(f)##]. The distributions of the three axes (Fig. SI4) of the nuclei segmented on the control tissue can be described by a single Gaussian function with average values:  = 1.4  0.4  = 2.2  0.4  = 2.9  0.6  for the control samples, and  = 2.3  0.4  = 3.12  0.5  = 4.26  0.6  for the Microatlas implanted at EID14. Systematically larger values are found for the embryos in which the microstructures were implanted (see SI4, “Analysis of the shape anisotropy of cell nuclei in CAM”).</p>", "<p>Interestingly, we find a correlation between the different axes (see Fig. SI4, off diagonal plots) that suggests the use of the axial ratios and as good parameters for identifying the cells in terms of their nuclear shape anisotropy. Large anisotropies fall in the third quadrant of the plot of as a function of . Indeed, granulocytes show a markedly higher anisotropy compared to all the other cells in control samples [##FIG##4##Fig. 4(g)##], with low values of the as and ratios. This is even more evident in implanted embryos as shown for EID14 in ##FIG##4##Fig. 4(h)##. Most of the granulocytes identified in implant regions are confined in the third quadrant of the vs correlation plot [##FIG##4##Fig. 4(h)##, dashed lines]. This and similar findings, that can be derived from high-resolution label-free optical images, can be the basis for an automated algorithm for the segmentation of granulocytes, in longitudinal studies of living avian embryos.</p>", "<p>The granulocytes are not uniformly distributed in the tissue. They occur in larger density in a region close [within from the microstructure, outer dashed box in ##FIG##4##Fig. 4(b)##] to the Microatlas. The fraction of granulocytes inside the Microatlas is almost of the total granulocytes number measured outside it, a result that is confirmed by the analysis of the images of the H&amp;E stained samples [##FIG##4##Fig. 4(i)##]. Also in blood vessels, the concentration of granulocytes is found to increase substantially, times [##FIG##4##Fig. 4(j)##], for implanted samples at EID14 as compared to the control samples. These phenomena can be considered a normal reaction to the injury on the CAM upon implant and do not indicate a massive reaction to the Microatlas itself.</p>", "<title>Vascularization in the Microatlas</title>", "<p>Neo-vascularization inside the Microatlas scaffolds [##FIG##5##Fig. 5(a)##] and in control samples [##FIG##5##Fig. 5(b)##] can be effectively followed by means of non-linear excitation microscopy (autofluorescence from the metabolic enzymes in the cytoplasm) and characterized in terms of cross section, length and branching ratios. Since the CAM has a very dense capillary network, it is commonly used to study <italic>in vivo</italic> angiogenesis as a response to a foreign body.##REF##33066403##35,36## Vessels grown in implanted embryos, around and within the scaffolds, can be mainly categorized as microcapillaries [compare for example ##FIG##5##Fig. 5(a)–5(b)##]. Their surface density in the Microatlas scaffolds reaches about six times the value measured in the control samples [##FIG##5##Fig. 5(c)##]. The distribution of the microvessels orientation angles is very wide (best fit Gaussian  = 60° ± 10°, indicating that there is not a marked preferential orientation with respect to the axes of the Microatlas [##FIG##5##Fig. 5(d)##]. The microcapillaries average size within the scaffold, estimated from the analysis of their 2D cross sections [##FIG##5##Fig. 5(e)##], is [##FIG##5##Fig. 5(f)##], less than one half the one measured in control samples that is 18  . Notably, the distribution of the microvessels within the Microatlas does also show the presence of a second, minor, component with larger vessels FWHM, [##FIG##5##Fig. 5(f)##], that is the value of newly formed vessels in the control tissue. The average value of number of branches of microvessels within the Microatlas is , with an average length per branch of .</p>", "<title>Collagen distribution in implants</title>", "<p>SHG is a second effective contrast parameter specific for the collagen I in tissue that we can exploit on non-linear excitation images. This parameter allows us also to directly visualize the collagen deposition within the Microtlas scaffold [##FIG##5##Fig. 5(g)##] and in control samples [##FIG##5##Fig. 5(h)##] <italic>in vivo</italic>. The formation of collagen I observed in implant regions <italic>in vivo</italic> is not more massive than the one quantified in control regions [##FIG##5##Fig. 5(i)##], a result confirmed on the TPEF and SHG images of the H&amp;E-stained tissue sections [##FIG##5##Figs. 5(k)–5(m)##]. Therefore, we have no evidence of the formation of a fibrotic capsule around the Microatlas implant. The orientation of collagen I fibers within the Microatlas grids was quantified by means of Fourier components analysis.##REF##23180029##37## The Fourier angular spectrum was fit to a Gaussian whose full width at half maximum [FWHM, ##FIG##5##Fig. 5(j)##] is taken as a measure of the collagen fibrils anisotropic distribution with respect to the edge of the Microatlas lattice. The FWHM of the orientation distribution in the Microatlas is [##FIG##5##Fig. 5(j)##], significantly lower than the values measured in control samples, , despite the fact that the collagen density in the implanted embryos was not significantly different than in the untreated embryos [##FIG##5##Fig. 5(i)##].</p>" ]
[ "<title>DISCUSSION</title>", "<p>In this work, we exploited a microgrid (Microatlas device) implanted in the chicken CAM and colonized by the cells and vessels of the host recipient, to quantify aspects of the FBR, by means of prolonged and repeated intravital microscopy observations. To do so, the microstructure must allow tissue and vessel reconstitution within it. However, our previous scientific efforts related to imaging the Microatlas device after its implantation in living chicken embryos,##UREF##10##23## indicated that angiogenesis in the microgrid was hindered in microstructures with a cubic pore size of about , even if host cell infiltration could occur physiologically. Here, by adopting a cubic pore size of we confirmed that the reduced neo-angiogenesis that we observed in previous work was due to the pore size. Indeed, the typical size of the microvessels in a chicken embryo lies in the range 40–100 .##REF##19081537##28,38–40## The translation of the technology presented here to higher level animals, such as rodents, should not be limited further by the microvessel size due to the low dependence ( of the vessel size on the animal mass M.##UREF##16##39##</p>", "<p>By devising a variable geometry multi-spot fabrication setup, we could fabricate in a limited time () a complex implantable microstructured chip that had four scaffolds for the host organism tissue regeneration (##FIG##0##Fig. 1##). The structural stability of the Microatlas was sufficient to stand the mechanical stresses of the growing tissue during its implant in chicken embryos up to EID14 (i.e., 7 days after the implant). This result was achieved by inserting thicker elements in the structure, with a X-shaped cross section that reduced the level of autofluorescence to a level that did not interfere significantly with the autogenous signals from the tissue, allowing <italic>ex vivo</italic> and <italic>in vivo</italic> imaging (##FIG##1##Fig. 2##).</p>", "<p>The highly porous lattice of the Microatlas scaffold clearly allowed the infiltration of cells (##FIG##1##Fig. 2##), vessels [##FIG##5##Figs. 5(a)## and ##FIG##5##5(b)##] and collagen fibers [##FIG##5##Figs. 5(g)## and ##FIG##5##5(h)##] in the structure, supporting the achievement of a mechanically guided FBR. By means of a direct and systematic comparison with histological images of H&amp;E stained tissue sections, we showed that high resolution TPEF microscopy images allow a detailed analysis of the cell and nuclei conformation, both outside and inside the Microatlas. In this way, we developed a dataset of avian cells [##FIG##3##Fig. 3(d)##] that we used for segmentation on fluorescence and SHG <italic>in vivo</italic> of non-linear excitation images, providing us with high resolution cellular details. The conclusions that we can draw from this analysis are remarkably comparable to those obtained from the analysis of H&amp;E-stained tissue sections, but without the necessity for time-consuming sample preparation (like tissue sectioning and H&amp;E staining).</p>", "<p>The dynamics of tissue regeneration is essential to characterize the effect of the implant. We characterized it in our living model exploiting <italic>in vivo</italic> fluorescence imaging. The physiological tissue density in the control regions was reached at EID10 [##FIG##1##Fig. 2(e)##], with non-significant variations up to EID14. In contrast, the infiltration in the Microatlas reached a tissue density comparable to the one of untreated embryos by EID11, increasing up to 1.8 times the density in the control regions at EID14, similarly to vessels that were denser in the Microatlas than in the control [##FIG##5##Fig. 5(c)##]. This result is in agreement with the histology and physiology of the host chicken embryo, whose angiogenesis is known to be very much responsive to foreign implant on the CAM.##UREF##17##41–43## The slightly larger value of the cell density in the histological images compared to fluorescence images [##FIG##1##Fig. 2(e1)## compared to ##FIG##1##Figs. 2(e2)## and ##FIG##1##2(e3)##], can be ascribed to the fact that in the first case we did not have access to the Microatlas and we were therefore probing the tissue in close vicinity to it. This effect is more evident for the granulocyte subpopulation [##FIG##4##Fig. 4(i)##] that was found within the Microatlas with  3.3 times lower density than outside. However, both histological and fluorescence images provided comparable values of the relative increase in the granulocytes in the samples implanted with Microatlas, with respect to the control samples [##FIG##1##Fig. 2(e3)##]. In the control samples, we confirmed on the histopathological images [##FIG##3##Fig. 3(a)## and Figs. SI3.1 and SI3.2] the presence of a wide population of cells, localized in the chorion layer and characterized by nuclei with shapes of varying eccentricity [##FIG##4##Figs. 4(g)## and ##FIG##4##4(h)##]. We found nucleated red blood cells in the vessels [##FIG##3##Fig. 3(a3)## and ##FIG##1##Figs. 3(c2)## and ##FIG##3##3(c4)##], and other types of cells (i.e., fibroblasts) in the dense mesoderm, in which secretory vesicles were also visible (Fig. SI3.1), likely with the function of calcium provision to the embryo.</p>", "<p>Regarding the reaction of the embryo to the implant of the Microatlas, it is known that in chicken embryos, as well as in other vertebrates, granulation tissue develops when the acute and chronic inflammatory responses are extinguished. On microscopy images we can identify granulocytes, fibroblasts, collagen and an increase in the neo-vascularization rate.##REF##18162407##5## Indeed, SHG imaging revealed the presence of fibrillar collagen I, which was an indication of an ongoing fibrous scarring process. Collagen I was found to be as dense in the Microatlas as in the control tissue, even though it appeared wrapped around in control tissue with a high angular dispersion of the fibers  50°, while it appeared more oriented, with an angular dispersion  25°, within the scaffold trusses [##FIG##5##Fig. 5(j)##]. As a matter of fact, extra-cellular matrix and collagen I production from fibroblasts invading a scaffold are known to depend both on the physical and chemical properties of the scaffold material##REF##35269536##44–46## and on the scaffold geometry and size##UREF##18##47–49## (i.e., fiber diameter or thickness, as in our case). Apart from these findings, we had no evidence of a fibrotic capsule comprising thick, densely packed bundles of collagen fibers that may hinder the diffusion of analytes into the microstructure,##REF##9599036##50## possibly leading to chronic inflammation states and to infections.##REF##18162407##5,46## Instead, our observations point toward a wound healing process, with a temporal progression of inflammatory states.##REF##21715002##51## Moreover, the presence of micro vessels regenerated within the Microatlas scaffolds would likely ensure an appropriate access to the inner pores of the Microatlas to small molecules to which the vessels are permeable.</p>", "<p>By comparing the microvascular network regenerated in the Microatlas with the ones reported in the literature for similar imaging windows or scaffolds,##UREF##5##9,17,23## we can confirm that a porous structure with pore size of about 50 , as the one used here, allows angiogenesis [##FIG##3##Figs. 3(c)## and ##FIG##3##3(d)##, ##FIG##4##Figs. 4(a)## and ##FIG##4##4(b)##, ##FIG##5##Fig. 5(a)##]. Indeed, apart from the increase in the vessel size around the site of the implant [##FIG##1##Figs. 2(f)## and SI3.2] due to hyperemia, we could observe neovascularization inside the Microatlas occurring primarily through small capillaries [##FIG##5##Figs. 5(e)## and ##FIG##5##5(f)##]. The Microatlas scaffolds enhanced the neo angiogenesis <italic>in situ</italic>, a strategy that could allow the regeneration of a functional microvascular network in close contact to a biomaterial coupled to the Microatlas. This outcome would let analytes diffuse into the device, with a minor impact of the overall collagen capsule thickness, which is however limited around and within the Microatlas [##FIG##5##Fig. 5(g)##]. This result is particularly relevant since the vascularization of an implanted device is a preliminary condition to ensure a limited inflammatory reaction by the host.</p>", "<p>As a result, we may presume that the formation of fibrotic tissue around the device is mild enough to allow trans-capsular moieties diffusion, as suggested by previous works on porous biomaterial implantation.##REF##9599036##50–52## To confirm this deduction, we will perform additional studies on the activity of the cells within the Microatlas. In fact, specific geometrical features in the substrates (such as channel width, pore size, etc.) could play important roles in the way fibroblasts orient and attach to the substrate,##REF##18162407##5,53–56## especially by controlling cell metabolic activity,##REF##18162407##5,57## and the interaction of the microstructure with other types of cells, like endothelial cells and macrophages.##UREF##20##53,58## We also plan to translate these studies to other animal models with more developed and known immune systems. In the chick embryo, in fact, the possibility to differentiate among different types of immune cells is limited, to our knowledge, to the monoclonal antibody <italic>KUL01</italic> that label mononuclear phagocyte cells like granulocytes, monocytes or macrophages, differentiating them from T and B lymphocytes.##REF##32688059##59## We could, in this way, determine whether T and B cells are recruited at the implant at later stages (i.e., EID14) when granulocytes recruitment is already fading. However, macrophages of different phenotypes, at least in the two wide classes of pro-inflammatory or pro-healing cells, cannot be tracked due to the absence of specific antibodies, which are not yet available for this species.</p>", "<p>The absence of the formation of a thick fibrotic capsule indicated a limited specific reaction due to the resin used for the fabrication of the microstructures. This could also be partially due to the fact that at the considered developing stages, chicken embryos immunocompetent system is not fully developed having rejection conditions not established yet.##REF##4734410##60## As a matter of fact, chicken embryos are protected by an effective immune system composed of both B and T cells, which control the antibody and cell-mediated immunity, respectively.##REF##12737220##61,62## However, until EID 10 the chicken embryo immune system cannot be considered completely developed: the presence of T cells can be first detected at EID 11, but B cells appear only at EID 12.##REF##34868080##63## After EID 15, B cell begins to diversify but chicken embryos do not become immunocompetent until EID 18.##REF##12737220##61##</p>", "<p>Even by basing on label-free images only, we can draw some relevant conclusions on immune cell recruitment. We observed that the density of granulocytes near to the Microatlas implanted in CAMs (i.e., within 200  from the Microatlas) was times at EID14, compared to control embryos [##FIG##4##Fig. 4(i)##]. Also, within the Microatlas, the density of the granulocytes was significantly greater than in controls, reaching a value of times the value found in control CAM samples. These findings are particularly relevant if one also considers the reduction due to the steric hindrance due to the excluded volume given by the pillars of the lattice and indicate that the Microatlas can be efficiently colonized by the host cells. Similarly, we found that the granulocyte concentration inside the vessels, being their primary route of recruitment, was times greater in the Microatlas implants than in the CAM membranes at EID14 [##FIG##4##Fig. 4(j)##]. Altogether, the limited deposition of collagen around the Microatlas, the high concentration of microvessels within the Microatlas, and the level of concentration of granulocytes indicate that, although a reaction is present, no acute inflammation is activated until EID14 in the implanted embryos. This particularly relevant since the reactions of chicken embryo to foreign bodies are significantly faster, compared to what observed in adult rodents or humans. Fibrotic capsules have been reported to form after four days from implantation of several types of artificial materials, i.e., in an interval between EID 9 and EID13.##UREF##22##64## Therefore, the microstructured device developed here can be successfully used as a reference frame for longitudinal observations in chicken embryos.</p>", "<p>Most importantly, even without species-specific antibodies for immune cells, we were able to identify and segment individual granulocytes [##FIG##3##Figs. 3(d)## and ##FIG##4##4(d)##] in and around the Microatlas [##FIG##5##Fig. 5(i)##] and in vessels [##FIG##5##Fig. 5(j)##] based on the dataset of cells developed on the non-linear excitation and confocal fluorescence images [##FIG##3##Fig. 3(d)##] and systematically validated against the H&amp;E histological analysis. This dataset could be used, once expanded to other types of immune cells in higher organisms and together with automatic feature recognition algorithms,##REF##34568816##65## to assess the amount of the different types of immune cells triggered by the reaction to an implant.</p>", "<p>Regarding the possibility to carry out similar studies <italic>in vivo</italic> on a higher organisms, like rodents, in a minimal invasive way, one should resort to through-skin non-linear optical microscopy. In this case, tissue induced scattering and spherical aberrations would reduce the image signal/noise ratio. Alternative to non-linear excitation microscopy, one could exploit photoacoustic microscopy.##REF##32637316##66–68## This technique exploits 10 ns pulsed laser sources to penetrate in the tissue and recover the spatial information from ultrasounds generated by the thermo-acoustic shock wave in the tissue. It is however limited in spatial resolutions by the ultrasound frequency bandwidth,##UREF##23##69## reaching typical values of about 20–100 , two orders of magnitude larger than the ones obtained with non-linear excitation microscopy.</p>", "<p>High resolution () optical microscopy in deep tissue can be obtained by physical corrections of the optical aberrations. Spherical aberrations could be corrected with conventional Zernike polynomials approaches.##UREF##24##70## High order optical modes correction methods must be implemented to tackle with tissue induced scattering, as recently proposed by Papadopolous <italic>et al.</italic>##UREF##25##71## and improved by May <italic>et al.</italic>##REF##34267207##72## However, this method of correction of the tissue scattering works on a very limited field of view, of the order of few Airy disks. Alternatively, one can resort to use the NIR-II optical window that spans the range of wavelengths 1000 nm  1700 nm,##REF##14595365##73–75## that features higher penetration depth and reduced tissue scattering, exploiting three-photon excitation fluorescence of red fluorescent proteins (dsRed and dTomato) and third harmonic generation (THG) scattering at 1700 nm.##REF##35166669##74,76,77## These or similar approaches could also be combined with the use of implantable micro-lenses coupled to the Microatlas as proposed recently.##UREF##26##78,79## By taking advantage of these additional technological advances, one can envision to extend the potential application of an imaging window, like the Microatlas presented here, to perform a full characterization of the immune reaction to the implant <italic>in vivo</italic> also in rodents.</p>" ]
[ "<title>CONCLUSIONS</title>", "<p>We developed and characterized a two-photon polymerized miniaturized implantable imaging window, the Microatlas, and tested the possibility to use it to recapitulate accurately the reaction of a living organism to the implant of an exogenous material. To this purpose, we implanted the Microatlas in a model of a living system, the chicken embryo, in which we could demonstrate its capability in stimulating cell infiltration and neovascularization with no massive deposition of collagen up to seven days of implantation, a duration of implant at which, in chicken embryos, foreign body reactions are already overcome the acute phase.##UREF##22##64##</p>", "<p>Validation of biomaterials for clinical use stems from the application of the ISO 10993 norm, that requires a simple classification of the extent of the capillaries proliferation in three classes: minimal, broad and extended (Table E.II in Ref. ##UREF##3##4##). However, not much attention is given to the size, the shape (ramification) and the state of the endothelium of the vessels grown around or infiltrated within the biomaterial. Most immunological response mechanisms (such as inflammation, allograft and xenograft preservation/reperfusion and rejection) are reflected by primary manifestations at the level of the microcirculatory system. Moreover, the successful application of regenerated tissues or even homologous implants, critically relies on the capability to elicit an effective and functional vascularization when embedded into the surrounding host tissue.##REF##33586357##80,81##</p>", "<p>It is then particularly relevant that we could exploit non-linear excitation imaging (TPEF and SHG) to develop a high resolution cell atlas that allows to quantify <italic>in vivo</italic> and <italic>ex vivo</italic> the reactions occurring inside MicroAtlas grids at different time points with no need of specific markers (label-free). In this way we could single out granulocytes from the tissue, quantify them in the control samples, close to (within 200 ) and in the Microatlas, as well as in the microvessels, and evaluate the density of collagen.</p>", "<p>Our next steps will be to integrate different materials with the Microatlas and implant the combination of the two in animal models to observe the immune cell recruitment at the biomaterial surface. With the purpose to use this technology, i.e., digital pathology of the biomaterial immune reaction based on fluorescence microscopy, for biomaterial validation as per ISO10993-6 norms, we will elaborate protocols for the implant and observation in rodents.</p>", "<p>Regarding the effective possibility to replace the standard histological analysis with non-linear excitation microscopy on microstructures guiding the reconstitution of tissue, one should consider that even if 2PP lithography is a costly and low throughput technology, the largest fraction of the cost of the tissue analysis with the non-linear excitation imaging on microstructures is given by the cost of the technician for the microscopy experiments. The cost of the microstructures for a massive production (about 180 000 pieces per year) is only 0.5 Euro per piece. The 3D optical sectioning by means of non-linear excitation microscopy allows to sample about 0.3  of tissue per day. We then estimate a cost of 1100 €/ for the non-linear microscopy analysis. The cost of conventional histopathology is again mostly determined by the cost of the technician. The volume of tissue sampled per day is limited by the number of stained tissue slices per day and by the thickness of the sections (typically . All together, we can estimate a cost of about 950 Euro/ of sampled tissue. The two approaches are therefore comparable in terms of costs.</p>", "<p>In conclusion, from the bio-engineering point of view, our results pave the way to a production of microfabricated structures for extended testing of implanted biomaterials. From the biological point of view, our results demonstrate the possibility to follow, in longitudinal studies, the immunological reaction to the implant based only on optical microscopy, opening the possibility of a direct monitoring of the reaction of the host animal to the implant <italic>in vivo</italic>. In addition, the potentiality of Microatlas proven here on chicken embryos, which is a simple animal model, its use in tissue imaging under high order nonlinear excitation (like three photons excitation and third harmonic generation), could allow to perform longitudinal studies of the foreign-body response also in higher animals, such as rodents.</p>" ]
[ "<p>Tissue histopathology, based on hematoxylin and eosin (H&amp;E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy <italic>in vivo</italic>, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&amp;E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image <italic>in vivo</italic> the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in i<italic>n vivo</italic> longitudinal studies.</p>" ]
[ "<title>SUPPLEMENTARY MATERIAL</title>" ]
[ "<title>ACKNOWLEDGMENTS</title>", "<p>This research has received funding from the European Union under the Horizon 2020 research and innovation program (G.A. No. 964481 – IN2SIGHT); ERC, project BEACONSANDEGG, G.A. 101053122.</p>", "<title>AUTHOR DECLARATIONS</title>", "<title>Conflict of Interest</title>", "<p>The authors have no conflicts to disclose.</p>", "<title>Ethics Approval</title>", "<p>In the experiments reported in this paper the embryonated eggs have been used by day 16 or earlier. Ethics approval is not required.</p>", "<title>Author Contributions</title>", "<p>Claudio Conci and Laura Sironi contributed equally to the work.</p>", "<p><bold>Claudio Conci:</bold> Data curation (equal); Formal analysis (equal); Writing – original draft (equal); Writing – review &amp; editing (equal). <bold>Giuseppe chirico:</bold> Conceptualization (equal); Data curation (equal); Formal analysis (equal); Writing – original draft (equal); Writing – review &amp; editing (equal). <bold>Manuela Raimondi:</bold> Conceptualization (equal); Writing – original draft (equal); Writing – review &amp; editing (equal). <bold>Laura Sironi:</bold> Conceptualization (equal); Data curation (equal); Formal analysis (equal); Writing – review &amp; editing (equal). <bold>Emanuela Jacchetti:</bold> Data curation (equal); Writing – original draft (equal); Writing – review &amp; editing (equal). <bold>Davide Panzeri:</bold> Data curation (equal); Formal analysis (equal); Software (equal). <bold>Donato Inverso:</bold> Conceptualization (equal); Data curation (equal); Methodology (equal). <bold>Rebeca Martinez Vazquez:</bold> Conceptualization (equal); Methodology (equal). <bold>Roberto Osellame:</bold> Conceptualization (equal); Writing – review &amp; editing (equal). <bold>Maddalena Collini:</bold> Supervision (equal); Writing – original draft (equal); Writing – review &amp; editing (equal). <bold>Giulio Cerullo:</bold> Conceptualization (equal); Writing – original draft (equal); Writing – review &amp; editing (equal).</p>", "<title>DATA AVAILABILITY</title>", "<p>The data that support the findings of this study are available from the corresponding author upon reasonable request.</p>" ]
[ "<fig position=\"float\" fig-type=\"figure\" id=\"f1\"><label>FIG. 1.</label><caption><p>Description of the implantable device constituted by four Microatlas structures and spacers. Panel (a) reports a low magnification scanning electron microscopy (SEM) micrograph of the whole device with indications of the Microatlas (M), the spacers (S), the frame of reference (F), and the reference cone (C). Panel (b) reports a SEM micrograph of one Microatlas lattice, with the details of the thicker pillars (P) and the microgrid. Panel (c) is a photograph of a whole device composed of four Microatlas (M), four spacers (S) on a 5 mm diameter glass slide. Panel (d) reports details of the pillar's cross section and the reference cone. Panel (e) reports a cross section (x–z) view and a perspective view (xyz) of one C-shaped spacer.</p></caption></fig>", "<fig position=\"float\" fig-type=\"figure\" id=\"f2\"><label>FIG. 2.</label><caption><p>Evaluation of the cellular density. Microatlas implanted <italic>ex ovo</italic> in CAM and comparison of H&amp;E histology and of fluorescence microscopy analysis. In panel (a), we show the implants in the embryo and a wide field of view of the implant taken under two-photon excitation ( = 800 nm). Panels (b)–(d) present details of a Microatlas grid, imaged by two-photon excitation, at EID10, EID11, and EID14 (top row, bar size = 50 ) and of control tissue (bottom row, bar size = 50 . Signals are fluorescence from cytoplasm and vessels (green, Exc: 800 nm, Em: 535/50 nm) and second harmonic generation from collagen I (blue, Exc: 800 nm; Em: 400/40 nm). Panels (e): cell counting in the Microatlas and control tissue: (e1) cellular volume density measured on 3D fluorescence images in the Microatlas (dark wine) and in the control samples (light orange); (e2) cellular volume density measured on H&amp;E stained tissue sections measured around the Microatlas (dark wine) and in the control samples (light orange); (e3) percentage increase in cellular density in the Microatlas as measured on fluorescence images (dark wine) and as measured on the H&amp;E sections around the Microatlas (pink); and (e4) ratio of the cell counting on the control samples as measured on the histological sections over the cell counting as measured on the fluorescence images. Panel (f) H&amp;E stained cross section (x–z plane) of a CAM. The pocket in which the Microatlas was implanted is indicated by a arrow. Tissue fixed at EID14. Panels (g) and (h) compare standard H&amp;E staining to confocal images. Panel (g) reports the area framed in panel (f), below the Microatlas implantation site (x–z section), in H&amp;E staining. Panel (h) shows the <italic>confocal image</italic> of the tissue area near [approximately the red boxed area in panel (g)] the Microatlas (the pillars of two microgrids are visible in the upper part of the image). The green and red framed boxes show a zoomed area of granulocytes visualized by means of standard H&amp;E and of confocal microscopy, respectively. Color codes are vessels, cells cytoplasm and Microatlas autofluorescence (Exc: 488 nm, Em: 500–550 nm, green) and nuclear fluorescence due to DRAQ5 (Exc: 633 nm; Em: 645–720 nm, red). Panel (i) illustrates the power of <italic>label-free histology</italic>. Confocal and TPEF images of the tissue surrounding a Microatlas are reported in the left (red framed) panel and in the right (light blue framed) panel. Panel (j) direct comparison of confocal and TPEF high resolution images of the field of view boxed in white dashed frames in panel (i) (x–y plane). Examples of granulocytes visualized by confocal (red framed) and TPEF (light blue framed) images are indicated by white arrows and in the corresponding blow-ups. The green framed box reports the detail of the x–z section corresponding to the confocal and TPEF images. For confocal images, color code is as in panel (h). For TPEF images, blue: second harmonic signal generated from collagen and Microatlas autofluorescence (Exc: 800 nm; Em: 400/40 nm); green: vessels, cells cytoplasm and Microatlas autofluorescence (Exc: 800 nm, Em: 535/50 nm).</p></caption></fig>", "<fig position=\"float\" fig-type=\"figure\" id=\"sch1\"><label>SCHEME 1.</label><caption><p>General scheme of the multimodal approach aimed to assess the possibility to perform histological analysis in chicken embryos through <italic>in vivo</italic> non-linear excitation microscopy. CAM = “Chorioallantoic membrane;” TPEF = “two-photon excitation fluorescence;” SHG = “Second Harmonic Generation.” The sections of this paper, the modalities (TPEF, SHG, etc.), the outputs (Results) and the corresponding figures are listed on the top bar of the scheme.</p></caption></fig>", "<fig position=\"float\" fig-type=\"figure\" id=\"f3\"><label>FIG. 3.</label><caption><p>Visualization of the chicken embryo foreign body reaction to the Microatlas implant by H&amp;E histology and fluorescence imaging. Panels (a) reports, at various levels of magnification (coded in colors), the H&amp;E images of a CAM cross section at EID14 of an embryo in which the Microatlas was implanted [the implant site is indicated by the fat gray arrow, panel (a1)]. Examples of mesothelium, chorionic ectoderm, and the vessels are identified by blue, red, and yellow dashed boxes [panel (a2)]. The increasing levels of magnification are identified by green boxes and provide examples of granulocytes (right, whose nuclei are segmented by an orange line), fibroblasts (left), and vessels (center) with nucleated red blood cells [panel (a3)]. Panels (b) and (c): Confocal fluorescence and non-linear excitation images are compared to H&amp;E one [panel (d), first column]. The mesothelium layer, the chorionic ectoderm and the vessels are identified in the H&amp;E image [panel (a2)] by a red, a blue, and a yellow dashed box, respectively. Confocal images [panels (b1), (b3), (c1), and (c2)] are collected on <italic>ex vivo</italic> CAM samples stained with DRAQ5 dye (nuclei, red channel) and exploiting the cytoplasm auto-fluorescence (green). <italic>In vivo</italic> (<italic>ex ovo</italic>) non-linear excitation images were collected upon excitation at [panels (b2), (b4), (c3), and (c4)]. Green and blue channels are the cell auto-fluorescence and the second harmonic signal coming from the collagen fibers. The color (red, blue, and yellow) of each image edge corresponds to the color of the boxes in panel (a2). Panels (c2) and (c4) report details of the red blood cells within capillaries in confocal fluorescence images (c2) and under non-linear excitation (c4). Panel (d): atlas of single cells as identified on the H&amp;E, the confocal and the non-linear excitation images (TPEF column).</p></caption></fig>", "<fig position=\"float\" fig-type=\"figure\" id=\"f4\"><label>FIG. 4.</label><caption><p>Characterization of the nuclear shape anisotropy. Panel (a): confocal images of control CAM (no implant) taken on a highly vascularized region. Panel (b): confocal image of a Microatlas implanted in a CAM at EID14. Pillars of the Microatlas are clearly visible together with vessels inside (inner dashed framed box) and outside (outer gray box) the Microatlas. Granulocytes were counted in 50 × 50  m<sup>2</sup> ROIs and averaged. Panel (c): three orthogonal cross sections of a granulocyte segmented on a fluorescence microscopy image, together with the definition of the ellipsoid axes. Panel (d): H&amp;E histological image of the x–z section of a CAM (mesothelium) with granulocytes and fibroblasts segmented around the Microatlas, marked in orange and cyan, respectively. Panels (e) and (f) report the detail of a granulocyte whose bilobate nucleus is segmented on the H&amp;E and the TPEF images, respectively. In the TPEF image the nucleus appears dark on the background of the cytoplasmic autofluorescence. Panels (g) and (h): distribution of the axial ratio B/C as a function of A/C for granulocytes compared to the other cells present in the tissue for a control sample [panel (g)] and samples in which the Microatlas was implanted and observed at EID14 in and around the Microatlas [panel (h)]. The dashed lines identify four quadrants in the correlation plot. Panel (i): percentage of the granulocytes counted on the optical microscopy images inside [inner box, panel (b)] and outside [outer gray region, panel (b)] the Microatlas and on the H&amp;E histological sections (outside the Microatlas). Panel (j): density of granulocytes inside and outside the vessels as measured on the optical microscopy images.</p></caption></fig>", "<fig position=\"float\" fig-type=\"figure\" id=\"f5\"><label>FIG. 5.</label><caption><p>Quantification of the angiogenesis and fibrotic reaction within and around the Microatlas at EID14 on non-linear excitation images. Panels (a) and (b): autofluorescence images of a Microatlas implanted in an embryo [panel (a)] and of a control embryo [panel (b)]. Segmented vessels are highlighted with red continuous lines. Bars are 100 [panel (a)] and 50  [panel (b)]. Panel (c): vessel density in Microatlas and control tissues. Panel (d): analysis of the orientation of the vessels within the Microatlas. The orientation angle is measured with respect to the side of the Microatlas lattice. The dashed line is a Gaussian fit to the data with a best fit Gaussian  = 60° ± 10°. Panel (e): examples of cross section profile of microvessels in the Microatlas drawn on 2D autofluorescence images [panel (b)]. Dashed lines are full width at half maximum (FWHM). Panel (f): distribution of the vessels' cross section FWHM. The lines are the best fit of the data to a two components (solid black lines, single components, dotted gray line, full fit) Gaussian function with best fit vessel size of and with FWHM =  and respectively. Panels (g) and (h): non-linear excitation images acquired at EID14 (blue: SHG; green: TPEF autofluorescence) of a Microatlas scaffold and a control tissue, respectively. A large ( cross section) vessel is clearly visible, with further ramifications in the second case. Bars are 100  in both panels. Panel (i) reports the density of the collagen fibers as from SHG imaging <italic>in vivo</italic>. Panel (j) reports the values of the FWHM of the angular distribution of fiber orientation angle as derived from Fourier analysis of the images. Panel (k) reports the evaluation of the collagen density on the H&amp;E stained tissue sections as done by counting the content of the pixels in the blue channel (SHG). Panels (l) and (m): non-linear excitation microscopy images where the green and the blue channels correspond to the emission of the Eosin-stained cells and the collagen I fibers, respectively. Bars indicate 100  size on both panels. Arrows in panel (m) indicate the presence of residuals of the explanted Microatlas.</p></caption></fig>" ]
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mathvariant=\"normal\">m</mml:mi><mml:mo>,</mml:mo><mml:mo> </mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mo>⟨</mml:mo><mml:mi>C</mml:mi><mml:mo>⟩</mml:mo></mml:mrow><mml:mrow><mml:mi mathvariant=\"italic\">CTLR</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M35\" display=\"inline\" overflow=\"scroll\"><mml:mo>±</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M36\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M37\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:msub><mml:mrow><mml:mo>⟨</mml:mo><mml:mi>A</mml:mi><mml:mo>⟩</mml:mo></mml:mrow><mml:mrow><mml:mi mathvariant=\"italic\">EID</mml:mi><mml:mn>14</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M38\" display=\"inline\" overflow=\"scroll\"><mml:mo>±</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M39\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mo>,</mml:mo><mml:mo> </mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mo>⟨</mml:mo><mml:mi>B</mml:mi><mml:mo>⟩</mml:mo></mml:mrow><mml:mrow><mml:mi mathvariant=\"italic\">EID</mml:mi><mml:mn>14</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M40\" display=\"inline\" overflow=\"scroll\"><mml:mo>±</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M41\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mo>,</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mo>⟨</mml:mo><mml:mi>C</mml:mi><mml:mo>⟩</mml:mo></mml:mrow><mml:mrow><mml:mi mathvariant=\"italic\">EID</mml:mi><mml:mn>14</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M42\" display=\"inline\" overflow=\"scroll\"><mml:mo>±</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M43\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M44\" display=\"inline\" overflow=\"scroll\"><mml:mi>A</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M45\" display=\"inline\" overflow=\"scroll\"><mml:mi>B</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M46\" display=\"inline\" overflow=\"scroll\"><mml:mi>B</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M47\" display=\"inline\" overflow=\"scroll\"><mml:mi>A</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M48\" display=\"inline\" overflow=\"scroll\"><mml:mi>B</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M49\" display=\"inline\" overflow=\"scroll\"><mml:mi>A</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M50\" display=\"inline\" overflow=\"scroll\"><mml:mi>B</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M51\" display=\"inline\" overflow=\"scroll\"><mml:mi>A</mml:mi><mml:mo>/</mml:mo><mml:mi>C</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M52\" display=\"inline\" overflow=\"scroll\"><mml:mn>200</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M53\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mn>4</mml:mn></mml:mrow></mml:mfrac></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M54\" display=\"inline\" overflow=\"scroll\"><mml:mn>4.5</mml:mn><mml:mo> ± </mml:mo><mml:mn>0.1</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M55\" display=\"inline\" overflow=\"scroll\"><mml:mi mathvariant=\"normal\">FWHM</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M56\" display=\"inline\" overflow=\"scroll\"><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M57\" display=\"inline\" overflow=\"scroll\"><mml:mn>7.2</mml:mn><mml:mo>±</mml:mo><mml:mn>1.5</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M58\" display=\"inline\" overflow=\"scroll\"><mml:mo>±</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M59\" display=\"inline\" overflow=\"scroll\"><mml:mn>6</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M60\" display=\"inline\" overflow=\"scroll\"><mml:mo>≃</mml:mo><mml:mn>12.5</mml:mn><mml:mo>±</mml:mo><mml:mn>2</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M61\" display=\"inline\" overflow=\"scroll\"><mml:mn>70</mml:mn><mml:mo>±</mml:mo><mml:mn>30</mml:mn><mml:mi>%</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M62\" display=\"inline\" overflow=\"scroll\"><mml:mn>9</mml:mn><mml:mo>±</mml:mo><mml:mn>2.4</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M63\" display=\"inline\" overflow=\"scroll\"><mml:mn>60</mml:mn><mml:mo>±</mml:mo><mml:mn>11</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M64\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M65\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M66\" display=\"inline\" overflow=\"scroll\"><mml:mi mathvariant=\"normal\">FWHM</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M67\" display=\"inline\" overflow=\"scroll\"><mml:mn>5.6</mml:mn><mml:mo>±</mml:mo><mml:mn>1</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M68\" display=\"inline\" overflow=\"scroll\"><mml:mn>12.8</mml:mn><mml:mo>±</mml:mo><mml:mn>3.5</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M69\" display=\"inline\" overflow=\"scroll\"><mml:mn>5.4</mml:mn><mml:mo>±</mml:mo><mml:mn>1.4</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M70\" display=\"inline\" overflow=\"scroll\"><mml:mn>3.8</mml:mn><mml:mo>±</mml:mo><mml:mn>2</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mo>,</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M71\" display=\"inline\" overflow=\"scroll\"><mml:mo>≅</mml:mo><mml:mn>75</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M72\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M73\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M74\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>25</mml:mn><mml:mo>°</mml:mo></mml:mrow><mml:mo>±</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>°</mml:mo></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M75\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>48</mml:mn><mml:mo>°</mml:mo></mml:mrow><mml:mo>±</mml:mo><mml:mrow><mml:mn>2</mml:mn><mml:mo>°</mml:mo></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M76\" display=\"inline\" overflow=\"scroll\"><mml:mn>20</mml:mn><mml:mo>×</mml:mo><mml:mn>20</mml:mn><mml:mo>×</mml:mo><mml:mn>20</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M77\" display=\"inline\" overflow=\"scroll\"><mml:mn>50</mml:mn><mml:mo>×</mml:mo><mml:mn>50</mml:mn><mml:mo>×</mml:mo><mml:mn>20</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mo>,</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M78\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M79\" display=\"inline\" overflow=\"scroll\"><mml:mo>≅</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mrow><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mn>12</mml:mn></mml:mrow></mml:mfrac></mml:mrow></mml:mrow></mml:msup></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M80\" display=\"inline\" overflow=\"scroll\"><mml:mo>≅</mml:mo><mml:mn>45</mml:mn><mml:mo> </mml:mo><mml:mtext mathvariant=\"normal\">min</mml:mtext></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M81\" display=\"inline\" overflow=\"scroll\"><mml:mo>≃</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M82\" display=\"inline\" overflow=\"scroll\"><mml:mo>≅</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M83\" display=\"inline\" overflow=\"scroll\"><mml:mo>≅</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M84\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M85\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M86\" display=\"inline\" overflow=\"scroll\"><mml:mn>6.3</mml:mn><mml:mo>±</mml:mo><mml:mn>1.5</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M87\" display=\"inline\" overflow=\"scroll\"><mml:mn>3.3</mml:mn><mml:mo>±</mml:mo><mml:mn>0.3</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M88\" display=\"inline\" overflow=\"scroll\"><mml:mn>4.2</mml:mn><mml:mo>±</mml:mo><mml:mn>0.5</mml:mn></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M89\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M90\" display=\"inline\" overflow=\"scroll\"><mml:mo>≃</mml:mo><mml:mn>1</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M91\" display=\"inline\" overflow=\"scroll\"><mml:mo>≤</mml:mo><mml:mi>λ</mml:mi><mml:mo>≤</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M92\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M93\" display=\"inline\" overflow=\"scroll\"><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M94\" display=\"inline\" overflow=\"scroll\"><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M95\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M96\" display=\"inline\" overflow=\"scroll\"><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M97\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">M</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M98\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M99\" display=\"inline\" overflow=\"scroll\"><mml:mn>37</mml:mn><mml:mmultiscripts><mml:mrow><mml:mo>°</mml:mo><mml:mi mathvariant=\"normal\">C</mml:mi></mml:mrow><mml:mprescripts/><mml:none/><mml:none/></mml:mmultiscripts></mml:math></inline-formula>", "<disp-formula id=\"d1\">\n<mml:math id=\"M100\" display=\"block\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mrow><mml:msub><mml:mrow><mml:mi>f</mml:mi></mml:mrow><mml:mrow><mml:mi>x</mml:mi><mml:mo> </mml:mo><mml:mtext mathvariant=\"normal\">axis</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:mo>=</mml:mo><mml:mn>1.6</mml:mn><mml:mo> </mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mtext mathvariant=\"normal\">pixel</mml:mtext></mml:mrow><mml:mrow><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow></mml:mfrac></mml:mrow><mml:mo>,</mml:mo></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:msub><mml:mrow><mml:mi>f</mml:mi></mml:mrow><mml:mrow><mml:mi>y</mml:mi><mml:mo> </mml:mo><mml:mtext mathvariant=\"normal\">axis</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:mo>=</mml:mo><mml:mn>1.7</mml:mn><mml:mo> </mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mtext mathvariant=\"normal\">pixel</mml:mtext></mml:mrow><mml:mrow><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:mrow></mml:mfrac></mml:mrow><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math>\n<label>(1)</label></disp-formula>", "<inline-formula>\n<mml:math id=\"M101\" display=\"inline\" overflow=\"scroll\"><mml:mo>≃</mml:mo></mml:math></inline-formula>", "<disp-formula id=\"d2\">\n<mml:math id=\"M102\" display=\"block\" overflow=\"scroll\"><mml:mi mathvariant=\"normal\">Cell</mml:mi><mml:mo> </mml:mo><mml:mtext mathvariant=\"normal\">density</mml:mtext><mml:mo>=</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">Cell</mml:mi><mml:mo> </mml:mo><mml:mtext mathvariant=\"normal\">number</mml:mtext></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">Volume</mml:mi></mml:mrow></mml:mfrac></mml:mrow><mml:mo>.</mml:mo></mml:math>\n<label>(2)</label></disp-formula>", "<inline-formula>\n<mml:math id=\"M103\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M104\" display=\"inline\" overflow=\"scroll\"><mml:mn>5</mml:mn><mml:mo> </mml:mo><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M105\" display=\"inline\" overflow=\"scroll\"><mml:mtext mathvariant=\"normal\">pixel</mml:mtext><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mo>,</mml:mo></mml:math></inline-formula>", "<inline-formula>\n<mml:math id=\"M106\" display=\"inline\" overflow=\"scroll\"><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">m</mml:mi></mml:math></inline-formula>" ]
[]
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[ "<supplementary-material position=\"float\" content-type=\"local-data\"><p>See the supplementary material for Microatlas design, on the two-photon laser polymerization setup and parallelization of the fabrication, on the histopathological analysis of CAM, on the analysis of the shape anisotropy of cell nuclei in CAM. Finally, details on the N2V denoising algorithm for linear and non-linear images.</p></supplementary-material>" ]
[]
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[{"label": ["1."], "mixed-citation": ["Market Data Forecast. \u201c\n"], "article-title": ["Global biomaterials market size, share, trends, COVID-19 impact & growth analysis report\u2014Segmented by material type, application and region\u2014Industry forecast (2023 to 2028)"], "ext-link": ["https://www.marketdataforecast.com/market-reports/biomaterials-market"]}, {"label": ["2."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["K.", "N. E.", "D. A.", "H.", "A.", "C.", "M.", "K. S. C. M."], "surname": ["Dzobo", "Thomford", "Senthebane", "Shipanga", "Rowe", "Dandara", "Pillay", "Motaung"], "article-title": ["Advances in regenerative medicine and tissue engineering: Innovation and transformation of medicine"], "source": ["Stem Cells Int."], "volume": ["2018"], "fpage": ["1"], "lpage": ["24"], "pub-id": ["10.1155/2018/2495848"]}, {"label": ["3."], "collab": ["Biomerics"], "article-title": ["Why Is Biocompatibility Testing Critical to Your Medical Device Project's Success?"], "ext-link": ["https://biomerics.com/BlogDetails/why-is-biocompatibility-testing-critical-to-your-medical-device-projects-success"], "year": ["2023"]}, {"label": ["4."], "mixed-citation": ["See "], "ext-link": ["https://www.Iso.Org/Standard/61089.Html"], "article-title": ["ISO 10993-6:2016\u2014Biological evaluation of medical devices\u2014Part 6: Tests for local effects after implantation"], "year": ["2016"]}, {"label": ["7."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "given-names": ["G.", "B.", "J. Y.", "J. P."], "surname": ["Yang", "Mahadik", "Choi", "Fisher"], "article-title": ["Vascularization in tissue engineering: Fundamentals and state-of-art"], "source": ["Prog. Biomed. Eng."], "volume": ["2"], "issue": ["1"], "fpage": ["012002"], "year": ["2020"], "pub-id": ["10.1088/2516-1091/ab5637"]}, {"label": ["9."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["E.", "B. M.", "S.", "S.", "D. W.", "P."], "surname": ["Dondossola", "Holzapfel", "Alexander", "Filippini", "Hutmacher", "Friedl"], "article-title": ["Examination of the foreign body response to biomaterials by nonlinear intravital microscopy"], "source": ["Nat. Biomed. Eng."], "volume": ["1"], "issue": ["1"], "fpage": ["0007"], "year": ["2017"], "pub-id": ["10.1038/s41551-016-0007"]}, {"label": ["10."], "mixed-citation": ["\n"], "string-name": ["\n", "\n"], "given-names": ["E.", "P."], "surname": ["Dondossola", "Friedl"], "article-title": ["Host responses to implants revealed by intravital microscopy"], "source": ["Nat. Rev. Mater."], "volume": ["7"], "issue": ["1"], "fpage": ["6"], "lpage": ["22"], "year": ["2022"], "pub-id": ["10.1038/s41578-021-00369-x"]}, {"label": ["18."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n"], "given-names": ["J.", "S.", "C.", "R. M.", "S."], "surname": ["Schreiter", "Meyer", "Schmidt", "Schulz", "Langer"], "article-title": ["Dorsal skinfold chamber models in mice"], "source": ["GMS Interdiscip. Plast. Reconstr Surg DGPW"], "volume": ["6"], "fpage": ["1"], "lpage": ["8"], "year": ["2017"], "pub-id": ["10.3205/iprs000112"]}, {"label": ["19."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "given-names": ["M. W.", "B.", "M. D."], "surname": ["Laschke", "Vollmar", "Menger"], "article-title": ["The dorsal skinfold chamber: Window into the dynamic interaction of biomaterials with their surrounding host tissue"], "source": ["Eur. Cells Mater."], "volume": ["22"], "fpage": ["147"], "lpage": ["167"], "year": ["2011"], "pub-id": ["10.22203/eCM.v022a12"]}, {"label": ["20."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["O.", "H. M.", "J. E.", "J. T.", "J.", "R. I.", "M. J.", "R."], "surname": ["Jonas", "Landry", "Fuller", "Santini", "Baselga", "Tepper", "Cima", "Langer"], "article-title": ["An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors"], "source": ["Sci. Transl. Med."], "volume": ["7"], "issue": ["284"], "fpage": ["284ra57"], "year": ["2015"], "pub-id": ["10.1126/scitranslmed.3010564"]}, {"label": ["23."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["C.", "E.", "L.", "L.", "G.", "R.", "G.", "M. T."], "surname": ["Conci", "Jacchetti", "Sironi", "Gentili", "Cerullo", "Osellame", "Chirico", "Raimondi"], "article-title": ["A miniaturized imaging window to quantify intravital tissue regeneration within a 3D microscaffold in longitudinal studies"], "source": ["Adv. Opt. Mater."], "volume": ["10"], "fpage": ["2101103"], "year": ["2022"], "pub-id": ["10.1002/adom.202101103"]}, {"label": ["25."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["E.", "M.", "K.", "G.", "B.", "R.", "P.", "P.", "S.", "A.", "K.", "L."], "surname": ["E\u017eerskyt\u0117", "Vengris", "Gineitis", "Merkininkait\u0117", "Leber", "Vargalis", "Stiegler", "Schemmer", "\u0160akirzanovas", "Kielait\u0117-Gulla", "Strupas", "Jonu\u0161auskas"], "article-title": ["Qualitative comparison between different biopolymers for usage in two-photon polymerization towards liver regeneration"], "source": ["Opt. Mater. Express"], "volume": ["12"], "issue": ["7"], "fpage": ["2550"], "year": ["2022"], "pub-id": ["10.1364/OME.459057"]}, {"label": ["27."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["L.", "A.", "U.", "J.", "Y.", "W.", "J.", "B. N."], "surname": ["Yang", "El-Tamer", "Hinze", "Li", "Hu", "Huang", "Chu", "Chichkov"], "article-title": ["Parallel direct laser writing of micro-optical and photonic structures using spatial light modulator"], "source": ["Opt. Lasers Eng."], "volume": ["70"], "fpage": ["26"], "lpage": ["32"], "year": ["2015"], "pub-id": ["10.1016/j.optlaseng.2015.02.006"]}, {"label": ["29."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["G.", "H.", "M.", "M.", "W.", "I.", "A."], "surname": ["Merckx", "Tay", "Lo Monaco", "Van Zandvoort", "De Spiegelaere", "Lambrichts", "Bronckaers"], "article-title": ["Chorioallantoic membrane assay as model for angiogenesis in tissue engineering: focus on stem cells"], "source": ["Tissue Eng., Part B"], "volume": ["26"], "issue": ["6"], "fpage": ["519"], "lpage": ["539"], "year": ["2020"], "pub-id": ["10.1089/ten.teb.2020.0048"]}, {"label": ["30."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["H. C.", "A.", "N.", "A. A.", "C. B.", "J. T."], "surname": ["Yalcin", "Shekhar", "Nishimura", "Rane", "Schaffer", "Butcher"], "article-title": ["Two-photon microscopy-guided femtosecond-laser photoablation of avian cardiogenesis: Noninvasive creation of localized heart defects"], "source": ["Am. J. Physiol.: Heart Circ. Physiol."], "volume": ["299"], "fpage": ["1728"], "lpage": ["1735"], "year": ["2010"], "pub-id": ["10.1152/ajpheart.00495.2010"]}, {"label": ["32."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "given-names": ["T. W.", "J. R.", "M."], "surname": ["Ecay", "Stewart", "Khambaty"], "article-title": ["Functional complexity in the chorioallantoic membrane of an oviparous snake: Specializations for calcium uptake from the eggshell"], "source": ["J. Exp. Zool., Part B"], "volume": ["338"], "fpage": ["331"], "year": ["2022"], "pub-id": ["10.1002/jez.b.23146"]}, {"label": ["39."], "mixed-citation": ["\n"], "string-name": ["\n"], "given-names": ["T. H."], "surname": ["Dawson"], "article-title": ["Scaling laws for capillary vessels of mammals at rest and in exercise"], "source": ["Proc. R. Soc. B"], "volume": ["270"], "issue": ["1516"], "fpage": ["755"], "lpage": ["763"], "year": ["2003"], "pub-id": ["10.1098/rspb.2002.2304"]}, {"label": ["41."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "given-names": ["K. M.", "J. M.", "R. O."], "surname": ["Marshall", "Kanczler", "Oreffo"], "article-title": ["Evolving applications of the egg: Chorioallantoic membrane assay and "], "italic": ["ex vivo"], "source": ["J. Tissue Eng."], "volume": ["11"], "fpage": ["204173142094273"], "year": ["2020"], "pub-id": ["10.1177/2041731420942734"]}, {"label": ["47."], "mixed-citation": ["\n"], "string-name": ["\n"], "given-names": ["M."], "surname": ["Ebrahimi"], "article-title": ["Porosity parameters in biomaterial science: Definition, impact, and challenges in tissue engineering"], "source": ["Front. Mater. Sci."], "volume": ["15"], "issue": ["3"], "fpage": ["352"], "lpage": ["373"], "year": ["2021"], "pub-id": ["10.1007/s11706-021-0558-4"]}, {"label": ["49."], "mixed-citation": ["\n"], "string-name": ["\n", "\n"], "given-names": ["Q. L.", "C."], "surname": ["Loh", "Choong"], "article-title": ["Three-dimensional scaffolds for tissue engineering applications: Role of porosity and pore size"], "source": ["Tissue Eng., Part B"], "volume": ["19"], "issue": ["6"], "fpage": ["485"], "lpage": ["502"], "year": ["2013"], "pub-id": ["10.1089/ten.teb.2012.0437"]}, {"label": ["53."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "given-names": ["C. E.", "D.", "T. H.", "K. L."], "surname": ["Witherel", "Abebayehu", "Barker", "Spiller"], "article-title": ["Macrophage and fibroblast interactions in biomaterial-mediated fibrosis"], "source": ["Adv. Healthc. Mater."], "volume": ["8"], "fpage": ["1801451"], "year": ["2019"], "pub-id": ["10.1002/adhm.201801451"]}, {"label": ["57."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "given-names": ["S.", "Y.", "C."], "surname": ["Wang", "Liang", "Dai"], "article-title": ["Metabolic regulation of fibroblast activation and proliferation during organ fibrosis"], "source": ["Kidney Dis."], "volume": ["8"], "issue": ["2"], "fpage": ["115"], "lpage": ["125"], "year": ["2022"], "pub-id": ["10.1159/000522417"]}, {"label": ["64."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "given-names": ["W.", "K. D.", "B.", "G."], "surname": ["Jakob", "Jentzsch", "Mauersberger", "Heder"], "article-title": ["The chick embryo chorioallantoic membrane as a bioassay for angiogenesis factors: Reactions induced by carrier materials"], "source": ["Exp. Pathol."], "volume": ["15"], "issue": ["5"], "fpage": ["241"], "lpage": ["249"], "year": ["1978"], "pub-id": ["10.1016/S0014-4908(78)80064-7"]}, {"label": ["69."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["E. A.", "M. O.", "G.", "M.", "A.", "M."], "surname": ["Vallicelli", "Cosmi", "Chirico", "Collini", "Baschirotto", "De Matteis"], "italic": ["IEEE Sensors"], "publisher-name": ["IEEE"], "year": ["2020"]}, {"label": ["70."], "mixed-citation": ["\n"], "string-name": ["\n", "\n"], "given-names": ["V.", "A."], "surname": ["Lakshminarayanan", "Fleck"], "article-title": ["Zernike polynomials: A guide"], "source": ["J. Mod. Opt."], "volume": ["58"], "issue": ["7"], "fpage": ["545"], "lpage": ["561"], "year": ["2011"], "pub-id": ["10.1080/09500340.2011.554896"]}, {"label": ["71."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "given-names": ["IN.", "J. S.", "J. F. A.", "B."], "surname": ["Papadopoulos", "Jouhanneau", "Poulet", "Judkewitz"], "article-title": ["Scattering compensation by focus scanning holographic aberration probing (F-SHARP)"], "source": ["Nat. Photonics"], "volume": ["11"], "issue": ["2"], "fpage": ["116"], "lpage": ["123"], "year": ["2017"], "pub-id": ["10.1038/nphoton.2016.252"]}, {"label": ["78."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n", "\n"], "given-names": ["M.", "A.", "R.", "C.", "M.", "M.", "R.", "G.", "B. S.", "M.", "E.", "M. T.", "G."], "surname": ["Marini", "Nardini", "Mart\u00ednez V\u00e1zquez", "Conci", "Bouzin", "Collini", "Osellame", "Cerullo", "Kariman", "Farsari", "Kabouraki", "Raimondi", "Chirico"], "article-title": ["Microlenses fabricated by two\u2010photon laser polymerization for cell imaging with non\u2010linear excitation microscopy"], "source": ["Adv. Funct. Mater."], "volume": ["33"], "fpage": ["2213926"], "year": ["2023"], "pub-id": ["10.1002/adfm.202213926"]}, {"label": ["79."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n", "\n"], "given-names": ["D.", "S.", "A.", "C.", "M."], "surname": ["Gonzalez\u2010Hernandez", "Varapnickas", "Bertoncini", "Liberale", "Malinauskas"], "article-title": ["Micro\u2010optics 3D printed via multi\u2010photon laser lithography"], "source": ["Adv. Opt. Mater."], "volume": ["11"], "issue": ["1"], "fpage": ["2201701"], "year": ["2023"], "pub-id": ["10.1002/adom.202201701"]}, {"label": ["82."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n", "\n"], "given-names": ["L.", "V.", "C.", "R."], "surname": ["Pertoldi", "Zega", "Comi", "Osellame"], "article-title": ["Dynamic mechanical characterization of two-photon-polymerized SZ2080 photoresist"], "source": ["J. Appl. Phys."], "volume": ["128"], "issue": ["17"], "fpage": ["175102"], "year": ["2020"], "pub-id": ["10.1063/5.0022367"]}, {"label": ["84."], "mixed-citation": ["\n"], "string-name": ["\n", "\n", "\n"], "given-names": ["A.", "T.-O.", "F."], "surname": ["Krull", "Buchholz", "Jug"], "article-title": ["Noise2Void\u2014Learning denoising from single noisy images"], "italic": ["IEEE/CVF Conference on Computer Vision and Pattern Recognition (CVPR),"], "publisher-name": ["IEEE"], "publisher-loc": ["Piscataway, NJ"], "year": ["2019"], "fpage": ["2124"], "lpage": ["2132"], "pub-id": ["10.1109/CVPR.2019.00223"]}]
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2024-01-14 23:43:49
APL Bioeng. 2024 Jan 9; 8(1):016102
oa_package/a2/a1/PMC10787586.tar.gz
PMC10787588
0
[ "<title>Introduction</title>", "<p>Rectal cancer is prevalent in the United States, with an estimated 43,340 new cases in 2020, and the incidence rate among people 50 and younger has been increasing for the past decade.##REF##32133645##1## Rectal cancer requires complex care coordinated among surgeons, oncologists, and radiation specialists to achieve the highest survival rate. In recent years, clinical trials have investigated a non-operative strategy called “watch and wait,” which allows patients with complete clinical responses to treatment to avoid surgical rectal resection and still achieve good long-term oncologic and functional outcomes.##REF##32686268##2##<sup>,</sup>##REF##34738104##3## “Watch and wait” has gained tremendous interest, particularly among patients wishing to avoid often highly morbid rectal surgery. However, the strategy relies on accurate assessment of tumor regression and confident identification of patients with a pathological complete response (pCR), i.e., no residual tumors. Current assessment tools include digital rectal examination, endoscopy with biopsy, and functional MRI (fMRI). Unfortunately, none of these modalities is particularly sensitive to residual tumor in the post-treatment rectum.##REF##27331883##4##<named-content content-type=\"online\"></named-content><named-content content-type=\"print\"><sup>–</sup></named-content>##REF##30528891##6##</p>", "<p>Photoacoustic imaging (PAI) is a promising functional imaging technique that offers tumor vasculature and blood perfusion information.##UREF##0##7## Utilizing the ultrasound (US) waves created by tissue’s optical absorption of short laser pulses, PAI can map tissue microvasculature patterns as well as blood flow profiles. Photoacoustic microscopy (PAM) is based on the PAI principle and classified as either optical-resolution (OR) PAM or acoustic-resolution (AR) PAM; AR-PAM can penetrate deeper than OR-PAM by using acoustic focusing rather than optical focusing. Recently, we have developed a co-registered AR-PAM and US system (AR-PAM/US) to assess post-treatment rectal cancer patients. It has been shown to offer residual tumor vasculature information valuable for post-treatment assessment of rectal cancer.##REF##33754826##8##</p>", "<p>Although it provides better resolution and vascular contrast than fMRI, AR-PAM has limitations in quantitatively assessing rectal tissue. The PA signal is the product of local optical fluence and tissue absorption, and optical fluence can vary with tissue depth due to inhomogeneous light absorption and scattering that contribute to changes in the PA signal amplitude.</p>", "<p>OR-PAM Doppler was introduced by Fang et al. in 2007.##REF##17995411##9##<named-content content-type=\"online\"></named-content><named-content content-type=\"print\"><sup>–</sup></named-content>##REF##20436589##11## Since then, several experimental studies using AR-PAM have been introduced, implemented, and evaluated.##REF##26892989##12##<sup>,</sup>##UREF##1##13## A particularly interesting application is the time domain correlational shift Doppler proposed by Brunker et al.##REF##22978905##14## In their work, they demonstrated the potential of calculating flow speed via A-line cross-correlation in the time domain and performing fixed angle correction to estimate the actual speed with solid and linear flow phantoms.##REF##26892989##12## Other methods of obtaining photoacoustic flowmetry information, such as the dual-pulse photoacoustic flowmetry, have also been implemented.##REF##31956486##15## However, the short delay required to take advantage of the Grueneisen relaxation effect renders it very challenging to adapt for AR-PAM application.</p>", "<p>In this study, our AR-PAM/US system implements a quantitative time domain cross-correlational Doppler algorithm to extract the flow signal independent of the signal amplitude. To extract blood perfusion information, the flowmetry processing consists of signal filtering and conditioning, A-line correlational processing, and angle compensation. We first validated our implementation with linear and spiral flow phantoms and contrast agents that we developed for AR-PAM imaging, obtaining measurements that agreed well with theoretical values. Then, to assess blood perfusion and correlate it with treatment response, we applied the Doppler algorithm to <italic>in vivo</italic> AR-PAM endoscopy data from patients who were treated with chemoradiation. To the best of our knowledge, this is the first application of AR-PAM Doppler for assessing the treatment responses of rectal cancer patients.</p>" ]
[ "<title>Materials and Methods</title>", "<title>ARPAE/US System</title>", "<p>The AR-PAM/US system has been described earlier.##REF##33754826##8## Briefly, the system comprises a 1064 nm Nd: YAG laser (DPS-1064-Q,##UREF##2##16##, China) operating at 1 kHz with pulse energy of , an US pulser-receiver, a function generator, a DAQ PC, and an imaging probe. The ultrasound transducer (US XDC) was custom made by Capistro Labs (San Clemente, California), using Polyvinylidene Fluorid (PVDF) material. The US transducer central frequency is 15 MHz with a 115% bandwidth. The US pulser receiver was a Panametric (currently Olympus, Tokyo, Japan) 5900PR, the function generator was a Tektronix (Beaverton, Oregon) AFG1022, and the DAQ was an Alazartech (Pointe-Claire, Quebec, Canada) ATS9462. To develop this system, the probe was modified to use a large 5 mm laser line mirror for better optical and mechanical stability. The function generator generates pairs of pulses with a short delay: the first triggers the laser to generate a PA pulse, and the second triggers the US pulser-receiver to generate a US pulse. Both signals are amplified by the pulser-receiver before being received by the DAQ PC and digitized at 180 MHz.</p>", "<p>Using a LabVIEW (National Instrument, Austin, Texas) code, the DAQ PC also synchronously drives a stepper motor that rotates the imaging head via a drive shaft embedded in the probe, shown in ##FIG##0##Fig. 1(c)##. Circular microsteps scan 1000 PA and US A-lines in a full rotation. The system is limited by the laser pulse repetition frequency at 1 kHz, giving a 1 kHz A-line scan rate and an B scan rate. The PA and US data obtained from scanning are processed in real time and displayed on the DAQ PC monitor for evaluation. The 6 dB US lateral resolution and axial resolution were characterized by diameter carbon fiber targets to be 83 and , respectively, at the transducer focus. The PA resolution, characterized with the same target, measured laterally and axially at the US focal distance.</p>", "<title>Spiral Phantom Preparation</title>", "<p>A tissue-mimicking phantom with embedded spiral tubes was produced to simulate the imaging setting of a colorectal tissue scan with blood flow. Specifically, a two-pour approach and assemblies of three-dimensional (3D) printed phantom molds shown in ##FIG##1##Fig. 2(a)## were used to first produce a tubular gelatin phantom and then to embed a spiral of tubing on the outside of the phantom. The phantom was made of 8%w gelatin solubilized in degassed deionized (DI) water to match the speed of sound in biological tissue’s while limiting optical absorbance. To retard degradation of the phantom, 1%v Germall Plus preservative was added.##REF##22076278##17## This mixture was degassed in a vacuum chamber to remove air bubbles formed during mixing. After the mold was configured as in ##FIG##1##Fig. 2(b)##, the gelatin mixture was added and allowed to cool to 10°C, forming a small hollow cylindrical phantom. Then, PTFE tubing with a ID was spiraled onto the phantom [##FIG##1##Fig. 2(c)##]. The outer wall mold is mounted around the phantom, as shown in ##FIG##1##Fig. 2(d)##, then the gelatin mixture is poured into the mold and allowed to cool to give the final spiral flow phantom shown in ##FIG##1##Fig. 2(e)##.</p>", "<title>Doppler Processing</title>", "<p>Doppler flowmetry in the phantom and in tissue utilizes a correlation Doppler algorithm with extensive noise rejection features. The PA signal is first passed through an FIR filter with a 3 to 30 MHz passband, then the average intensity of the noisy regions is analyzed and used to apply amplitude-based thresholding to the signal to avoid noise correlation. The signals are interpolated by a factor of 3 to allow finer correlation shift calculation as: where is the velocity resolution, is the speed of sound in the medium, is the pulse repetition frequency of the laser, is the sampling frequency, is the signal interpolation factor.</p>", "<p>For adjacent A-lines, a 25-point sliding window cross-correlation is calculated, and their correlation shift and correlation amplitude are recorded: where is the actual velocity, is the velocity in A-line direction, is the angle between flow direction and A-line direction, is the shift in pixels calculated by cross-correlation.</p>", "<p>The correlation amplitude is used to filter out remaining noisy correlations. After filtering, the correlation shift can be converted to a velocity map based on Eqs. (1) and (2).##UREF##3##18## Equation (1) calculates the Doppler velocity resolution, . Because the direction of the scanning and the direction of the flow are at an angle relative to one another, angle compensation is added to the velocity calculation by using Eq. (2).</p>", "<p>Angle compensation is conventional in various Doppler velocimetry applications, where it is used to calculate the velocity component along the desired direction from the velocity measured along the A-line. ##FIG##2##Figure 3(b)## shows the angle and the true versus measured velocities. We implemented angle compensation for <italic>in vivo</italic> images by calculating the angle between the imaging and flow direction, using the estimated time of arrival from Sec. <xref rid=\"sec2.5\" ref-type=\"sec\">2.5</xref> and the transducer surface offset of 5.57 mm, a process shown in ##FIG##3##Figs. 4(l)##–##FIG##3##4(n)##. The formula for the angle calculation is Eq. (3). Since the compensation factor approaches infinity as the angle between the flow direction and the scanning direction approaches 90 deg, the compensation angle is limited to 70 deg to prevent over-compensation at small angles: Equation (3) is used for calculating the angle compensation, where is the angle between the scanning A-line’s flow and imaging directions, is the angle between the previous scanning A-line’s flow and imaging directions, , are the times of arrival of adjacent A-lines, offset is the distance from the transducer surface to the rotation axis.</p>", "<p>To display the flowmetry, the velocity map is re-plotted with a color Doppler color scheme, in which black represents stationary regions, red to orange represents regions of increasing positive velocity, and blue to cyan represents regions of negative velocity with increasing speed. Finally, the color Doppler map is superimposed on the US image. For <italic>in vivo</italic> and spiral phantom images, the images are converted to a polar display for co-registered geometrical representation. For <italic>in vivo</italic> data, B scan images are uniformly divided into 20 regions, and the Doppler flow speeds in the regions are calculated for statistical analysis. One limitation of this correlational Doppler method is that it can detect flow only if absorbers move along the A-line direction. If they flow orthogonal to the A-line direction, they cannot be measured. In this research, the probe is almost always off-center, and the vasculature directions are highly variable, so the flow direction is usually not normal to the scan direction, which makes angle compensation particularly important in calculating flow velocities.</p>", "<title>Phantom Imaging Setup</title>", "<p>##FIG##2##Figure 3(a)## shows the open loop flow setup for imaging the linear flow and spiral flow phantoms. The AR-PAM/US imaging system is mounted on a manual two-dimensional (2D) translation stage to allow fine adjustment of the scanning location. Because no appropriately sized microspheres absorbing at 1064 nm were available, we produced our own contrast agent by sonicating a mixture of 60%w glass beads infused with 40%w hydrophobic black dye. A 2%w dilution of the contrast agent was found to be optimal for PA imaging, with only moderate photothermal damage to the flow tubing. The imaging setup and scanning geometry for the linear flow phantom experiment are shown in ##FIG##2##Fig. 3(a)##. The imaging head is held stationary for maximum consistency in imaging the ID PTFE tubing, and both are submerged in degassed DI water for acoustical coupling. The axis of the imaging head is held at 60 deg to the tubing bore. To study the angle dependence of flow measurements, we wrote a LabVIEW program to rotate the imaging head, allowing fine adjustment of the scanning angle.</p>", "<p>To simulate the <italic>in vivo</italic> imaging conditions, we imaged the spiral flow phantom, as shown in ##FIG##2##Fig. 3(a)##. The AR-PAM imaging probe is mounted on a two-axis translational stage, with its imaging head submerged in degassed DI water. The processing for the spiral flow phantom is special because the imaging direction is perpendicular to the spiral flow direction, thus constant angle correction was applied. Due to tissue motion and the handheld probe consideration, our application of Doppler velocimetry employs limited averaging of 100 A-lines to the spiral flow phantom and <italic>in vivo</italic> data.</p>", "<title>Time of Arrival Correction for <italic>In Vivo</italic> and Spiral Flow Phantom Data Processing</title>", "<p>Time of arrival correction, shown in ##FIG##3##Fig. 4##, compensates for shifts in the signal due to shifts in the physical location of the rectal tissue, allowing correlational Doppler to more accurately estimate shifts in the optical absorber locations. Assuming the PA and US A-lines overlap, we developed an algorithm for aligning the individual PA A-lines. Because the physical boundary between the coupling DI water and the phantom/tissue is well defined in the US image, rising edge-based US time of arrival detection was applied to the US A-lines, yielding ##FIG##3##Fig. 4(c)##, in which the red line represents the time of arrival. To eliminate noise points in the rising edge detection, the threshold was manually set to 10 dB above noise floor, yielding ##FIG##3##Fig. 4(b)##. To remove outliers, the calculated time of arrival locations were passed through an outlier rejection algorithm. Based on their times of arrival, PA A-lines are shifted in time for correlational Doppler calculations and shifted back to their original location for coregistration with the US image, as shown in ##FIG##3##Figs. 4(e)##–##FIG##3##4(i)##. The PA Doppler image co-registered with the US image is then warped for display in polar coordinates, as shown in ##FIG##3##Fig. 4(k)##.</p>", "<title><italic>In Vivo</italic> Imaging of Post Treatment Colorectal</title>", "<p>For <italic>in vivo</italic> imaging, patients with rectal adenocarcinoma who had undergone radiation and chemotherapy were imaged pre-operatively before rectal tissue resection. Patients were first put under anesthesia per the standard of care, a colonoscopic exam was performed to localize the cancer, and our AR-PAM/US probe was then used to scan the patient’s rectum. The findings from five patients are included in this paper: three patients (P1, P2, P3) with residual adenocarcinoma, one with complete response (P4) of no remaining cancerous tissue, and one patient (P5) with a stricture below the tumor so only lesion-free tissues were imaged. This study was approved by the Institutional Review Board and was Health Insurance Portability and Accountability Act (HIPAA) compliant (ClinicalTrials.gov identifier NCT04339374), and all patients provided written informed consent.</p>" ]
[ "<title>Results</title>", "<title>Linear Phantom Flow Experiment</title>", "<p>A set of flow speeds, ranging from 0.63 to 6.3 mm/s in increments of 0.63 mm/s were measured with the linear flow imaging setup to observe the flow profile. Twenty US and PA coregistered B Scans were acquired for each flow speed setting, with the results shown in ##FIG##4##Fig. 5(a)##. The measurements demonstrate good linearity across the flow speeds in the measurement interval, and even though a slight discrepancy between the theoretical and measured values can be observed, the differences are within 15%.</p>", "<p>To study the effect of angular changes on the flow speed measurement, we varied the angle between the imaging head and flow direction from 70 deg to 50 deg, in 10 deg increments, while maintaining the flow speed at . As ##FIG##4##Fig. 5(b)## shows, the angle compensation procedure can compensate for large angles between the flow direction and imaging direction, but at angles , over-compensation occurs because the compensation factor increases exponentially. Consequently, we limited the angle compensation to 70 deg.</p>", "<title>Spiral Doppler Flow Experiment</title>", "<p>##FIG##5##Figure 6(a)## shows a 3D PA scan of the spiral flow phantom. This image allowed us to identify the turn of the tubing spiral that overlapped the most with the 2D imaging plane and consequently provided the most flow speed data. It also confirmed that the tube had no obstructions or kinks to cause flow speed irregularities. The flow speed in the spiral phantom was calculated from AR-PAM/US system data. Because the PA signals from the inner surface of the tubing are strong, as shown in ##FIG##5##Fig. 6(b)##, only a weak signal can be observed inside the tubing. The Doppler image coregistered with the US image, ##FIG##5##Fig. 6(c)##, appears quite similar, with measurable flow near the inner tubing surface. At the same time, in ##FIG##5##Fig. 6(c)##, we observe flow inside the tubing highlighted by Doppler processing, in contrast to the low signal amplitude in ##FIG##5##Fig. 6(b)##. These sets of images emphasize Doppler processing’s strength in providing perfusion information with little dependence on signal amplitude, which is valuable for <italic>in vivo</italic> data. Due to the difficulty of aligning the PA data with the low US signal, PA Doppler could be calculated only for regions with a strong US signal, but this should not be an issue for <italic>in vivo</italic> data since the water–tissue interface generates a strong US reflection.</p>", "<p>Statistically, spiral flow signals exhibit linear trends similar to those linear flow, but with larger deviations, which is expected since only 100 A-line ROI averaging was applied to the data as compared to averaging across 1000 A-lines in ##FIG##4##Fig. 5(a)##, and the signal-to- noise ratio is poorer because a portion of the tube does not exhibit a strong PA signal. Still, the actual speed and estimated speed are approximately linearly correlated with an value of 0.913, demonstrating the utility of flowmetry in spiral imaging scenarios.</p>", "<title><italic>In Vivo</italic> Processing Results</title>", "<p>Doppler processing was applied to <italic>in vivo</italic> images to test velocity estimation. Six rectal images were processed for five different patients: three patients with residual cancer (P1-3), one treatment responder (P4) with no residual cancer, and one patient (P5) with normal rectal tissue.</p>", "<p>##FIG##6##Figures 7(a)##–##FIG##6##7(c)## show a polar scan, co-registered US and AR-PAM of a tumor bed, and a flow map of the same region, respectively, demonstrating that correlational Doppler can highlight low flow in a tumor bed (the darker regions in the red box) as distinct from the nearby higher flow regions. The residual tumor in Figs. 7(a)–7(c) displays perfusion on the surface, with no perceivable flow inside the tumor, possibly due to necrotic tissue, but higher velocity and densely packed vessels are observed adjacent to the residual tumor. In the treatment responder [Figs. 7(e)–7(g)] and normal tissue [Figs. 7(i)–7(k)], a more uniform blood flow profile across the rectal cross section is observed. For a more quantitative analysis, we averaged the Doppler flow velocity of the cancerous patients’ images in cancerous and normal regions, the treatment responder’s images in treatment scar and normal regions, and the normal patient’s images in normal regions, with a sample size of 6 for each patient and type of tissue. A Student’s -test was used to evaluate the difference of P1 to P4’s cancerous or responding tumor bed, versus normal tissues. As shown in the box plot ##FIG##6##Fig. 7(m)##, there are clear statistically significant lower velocities in the cancer regions compared to normal regions in all three cancer patients. The responding tumor bed regions with scarring exhibited higher flow velocities than normal tissue regions, though not statistically significant. And even though other confounding factors including inter-patient variability and tissue heterogeneity influence flow velocities in patient rectums, the flow velocities of normal regions and responding tissue region are generally higher across all five patients.</p>" ]
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[ "<title>Summary and Conclusion</title>", "<p>In this study, we implemented acoustic resolution PA correlational Doppler flowmetry with an AR-PAM/US system. Linear and spiral flow phantoms were constructed and studied with a novel PA contrast made from glass microbeads and hydrophobic black dye. The results showed that correlational Doppler flowmetry has good linearity, its results agree well with theoretical values for linear flow and have a linear relationship with theoretical spiral flow values. A signal alignment and processing pipeline were developed for acquiring Doppler flowmetry images that are similar to conventional US Doppler images. Patient examples demonstrate correlational Doppler flowmetry’s ability to extract blood perfusion information, quantifying the expected lower flow rate inside treated cancerous tissues and relatively higher flow rate in adjacent normal tissues. In addition, the imaged differences among cancerous tissue, tumor bed tissue with no residual cancer, and normal rectal tissue suggest that flowmetry is a promising complementary modality for accurate diagnosis of residual cancer.</p>", "<p>To our knowledge, this is the first <italic>in vivo</italic> application of AR-PAM Doppler flowmetry. Blood flow velocity calculated from AR-PAM data provides additional functional information to complement AR-PAM-based optical absorption imaging and improve the cancer diagnosis and cancer treatment response assessment. Similar to Doppler ultrasound, the AR-PAM Doppler mode can be turned on to quantify the local blood flow in a selected region of interest. In addition, power Doppler can be implemented, which should be more sensitive than the flow velocity estimation. Currently, the AR-PAM Doppler flow calculation is done off-line because the algorithm is computationally expensive. However, real-time flow velocity estimation and display can be done by using a computer with advanced GPU after further validation of the algorithm reported in this study. We anticipate that the AR-PAM Doppler mode will be implemented in real-time to improve colorectal cancer imaging and in other oncology applications.</p>", "<p>One limitation of our technique is that area-based angle compensation cannot compensate for the flow directions of individual vessels. A potential solution is to implement a detailed vessel tracking algorithm to process rectal vasculature images from a 3D mechanically scanning probe, yielding the flow directions of individual blood vessels for more thorough and accurate angle compensation in computing flow velocities.</p>", "<p>Overall, through this series of experiments, we demonstrate the potential of correlational Doppler processing in quantifying blood flow. Pilot patient data showed statistically significant increased blood perfusion around the tumor periphery and more organized and distributed flow in the treated tumor bed without residual cancer and in the normal rectal tissue. This additional blood perfusion parameter can be combined with the rectal tumor morphology seen by co-registered US and the absorption profile provided by AR-PAM to more the accurately assess treatment response and optimize treatment outcomes. Currently, we are improving the prototype system and planning to recruit a large patient cohort to validate the preliminary results.</p>" ]
[ "<title>Abstract.</title>", "<title>Significance</title>", "<p>Photoacoustic Doppler flowmetry offers quantitative blood perfusion information in addition to photoacoustic vascular contrast for rectal cancer assessment.</p>", "<title>Aim</title>", "<p>We aim to develop and validate a correlational Doppler flowmetry utilizing an acoustic resolution photoacoustic microscopy (AR-PAM) system for blood perfusion analysis.</p>", "<title>Approach</title>", "<p>To extract blood perfusion information, we implemented AR-PAM Doppler flowmetry consisting of signal filtering and conditioning, A-line correlation, and angle compensation. We developed flow phantoms and contrast agent to systemically investigate the flowmetry’s efficacy in a series of phantom studies. The developed correlational Doppler flowmetry was applied to images collected during <italic>in vivo</italic> AR-PAM for post-treatment rectal cancer evaluation.</p>", "<title>Results</title>", "<p>The linearity and accuracy of the Doppler flow measurement system were validated in phantom studies. Imaging rectal cancer patients treated with chemoradiation demonstrated the feasibility of using correlational Doppler flowmetry to assess treatment response and distinguish residual cancer from cancer-free tumor bed tissue and normal rectal tissue.</p>", "<title>Conclusions</title>", "<p>A new correlational Doppler flowmetry was developed and validated through systematic phantom evaluations. The results of its application to <italic>in vivo</italic> patients suggest it could be a useful addition to photoacoustic endoscopy for post-treatment rectal cancer assessment.</p>", "<title>Keywords:</title>" ]
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[ "<title>Acknowledgments</title>", "<p>We gratefully acknowledge funding support from the National Institutes of Health (NIH), grant no. R01 R01EB034398, and the institutional support of the Siteman Cancer Center. We thank Dr. James Ballard for providing editing support.</p>", "<p><bold>Sitai Kou</bold> received his BSc degree in bioengineering from the University of Washington, Seattle, Washington, in 2016. He is currently working towards his PhD at Washington University in St. Louis, Missouri, in the biomedical engineering program. His research interests include ultrasound and photoacoustic microscopy and tomography, photoacoustic imaging system development, and quantitative photoacoustic imaging of human tissue.</p>", "<p><bold>Xiandong Leng</bold> currently serves as a staff optical system engineer at Seyond. He boasts a solid academic foundation with BS and MS degrees in applied physics and optics from Harbin Institute of Technology, earned in 2014 and 2016, respectively. He received his PhD in 2021 from the Biomedical Engineering Department at Washington University in St. Louis. His work spans various applications, such as biomedical imaging, LiDAR, semiconductors, and nanophotonics.</p>", "<p><bold>Hongbo Luo</bold> joined Dr. Quing Zhu’s research group in 2018 and received his PhD in 2023 from the Electrical and System Engineering Department at Washington University in St. Louis. He is currently a system engineer at Changzhou United Imaging Healthcare Surgical Technology Co., Ltd. His main research interests are optical coherence tomography endoscopy and photoacoustic endoscopy for cancer imaging and diagnosis.</p>", "<p><bold>Haolin Nie</bold> received her BS degree in biomedical engineering and her MS degree in computer science from Northwestern University. She is a PhD candidate in biomedical engineering at Washington University in St. Louis. Her research interests include computer-aided diagnosis, optical and ultrasound microscopy and tomography, cancer imaging, and software engineering.</p>", "<p><bold>Quing Zhu</bold> is the Edwin H Murty Professor of Engineering in Washington University in St. Louis. Her research interests are focused on multimodality photoacoustic, ultrasound, optical coherence tomography, and structured light imaging techniques and machine learning algorithms for cancer detection and treatment assessment and prediction.</p>", "<title>Disclosures</title>", "<p>The authors declare that there are no conflicts of interest related to this article.</p>", "<title>Code and Data Availability</title>", "<p>Associated code is available at <ext-link xlink:href=\"https://github.com/OpticalUltrasoundImaging/Photoacoustic_dopplor_code\" ext-link-type=\"uri\">https://github.com/OpticalUltrasoundImaging/Photoacoustic_dopplor_code</ext-link>. Data are available from the corresponding author upon reasonable request.</p>" ]
[ "<fig position=\"float\" id=\"f1\"><label>Fig. 1</label><caption><p>AR-PAM/US system components (a) Probe body picture. (b) Probe body CAD. (c) Probe body with transparent parts. The stepper motor, at the extreme left, turns the drive shaft in small radial increments. The drive shaft and probe body house the US XDC wires (not shown) and the optical fiber, which extends along the center of the probe. (d) Cross-sectional view of the imaging head. The drive shaft is fixed to the imaging head with set screws. The optical fiber passes through a small opening in the waterproof housing holding the laser line mirror that reflects light into the short fiber fixed in the center hole of the US XDC.</p></caption></fig>", "<fig position=\"float\" id=\"f2\"><label>Fig. 2</label><caption><p>Drawings illustrating the preparation of a spiral flow phantom. (a) Individual molds. (b) Mold assembly for making a small cylindrical phantom. (c) Resulting small cylindrical phantom with spiral tubing on its exterior. (d) Mold with sleeve for encasing the spiral tubing to produce the final phantom. (e) Image of completed spiral flow phantom with embedded tubing.</p></caption></fig>", "<fig position=\"float\" id=\"f3\"><label>Fig. 3</label><caption><p>(a) Experimental flow phantom setup with linear tubing suspended in DI water (left) and with spiral tubing gelatin phantom fixed in DI water (right). Contrast agents suspended in DI water flow from the syringe pump through the tubing, which is imaged by the endoscopic imaging probe. (b) Illustration of the linear tubing being scanned. Upon laser exposure, optical contrast agents traveling in the tubing generate PA signals that are picked up by the US XDC. Angle for angle correction between the true velocity direction and measured velocity direction is illustrated in the zoomed-in figure.</p></caption></fig>", "<fig position=\"float\" id=\"f4\"><label>Fig. 4</label><caption><p>Time of arrival correction algorithm workflow and PA/US coregistration workflow. (a) The original US image is denoised at 10 dB above the noise floor to give (b). To compensate for tissue displacement, the rising edge in (c) is aligned with (d) the original PA image to yield (e) an aligned PA image, where the time of arrival is the same for all A-lines. Correlational Doppler processing yields (g). The Doppler data are subsequently backshifted (h) and coregistered with the original US image (j) to give (i) the Doppler and US coregistration image. This coregistered image is then converted to (k) polar display to better represent the scanning. (l) The rising edge time of arrival is changed to (m) polar display and used in angle calculation (n) for angle compensation.</p></caption></fig>", "<fig position=\"float\" id=\"f5\"><label>Fig. 5</label><caption><p>(a) Flow speed measurements from 0.01 to . A fitted line with value of 0.965 and theoretical velocity is shown. (b) Experimental angle compensation results, where blue is the calculated velocity, red is the compensated velocity, and yellow is the actual flow velocity.</p></caption></fig>", "<fig position=\"float\" id=\"f6\"><label>Fig. 6</label><caption><p>Spiral flow phantom imaging. (a) 3D AR-PAM endoscopic image of the spiral phantom. Contrast dye deposited on the inner radius of the spiral tubing wall generated a higher PA signal than dye on the outer radius. (b) PA coregistered with the US image of the scan plane that maximally intersects the tubing bore (c) Doppler image coregistered with US image. (d) Variation of Doppler-calculated flow speed with actual speed. 100 A-line ROI averaging is applied to the data, and a fitted curve with value of 0.913 is shown.</p></caption></fig>", "<fig position=\"float\" id=\"f7\"><label>Fig. 7</label><caption><p>Examples of PA images coregistered with US, Doppler coregistered with US, and flow speed across a rectal cross-section. (a) Polar and (b) linear US coregistered with PA scan of a patient with cancerous tissue bordered in the red rectangle. (c) US coregistered with a Doppler scan of the same patient (d) Doppler flow speed bar chart across rectal tissue of the same patient, with cancerous tissue bordered in red. (e) Polar and (f) linear US coregistered with a PA scan of a patient with treatment scarring, bordered in green (g) US coregistered with Doppler scan of the same patient with treatment scar, labeled in green (h) Doppler flow speed bar chart across rectal tissue of patient with treatment scar, labeled in green (i) polar and (j) linear US coregistered with PA scan of patient with normal rectal tissue (k) US coregistered with Doppler scan of the patient with normal tissue (l) Doppler flow speed bar chart across rectal tissue of the same patient. (m) Box plot of average Doppler velocity across patients, analyzed with a -test, the cancer regions exhibit statistically significant lower velocity than normal regions in all three cancer patients (P1–P3), and the responding tumor bed region exhibits slightly higher flow velocities than normal regions, but these are not statistically significant.</p></caption></fig>" ]
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[{"label": ["7."], "surname": ["Yao", "Wang"], "given-names": ["J.", "L. V."], "article-title": ["Photoacoustic microscopy"], "source": ["Laser Photonics Rev."], "volume": ["7"], "issue": ["5"], "fpage": ["758"], "lpage": ["778"], "year": ["2013"], "pub-id": ["10.1002/lpor.201200060"]}, {"label": ["13."], "surname": ["B\u00fccking", "van den Berg", "Balabani"], "given-names": ["T. M.", "P. J.", "S."], "article-title": ["Processing methods for photoacoustic Doppler flowmetry with a clinical ultrasound scanner"], "source": ["J. Biomed. Opt."], "volume": ["23"], "issue": ["2"], "fpage": ["026009"], "year": ["2018"], "pub-id": ["JBOPFO", "10.1117/1.JBO.23.2.026009"], "issn": ["1083-3668"]}, {"label": ["16."], "ext-link": ["CNI Laser.com"]}, {"label": ["18."], "surname": ["Jensen"], "given-names": ["J. A."], "source": ["Estimation of Blood Velocities Using Ultrasound: A Signal Processing Approach"], "publisher-name": ["Cambridge University Press"], "year": ["1996"]}]
{ "acronym": [], "definition": [] }
18
CC BY
no
2024-01-14 23:43:49
J Biomed Opt. 2024 Jan 13; 29(Suppl 1):S11517
oa_package/11/db/PMC10787588.tar.gz
PMC10787589
0
[ "<title>Introduction</title>", "<p>In photoacoustic (PA) imaging, short-pulsed near-infrared light is applied to tissue. Various tissue chromophores absorb the light to trigger thermoelastic expansion and pressure wave generation. Detection of these waves, which have frequencies in the ultrasound (US) regime, allows for image reconstruction of the absorbed optical energy.##REF##27086868##1## A strongly absorbing chromophore is hemoglobin, which enables PA imaging to accurately depict blood vessels. Further, due to the specific absorption spectra of hemoglobin in its two states, oxygenated and deoxygenated, the method can potentially report on blood oxygen saturation in tissue. It is known that vascularization associated with cancer is more abundant and structurally different than in healthy tissues, and oxygen saturation is an important physiological biomarker in tumor hypoxia. This makes PA imaging highly attractive for oncology.##REF##35322236##2## Furthermore, the method is noninvasive, contrast agent-free, relatively cheap, and with image acquisition times lower than conventional magnetic resonance imaging (MRI) protocols.</p>", "<p>In the context of breast cancer, PA tomography (PAT) has shown encouraging results with multiple proposed devices exploiting different light delivery systems and US detection geometries.##REF##31871887##3## Promising PAT applications for breast cancer span tumor detection,##REF##29907740##4## diagnosis,##REF##28899968##5##<sup>,</sup>##REF##28169313##6## and neoadjuvant chemotherapy (NAC) monitoring.##REF##28169313##6##<sup>,</sup>##UREF##0##7## The latter is a systemic therapy administered before surgery, with the goals of (1) reducing tumor size enabling breast-conserving surgery; (2) treating possible future metastatic disease, even if undetectable in preoperative staging; and (3) tailoring future chemotherapeutic decisions.</p>", "<p>It is important to monitor response to NAC, to spare nonresponding patients the toxicity (and expenses) of ineffective treatments, and to prevent further tumor progression due to postponed surgery. Repetitive imaging at optimal time-points during NAC is conducted using MRI techniques, radionuclide (positron emission tomography) imaging, and hybrid imaging.##REF##25727555##8## This longitudinal imaging enables tumor assessment by evaluating the changes in size, shape, and characteristics of the tumor, and helps to gauge the effectiveness of NAC. Compared to the standard of care imaging modalities, PAT has advantages for repeat measurements and could play an important role in NAC response monitoring by providing a safe and noninvasive method to visualize biological and functional response in the tumor microenvironment.</p>", "<p>One of the requirements in longitudinal imaging is that data between imaging sessions are comparable, from the point of view of patient positioning. In particular in PAT, repositioning of the breast inside the recording aperture can cause changes in the field-of-view as well as geometric misalignment of blood vessels between PAT scans, which challenges the measurement of qualitative and quantitative vascular changes.##REF##33473348##9## An available solution to this problem is the use of breast-supporting cups, which partially enable reproducible repositioning, but their use is inherently prone to human error and dependent on the operator’s experience.##REF##33473348##9## Further, such solutions can be complex to implement due to large variability in breast size, morphology, and composition.</p>", "<p>Approaches based on image co-registration can constitute an effective and easy-to-implement solution to the problem of geometric misalignment between repeated scans. In this case, PAT scans acquired before and at different time points during the therapeutic regimen can be aligned in the same reference coordinate system such that the same anatomical structures become geometrically matched. This would allow the radiologist to conveniently focus on observing locoregional evolution in time or also, to develop and train computer-aided systems to extract changes in quantitative imaging biomarkers to monitor or predict the tumor response.</p>", "<p>Several image registration algorithms have been proposed for PA imaging but most of them aimed at registering PA images with conventional imaging modalities such as MRI.##REF##28169313##6##<sup>,</sup>##REF##37397508##10##<named-content content-type=\"online\"></named-content><named-content content-type=\"print\"><sup>–</sup></named-content>##REF##35336570##13## To the best of our knowledge, only two research studies deal with the problem of unimodal registration of serial PA images.##UREF##1##14##<sup>,</sup>##UREF##2##15## A parametric approach using an intensity-based scheme and gradient descent optimizer was proposed to co-register PAT images of hands.##UREF##1##14## However, one major limitation of parametric registration algorithms is the requirement to choose the anticipated deformation field parametrization (examples are rigid, affine, or B-splines). This can be problematic in the case of complex and unknown deformations, i.e., the breast repositioning within the recording aperture. In a second study,##UREF##2##15## a registration method based on PAT image decomposition (into foreground, noisy background, and corrupted foreground) and coarse-to-fine alignment was proposed. This was intended to correct body motion in <italic>in vivo</italic> PAT images by co-registering multiple shot-volumes before averaging.##UREF##2##15## Integrating image decomposition with a multiscale approach was a key to cope with the sparse nature of PAT images, presence of noise, and variety in sizes of blood vessels. However, the algorithm is only able to correct for global affine transformations reducing its use to a limited number of applications. Several deep learning approaches have been proposed for medical image registration.##UREF##3##16## However, most of these require a pretraining phase, and their generalization ability strongly depends on the amount of available training data. This makes their application inconvenient in PAT image registration owing to the general paucity of standardized datasets. Therefore, we believe that there is a strong need in this research field for an image registration framework able to align <italic>in vivo</italic> three-dimensional (3D) PAT images affected by complex and nonlinear deformations.</p>", "<p>In this work, we propose a registration framework based on the use of coordinate-based neural networks and multiscale Frangi vesselness filtering to co-register longitudinal 3D PAT scans. The algorithm does not require any training data, <italic>a priori</italic> parametric deformation model, or manual landmark annotation. Following the recent study by Wolterink et al.,##UREF##4##17## a multilayer perceptron (MLP) network is used to transform coordinates of the fixed image domain to the moving image domain, and hence, implicitly represent deformation fields. Thanks to the use of sinusoidal activation functions, the network is able to represent local and nonlinear deformation fields. We show the effectiveness of the framework in aligning repeated PAT scans acquired with the recently developed hybrid PA-US system [Photoacoustic Mammoscope 3 (PAM3)]##UREF##5##18## under various conditions such as different illumination wavelengths and different breast supporting cups. Comparison with conventional image registration approaches shows the superiority of the proposed framework in serial PAT image alignment. We refer to the proposed framework as MUVINN-reg, which stands for multiscale vesselness-based image registration using neural networks. The proposed framework is implemented in Python and fully available together with data on GitHub (available at <ext-link xlink:href=\"https://github.com/brunodesanti/muvinn-reg\" ext-link-type=\"uri\">https://github.com/brunodesanti/muvinn-reg</ext-link>).</p>" ]
[ "<title>Methods</title>", "<title>MUVINN Image Registration Framework</title>", "<p>The proposed MUVINN framework aims to co-register serial PAT scans of the breast. A coordinate-based MLP is optimized to find the displacement field between a first or reference PAT scan called the fixed image, and a second PAT scan called the moving image, acquired after breast repositioning inside the recording aperture. The framework is based on Wolterink et al.##UREF##4##17## who proposed the use of coordinate-based neural networks for co-registration of inspiration and expiration 3D computed tomography lung images. The algorithm was modified to adapt its use on PAT images of the breast, mainly by implementing a coarse-to-fine strategy with the use of multiscale Frangi vesselness filtering within the network optimization. An overview of the proposed framework is shown in ##FIG##0##Fig. 1##. The entire framework was implemented in CUDA-enabled PyTorch.</p>", "<title>Coarse-to-fine similarity term</title>", "<p>In the context of PAT image registration, vascular structures are a valuable source of common information, since PAs are sensitive to blood. For this reason, the similarity metric was computed on Frangi vesselness filtered versions of the original images. A coarse-to-fine strategy was adopted to favor alignment of vessels with different sizes; network optimization starts with a higher value of the standard deviation of the gaussian kernel to enhance only the main vascular structure, then, is progressively decreased in a step-wise fashion during training iterations to enhance smaller vessels. Using a higher provides the network with low noise and less sparse (in spatial domain) data in the first iterations, which has the positive effects of improving robustness to noise and improving gradients for the network optimization (see section 1 in the <ext-link xlink:href=\"https://doi.org/10.1117/1.JBO.29.S1.S11515.s01\" ext-link-type=\"uri\">Supplementary Material</ext-link>). For each experiment, five values of sigma were used during optimization, , 4000 training iterations each. Intensities of the Frangi filtered images were adaptively modulated to improve image contrast.##REF##33563996##24##<sup>,</sup>##REF##36862628##25## Normalized cross correlation (NCC), which is more robust compared to intensity difference-based metrics such as L1 or mean square error, was used as the similarity metric to intensity shifts that might be caused by changes in light fluence due to breast repositioning.</p>", "<title>Regularization and activation function</title>", "<p>Image registration is an ill-posed problem, hence, regularization is fundamental to avoid instabilities and converge to smooth deformation fields.##UREF##10##26## Jacobian regularization was used to penalize excessive expansion and shrinkage of vascular features. It is reasonable to assume that breast repositioning will not heavily affect the size of biological structures in the tissue. Furthermore, any expansion and shrinkage is of interest when capturing biological changes over time due to therapy or disease progression, thus, not to be corrected. The weighting parameter was set to 0.95 for all experiments.</p>", "<p>Activation functions of the MLP have a direct effect on the ability of the network to accurately represent the signal (in this case, the displacement field). It has been shown that sinusoidal activation function has a superior ability to represent high frequency content than rectified linear unit.##UREF##11##27## To correct for small and local misalignments of vessels, sinusoidal activation functions were employed in this study with an angular frequency equal to 30.</p>", "<title>Training and implementation details</title>", "<p>As usually done for INRs,##UREF##11##27## image coordinates were normalized in the range . A coarse segmentation mask of vessels was obtained on the fixed image by Frangi vesselness filtering and adaptive thresholding (threshold map exponentially decaying as a function of depth). In each training epoch, a batch of 200 coordinates was randomly sampled from the set of coordinates belonging to the mask. A cubical patch was defined leading to a total number of coordinates for each training epoch. The side length of the patch was changed according to the scale of the Frangi filtering in the iteration, according to (in normalized units). This allowed to compute NCC in local patches with a size depending on the enhanced vessels around the sampled points during the optimization process. After transforming the coordinates through the network, intensity values were sampled from the fixed and moving images by linear interpolation and the loss was computed. Regarding model hyperparameters, an MLP was used with six hidden layers, each of which contained 300 units with sinusoidal activation function (except for the last one which uses linear activation functions to ensure linear mapping). Adam optimizer with learning rate of and 20,000 epochs was used. Training and tests were done on a Windows 11 machine with an Intel Core i9-11900K @ 3.5 GHz, 128 GB RAM and NVIDIA RTX3090 24 GB.</p>", "<title>Experimental Protocol</title>", "<p>The study was approved by the Medical Ethics Review Board and acknowledged by the Dutch Central Committee for Research on human subjects. A 59-year-old woman volunteered in the study. She was classified with Fitzpatrick skin type 2 and wore brassiere size 80E. The volunteer was informed about the study, and informed consent was obtained.</p>", "<title>Imaging session</title>", "<p>A total of 15 scans were acquired in the imaging session. The imaging session was divided into two phases. In phase A, seven repeated PAM3 scans (S1 to S7) of the left breast were performed by using the breast supporting cup with the optimal size equal to 7 out of 8 (largest size). The optimal cup size was determined through test-fitting various cup sizes on the volunteer by the operator (author R.F.G.B.) prior to the start of the imaging session. Between each pair of repeated scans, the volunteer was asked to stand up and then lie down on the imager to reposition the breast in the cup. Scans S1 to S3 were acquired with dual wavelength (720 and 870 nm) while the remaining four (S4 to S7) were acquired with a single wavelength (720 nm). For scans S6 and S7, the breast was deliberately mispositioned in the cup to test the algorithm performance when a more severe deformation occurs. In phase B, the right breast was scanned eight times (S8 to S15) with a single wavelength (720 nm) using different breast-supporting cups (S8 and S9: cup size 7, S10 and S11: cup size 5, S12 and S13: cup size 4, and S14 and S15: cup size 8).</p>", "<title>MUVINN-reg registration experiments</title>", "<p>The performances of the proposed image registration framework were evaluated under different serial imaging scenarios: experiment 1–standard breast repositioning; experiment 2–breast repositioning and different illumination wavelength; and experiment 3–breast repositioning and different breast-supporting cups. ##TAB##0##Table 1## shows an overview of the registration experiments conducted.</p>", "<title>Baseline methods</title>", "<p>We compared the proposed MUVINN-reg algorithm with two conventional image registration approaches: a parametric approach using Elastix##REF##19923044##28## and non-parametric approach with the diffeomorphic demons algorithm.##REF##19041946##29## For the comparison, we used the dataset of experiment 1 consisting of seven pairs of repeated scans: S1 and S2, S1 and S3, S1 and S4, S1 and S5, S1 and S6, and S1 and S7. For a fair comparison, images were preprocessed using Frangi vesselness filtering with same standard deviation values used for MUVINN-reg () and adaptive intensity modulation before image registration. Also, since the performance of these algorithms can highly depend on parameters, experimental tuning was performed to find a parameter configuration yielding accurate registration results in similar computational times of MUVINN-reg. For more information regarding the implementation and tuning of these methods, the reader can refer to section 2 in the <ext-link xlink:href=\"https://doi.org/10.1117/1.JBO.29.S1.S11515.s01\" ext-link-type=\"uri\">Supplementary Material</ext-link>.</p>", "<title>Evaluation metrics</title>", "<p>Peak signal-to-noise ratio (PSNR), NCC, Dice similarity coefficient (DSC), and target registration error (TRE) were computed to quantify performance of the proposed framework. While PSNR and NCC are based on similarity between image intensities,##UREF##12##30## DSC and TRE measure the geometric overlap between vascular structures. DSC was computed between binary masks of vessel segmentation (performed on both fixed and moving images as described in Sec. <xref rid=\"sec3.1.3\" ref-type=\"sec\">3.1.3</xref>.) before and after alignment. Regarding the TRE analysis, matching landmarks in correspondence of vessel branching points were manually annotated on both fixed and moving images using the open source software MeVisLab.##REF##22147070##31## After the co-registration, coordinates of the annotated points on the fixed image were transformed by feedforwarding them through the trained network into the moving coordinate system. Distances between corresponding points were computed before and after co-registration. Registration results were also evaluated qualitatively by plotting maximum intensity projections (MIPs) of the image pair overlay. The fixed image was encoded in the blue channel, whereas the moving one in the red channel. Overlapping structures appear as magenta. For better visualization of vascular features, images were processed using Frangi vesselness filtering and intensity adaptive modulation, similarly to previous studies.##REF##29907740##4##<sup>,</sup>##UREF##0##7##<sup>,</sup>##REF##33563996##24##<sup>,</sup>##REF##36862628##25##</p>" ]
[ "<title>Results</title>", "<title>Experiment 1: Performance of MUVINN-reg in Correcting Misalignments due to Breast Repositioning</title>", "<p>Despite the use of breast supporting cups, misalignment of vascular structures always occurred after breast repositioning inside the recording aperture. In ##FIG##1##Fig. 2(a)##, MIPs of the overlay of pair S1 and S3 are shown. Misalignment was observed also in deeper regions of the breast, as shown in the depth-layered MIPs in ##FIG##1##Fig. 2(b)##. TRE analysis showed a displacement equal to [##FIG##1##Fig. 2(c)##]. As shown in the bottom row of ##FIG##1##Fig. 2(a)##, co-registration by MUVINN-reg improved alignment of vascular features. Also, ##FIG##1##Fig. 2(b)## shows the improvement in the alignment of a vessel deeper than 4 cm (pointed by the black arrows). After co-registration, TRE decreased to , which represents a mean error of less than 2 voxels [bottom row of ##FIG##1##Fig. 2(c)##]. Similar results were obtained also in other cases (##TAB##1##Table 2##).</p>", "<p>For deliberate mispositioning, the breast was intentionally mispositioned inside the cup to cause larger and more complex deformation. In fact, this produced a greater initial displacement (maximum of mean displacements equal to 33.31 mm compared to 10.97 mm for the normal repositioning as shown in ##TAB##1##Table 2##). This was also prominent in the MIPs of the overlay [pair S1 and S6 before co-registration shown in ##FIG##2##Fig. 3(a)##]. For the specific case of pair S1 and S6, TRE analysis resulted in an initial displacement of . Despite the larger displacement, MUVINN-reg was able to successfully co-register this pair of scans. MIPs of the overlay showed the presence of vessels missing in one of the two scans due to different positioning of the breast inside the cup [shown by the black arrows in ##FIG##2##Fig. 3(a)##, after co-registration]. TRE analysis showed a decrease of the displacement to . In general, for every case of experiment 1, we observed an improvement of all the metrics from before to after co-registration (##TAB##1##Table 2##).</p>", "<title>Experiment 2: Testing MUVINN-reg with Different Illumination Wavelengths</title>", "<p>For this experiment, scan S1 at 720 nm was defined as fixed, and scans S2 and S3 at 870 nm as moving. ##FIG##3##Figure 4## shows the performance of MUVINN-reg in co-registering pair S1 and S2. MIPs of PAT scans [shown in ##FIG##3##Fig. 4(a)##] show differences in intensity due to different absorption of tissue chromophores at the two wavelengths 720 and 870 nm (e.g., skin intensity is higher in the 720 nm PAT scan due to higher absorption of melanin). To enable accurate comparison between co-registered images, the displacement field represented by the network was applied to the corresponding 720 nm version of the moving scans. MIPs of the overlays before and after co-registration are shown in ##FIG##3##Fig. 4(b)##. As a result, the proposed framework was able to successfully co-register PAT scans with different illumination wavelengths and decreasing the TRE from and to and , respectively for pairs S1 and S2 as well as S1 and S3. Values of displacement after co-registration were similar to those obtained in experiment 1 for pairs S1 and S2 (both 720 nm) as well as S1 and S3 (both 720 nm) showing the robustness of MUVINN-reg to different illumination wavelengths. Image similarity always increased from before to after co-registration (see ##TAB##1##Table 2##).</p>", "<title>Experiment 3: Testing MUVINN-reg with Different Breast Cup Sizes</title>", "<p>The Twente PAM3 uses breast-supporting cups, which help to stabilize the breast during the image acquisition. The selection of the appropriate cup size is determined by the breast size, which is assessed through a fitting process prior to the acquisition. If the cup is inadvertently chosen smaller than the breast, the outer regions of the breast closer to the chest wall will extend beyond the cup and fall out of the field-of-view. On the other hand, choosing a cup bigger than the breast will not stabilize the breast appropriately. Although it is unlikely that different cups would be chosen for repeated measurements during NAC monitoring, it is still possible for the breast to undergo morphological changes that would alter the field-of-view. So, it is important to test the capabilities of the framework in case of field-of-view inconsistency between scans.</p>", "<p>##FIG##4##Figure 5## shows the MIPs of overlay of the fixed image S8 (cup size 7) with three scans acquired with different cup sizes: (a) S11 (cup size 5), (b) S13 (cup size 4), (c) S15 (cup size 8) before [panel (a)] and after [panel (b)] co-registration by MUVINN-reg. TRE analysis showed larger initial displacements than the other experiments. MUVINN-reg was able to retrieve the deformation field accurately for each pair. After co-registration, from the MIPs of overlay, the inconsistency of field-of-view between different cup sizes was clearer to notice: for pairs S8 and S11 as well as S8 and S13, deeper vascular structures of the breast were mostly blue due to their absence in the smaller cup scan [see ##FIG##4##Fig. 5(b)##]. Comparing S8 and S15 after co-registration, vessels closer to the chest wall were imaged in S15 and not in S8. TRE analysis after co-registration showed a decrease of the displacement from , , and to , , and , respectively, for pairs S8 and S11, S8 and S13, as well as S8 and S15, respectively. Also for this experiment, the result of the quantitative analysis is an increase of all similarity metrics (##TAB##1##Table 2##).</p>", "<title>Comparison with Baseline Methods</title>", "<p>Optimal configuration of parameters was selected for each baseline method after experimental tuning (data shown in section 2 in the <ext-link xlink:href=\"https://doi.org/10.1117/1.JBO.29.S1.S11515.s01\" ext-link-type=\"uri\">Supplementary Material</ext-link>). ##FIG##5##Figure 6## shows the qualitative comparison of MIPs of the overlay after co-registration by Elastix, Demons, and MUVINN-reg for two pairs of repeated PAT scans: S1 and S2 (normal repositioning) as well as S1 and S6 (deliberate mispositioning). Although, for pair S1 and S2 (normal repositioning), major vascular structures were found to be aligned after co-registration, the black arrows point to regions where misalignment of vascular structures was present. In contrast, this was not the case for MUVINN-reg. Regarding the S1 and S6 pair (deliberate mispositioning), both Elastix and Demons were not able to correctly align the main vascular structure, in contrast to our proposed algorithm. The weak performance of the parametric approach by Elastix might be attributed to two main factors: (1) the parameterization of the transformation model (rigid + B-spline) may not be appropriate for the deformation that occurs in the case of breast repositioning, and (2) a too low number of iterations in order to achieve the global minimum. Regarding to the nonparametric approach by Demons, one possible reason could be the use of an intensity-based metric such as the sum of squared differences that does not take into account variations in intensity between the two images due to different light distribution. Regarding computational times, these are heavily dependent on the parameter selection (mainly the number of iterations and the stopping condition if present). To avoid excessively long computational times, only configurations with a reasonable number of iterations were tested (see section 2 in the <ext-link xlink:href=\"https://doi.org/10.1117/1.JBO.29.S1.S11515.s01\" ext-link-type=\"uri\">Supplementary Material</ext-link>). However, with the selected configurations of parameters, MUVINN-reg showed better performance at the cost of slightly higher computational times (e.g., pair S1 and S3, MUVINN-reg: 21.08 min, Elastix: , Demons: ).</p>" ]
[ "<title>Discussion</title>", "<p>Promising developments have been presented in the research into PA imaging for NAC monitoring.##REF##28169313##6##<sup>,</sup>##UREF##0##7## However, the analysis of longitudinal PA images in this setting has hitherto been qualitative. Issues related to organ repositioning in the imaging volume cannot be discounted, which can result in nonoptimal correlation between images and consequently complications in their comparison to extract subtle biological changes. Specifically, repositioning affects the position of vascular structures relative to the coordinate system of the device, posing challenges to identify the same structure-of-interest across multiple serial images. The use of a breast-supporting cup makes the repositioning procedure more reproducible##REF##33473348##9## as it provides a rigid containment structure for the breast. Nevertheless, the present study showed that despite the use of cups, mean displacements in the range of 5.99 to 10.97 mm were found between images.</p>", "<p>To address these problems, we developed MUVINN-reg, a framework that uses coordinate-based neural networks and multiscale Frangi vesselness filtering to co-register longitudinal PAT scans in three dimensions. The effectiveness of the framework was investigated in correcting for geometric misalignments that could potentially take place in subject repositioning. MUVINN-reg demonstrated a drastic reduction of displacement and increase in image similarity after co-registration for several tested scenarios (##TAB##1##Table 2##).</p>", "<p>The use of a coordinate-based neural network makes the proposed registration framework unique in that it is capable of being able to represent deformation fields in a continuous domain. This allows the deformation field to be known at any coordinate in the domain and avoids the use of finite-differences techniques to calculate spatial derivatives of the deformation field.##UREF##4##17## In addition, the approach gives the possibility to integrate similarity metrics, tailored for the image registration problem, as long as they are differentiable. For the specific case of PA images, using a similarity term based on vesselness features allows only relevant structures within images to be considered, and potential noise to be excluded. This feature combined with the use of NCC allows the method to be robust to variations in image intensities (as shown in ##FIG##3##Fig. 4## for experiment 2), which might occur in repeated PA imaging due to changes in light distribution. The implementation of a coarse-to-fine strategy is critical to the success of the framework. As already shown in other studies,##UREF##2##15## decomposing the image in different scales facilitates the registration process, which is the reason why image registration frameworks often work on multiple pyramidal levels. We observed that the use of a coarse-to-fine strategy crucially improves accuracy and consistency of the framework (see section 1 in the <ext-link xlink:href=\"https://doi.org/10.1117/1.JBO.29.S1.S11515.s01\" ext-link-type=\"uri\">Supplementary Material</ext-link>). Finally, the contribution of the Jacobian regularization in the loss function helps to represent regular deformation fields and avoids that vessels are excessively shrunk or expanded. The combination of all these features makes the algorithm capable of co-registering pairs of images at different displacement magnitudes (experiment 1), affected by inconsistencies in image intensity (experiment 2), and field-of-view (experiment 3).</p>", "<p>In experiment 3, the algorithm was subjected to pairs of images with different fields-of-view, where some vessels were present in only one of the two images. This is important in the context of NAC monitoring as it is very hard to image the same breast volume in longitudinal imaging. There is also potential utility of the algorithm in image mosaicking or image stitching,##REF##19923044##28## in scanning imaging systems where multiple smaller and overlapping images are compounded. Here, MUVINN-reg can be applied to perform the mosaicking based on overlapping regions.</p>", "<p>Comparison with baseline methods showed the superiority of MUVINN-reg over the existing conventional image registration algorithms based on parameterized transformations and intensity-based metrics. Adaption of these methods to PA imaging would require more intensive image preprocessing and accurate parameter tuning to cope with the sparse nature of PA data, and the complexity of the deformation fields.</p>", "<p>One limitation of the proposed framework lies in the necessity of parameter tuning to ensure optimal performance. The most critical parameters are the number of iterations and the values of the Frangi scales during the optimization. A higher number of the iterations gives better results in terms of co-registration accuracy but leads to higher computational times. Regarding the choice of the scales, higher standard deviations produce more regular displacement fields with the risk of not aligning fine structures in the image. On the other hand, the use of very low standard deviations lead to the risks of noise being considered in the optimization process which would ultimately produce wrong displacement fields and irregular distortion of structures in the transformed image. Also, starting with larger values allows to mitigate the sparsity of the image, therefore improving gradients for the network optimization. At this stage, parameter selection was performed using a trial and error approach and a good parameter configuration was found which can work in every scenario. However, the presence of inaccuracies in the alignment of some vessels was noted in certain cases (Fig. S3 in the <ext-link xlink:href=\"https://doi.org/10.1117/1.JBO.29.S1.S11515.s01\" ext-link-type=\"uri\">Supplementary Material</ext-link>). We believe this is because the misalignment of these small vascular structures did not have a significant contribution to the loss function during optimization. This can be solved by increasing either the number of iterations or the number of points sampled in each iteration, at the cost of increasing the computational expenses. Also, in future, automated parameter optimization and a user-friendly interface will be implemented to make the framework usable in a practical setting. Another limitation lies in computational time. While the fact that INRs are self-supervised techniques is a big advantage because they do not require a training set, each pair of images requires optimization of a new neural network. This results in higher computational times compared to a supervised technique. The computational time for each pair of image was . These are acceptable computational times when considering the context of disease or treatment monitoring but become less convenient in the case of real time applications.</p>", "<p>Finally, the effects of NAC on the patient’s breast can be heterogeneous, and it is largely unknown how the therapy changes the morphology of the breast and blood vessels. For this reason, it is essential to evaluate the framework in actual treatment monitoring conditions. It is worth noting that the dataset at this point is limited to only one PAT device with its own recording aperture geometry and breast-supporting system. We encourage researchers in the community to consider applying our method to their own image datasets for further validation and exploration.</p>" ]
[ "<title>Conclusions</title>", "<p>We presented MUVINN-reg, an automatic 3D image registration framework that can address the challenges of geometric misalignment in longitudinal PAT breast scans. We demonstrated the robust performance in co-registering images under different unfavorable repositioning scenarios. MUVINN-reg can align vessels deeper than 4 cm. This advancement holds significant promise for enabling reproducible and quantitative monitoring of disease progression and treatment response in breast cancer using PAs.</p>" ]
[ "<title>Abstract.</title>", "<title>Significance</title>", "<p>Photoacoustic tomography (PAT) has great potential in monitoring disease progression and treatment response in breast cancer. However, due to variations in breast repositioning, there is a chance of geometric misalignment between images. Further, poor repositioning can affect light fluence distribution and imaging field-of-view, making images different from one another. The net effect is that it becomes challenging to distinguish between image changes due to repositioning effects and those due to true biological variations.</p>", "<title>Aim</title>", "<p>The aim is to develop a three-dimensional image registration framework for geometrically aligning repeated PAT volumetric images, which are potentially affected by repositioning effects such as misalignment, changed radiant exposure conditions, and different fields-of-view.</p>", "<title>Approach</title>", "<p>The proposed framework involves the use of a coordinate-based neural network to represent the displacement field between pairs of PAT volumetric images. A loss function based on normalized cross correlation and Frangi vesselness feature extraction at multiple scales was implemented. We refer to our image registration framework as MUVINN-reg, which stands for multiscale vesselness-based image registration using neural networks. The approach was tested on a longitudinal dataset of healthy volunteer breast PAT images acquired with the hybrid photoacoustic-ultrasound Photoacoustic Mammoscope 3 imaging system. The registration performance was also tested under unfavorable repositioning conditions such as intentional mispositioning, and variation in breast-supporting cup size between measurements.</p>", "<title>Results</title>", "<p>A total of 13 pairs of repeated PAT scans were included in this study. MUVINN-reg showed excellent performance in co-registering each pair of images. The proposed framework was shown to be robust to image intensity shifts and field-of-view changes. Furthermore, MUVINN-reg could align vessels at imaging depths greater than 4 cm.</p>", "<title>Conclusions</title>", "<p>The proposed framework will enable the use of PAT for quantitative and reproducible monitoring of disease progression and treatment response.</p>", "<title>Keywords:</title>" ]
[ "<title>Background</title>", "<title>Image Registration Algorithm</title>", "<title>Image registration as a minimization problem</title>", "<p>The image registration problem can be defined as finding the optimal transformation such that where and are the moving and fixed image, respectively, and and are the moving and fixed image domains, respectively. The optimal transformation can be found by solving the following minimization problem: where is a similarity measure between two images, is a regularization term to encourage regular deformation field, and is a non-negative weighting parameter.</p>", "<title>Coordinate-based networks and their use for image registration</title>", "<p>According to the universal approximation theorem, any continuous function can be approximated by a multilayer neural network with nonlinear activation functions.##UREF##6##19## This allows a MLP network to represent a signal (e.g., image) by mapping its coordinate domain ( and pixel coordinates) to the signal intensity (pixel intensity values). These networks can be referred to as coordinate-based neural networks and are the basis of a fast growing category of deep learning techniques called implicit neural representations (INRs). Excellent results have been demonstrated using INRs in many applications such as scene rendering,##UREF##7##20## image reconstruction,##UREF##8##21## and recently image registration.##UREF##4##17##<sup>,</sup>##UREF##9##22##</p>", "<p>In image registration, an MLP network can be optimized to represent the displacement field such that coordinate in the fixed image geometrically corresponds to coordinate in the moving image.##UREF##4##17## A loss function based on a data term and a regularization term is used to maximize similarity between the images and encourage smooth displacement fields [see Eq. (2)]. The network is trained by using batches of coordinates from the fixed image domain as input. Using coordinate-based networks for image registration has several advantages compared to traditional or convolutional-based approaches. First, they allow to represent the deformation field continuously over the image domain with a low and fixed number of parameters (network weights). Second, they do not require any <italic>a priori</italic> information regarding the transformation model. Third, they allow derivatives to be computed analytically making the regularization task free of finite differences computations. Finally, these methods are highly flexible and extendable to different registration tasks, by changing the similarity metric, regularization term, or activation function.</p>", "<title>PAM3 Imaging System</title>", "<p>In this study, the hybrid PA-US PAM3 imaging system was used.##UREF##5##18## The system is embedded in a custom-designed bed, which includes the laser system and the hemispherical US recording aperture. The subject lies prone on the bed with her breast positioned within the imaging bowl (26 cm diameter). The bowl accommodates the water-coupled US aperture, which comprises 512 single-element US transducers flush with the inside of the bowl. For PA excitation, the bowl has 40 optical fiber bundle terminations distributed on the inner surface of the bowl. Acquisition parameters such as number of bowl rotations, number of wavelengths, number of averages, and number of US shots can be programmed for each individual acquisition. Eight breast-supporting cups with different sizes are available and used to stabilize the breast during the measurements. The operator chooses the appropriate cup size before the acquisition.##UREF##5##18## An iterative full-wave model-based image reconstruction method is to reconstruct PA images, which can make use of different speed of sound (SOS) models.##REF##27910824##23## Number of iterations was equal to 10 and voxel size equal to 0.4 mm.##UREF##5##18## In the present work, we used a 2-SOS model with the known SOS for the coupling water and a single SOS for the whole breast, which is manually chosen to maximize the sharpness of the reconstructed blood vessels.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgments</title>", "<p>This work was supported by the REACT-EU project “Foto-akoestische mammografie naar de kliniek met de PAM3+.” Partial funding was also received from the 4TU Precision Medicine program (4tu.nl/precision-medicine) and the Pioneers in Healthcare Innovation (PIHC) fund 2020 for project “Photoacoustic breast tomography: Towards monitoring of neoadjuvant chemotherapy response.” Authors are grateful to Dr. Jelmer Wolterink for his help during the development of the algorithm and for making his codes available at the following GitHub: <ext-link xlink:href=\"https://github.com/MIAGroupUT/IDIR\" ext-link-type=\"uri\">https://github.com/MIAGroupUT/IDIR</ext-link></p>", "<p><bold>Bruno De Santi</bold>, PhD, earned his master’s degree in biomedical engineering from the Polytechnic University of Turin, Turin, Italy. During his PhD, he developed interest in designing and implementing algorithms for multimodal image processing and image registration. His current research primarily focuses in developing deep learning-based solutions applied to breast photoacoustic imaging for image registration, motion compensation, and image denoising, with the ultimate objective of clinically translating the Twente Photoacoustic Mammoscope 3 (PAM3).</p>", "<p><bold>Lucia Kim</bold> is a biomedical engineering master’s student at the University of Twente. Throughout her academic journey, she developed interest in deep learning applications in medical imaging. Her thesis focuses on this area, which is about machine learning-based image registration for serial photoacoustic imaging.</p>", "<p><bold>Rianne F. G. Bulthuis</bold>, MSc, received her MSc degree in biomedical engineering from the University of Twente, Enschede, The Netherlands, in 2021. She subsequently received her MSc degree in industrial engineering and management from the same university. She is currently a PhD candidate in the Department Multi-Modality Medical Imaging (M3I) at the University of Twente. Her interest is in clinical translation of novel medical technologies. Currently, she focuses on clinical studies and phantom development for photoacoustic tomography of the breast.</p>", "<p><bold>Felix Lucka</bold>, PhD, is a senior researcher in the Computational Imaging Group at the Centrum Wiskunde &amp; Informatica. After a first degree in mathematics and physics in 2011, he completed a PhD in applied mathematics at WWU Münster (Germany) in 2015 followed by a postdoc at the University College London until 2017. His main interests are mathematical challenges arising from biomedical imaging applications that have an inverse problem described by partial differential equations at their core.</p>", "<p><bold>Srirang Manohar</bold>, PhD, is a full professor and chair of the newly formed Multi-Modality Medical Imaging (M3I) Group at the University of Twente. He received his PhD from the Indian Institute of Science, Bangalore, India. His research expertise is in photoacoustic imaging and spans technology development to early clinical assessment. The intended applications of the technologies span the range of <italic>ex vivo</italic> tissue imaging, minimally invasive imaging to noninvasive imaging.</p>", "<title>Disclosures</title>", "<p>Authors have no relevant financial interests and no potential conflicts of interest to disclose.</p>", "<title>Code and Data Availability</title>", "<p>All data and codes presented in this study are publicly available in the following GitHub repository: <ext-link xlink:href=\"https://github.com/brunodesanti/muvinn-reg\" ext-link-type=\"uri\">https://github.com/brunodesanti/muvinn-reg</ext-link></p>", "<title>Author Contributions</title>", "<p>B. De Santi conceived and designed the study, developed software, interpreted data, and authored the manuscript. L. Kim contributed to software development, designed and conducted the volunteer study, and provided manuscript input. R.F.G. Bulthuis contributed to the design and execution of the volunteer study and provided manuscript input. F. Lucka was responsible for data reconstruction and provided manuscript input. S. Manohar secured funding, contributed to study conception and design, contributed to the manuscript, and provided guidance. All authors reviewed and edited the manuscript.</p>" ]
[ "<fig position=\"float\" id=\"f1\"><label>Fig. 1</label><caption><p>Overview of MUVINN image registration framework. (a) Coordinate sampling from the fixed image domain. (b) MUVINN uses a coordinate-based MLP with sinusoidal activation functions to represent the displacement field, such that transformed coordinates on the moving image anatomically correspond to coordinates . in the fixed image. NCC between multiscale Frangi vesselness filtered images are used as data similarity term. Jacobian regularization is used to find smooth and regular deformation fields. (c) Coordinate transformation into the moving image domain.</p></caption></fig>", "<fig position=\"float\" id=\"f2\"><label>Fig. 2</label><caption><p>Performances of MUVINN-reg in correcting misalignments due to normal breast repositioning. (a) Coronal and axial MIPs of the overlay of pairs of PAT repeated scans (b) Sagittal MIPs of the overlay at different depths from the breast surface (&gt;0, 2, and 4 cm). (c) 3D rendered vascular network of the moving image; before co-registration: green spheres represent the ground-truth, namely the annotated points on the moving image, and blue spheres are the annotated points on the fixed image; after co-registration: red spheres are the annotated points on fixed image after transformation to the moving coordinate system. The black arrow indicates the alignment of a vessel deeper than 4 cm. TRE is reported as mean and standard deviation of the distances between corresponding points.</p></caption></fig>", "<fig position=\"float\" id=\"f3\"><label>Fig. 3</label><caption><p>Performances of MUVINN-reg in correcting misalignments due to deliberate breast mispositioning. Refer to ##FIG##1##Fig. 2## for explanation. The black arrows show vessels missing in one of the two repeated scans due to the different positioning of the breast. TRE is reported as mean and standard deviation of the distances between corresponding points.</p></caption></fig>", "<fig position=\"float\" id=\"f4\"><label>Fig. 4</label><caption><p>Performances of MUVINN-reg in co-registering repeated PAT scans with different illumination wavelengths. (a) MIPs of fixed and moving PAT images for pair S1 and S2. (b) Coronal and axial MIPs of the overlay of pairs of PAT repeated scans showing misalignment before and after co-registration. TRE is reported as mean and standard deviation of the distances between corresponding points.</p></caption></fig>", "<fig position=\"float\" id=\"f5\"><label>Fig. 5</label><caption><p>Performances of MUVINN-reg in co-registering repeated PAT scans with different breast-supporting cups. (a) MIPs of the overlay before co-registration for pairs S8 (cup size 7) and S11 (cup size 5), S8 (cup size 7) and S13 (cup size 4), as well as S8 (cup size 7) and S15 (cup size 8). (b) MIPs of the overlay after co-registration for the same pairs. TRE is reported as mean and standard deviation of the distances between corresponding points.</p></caption></fig>", "<fig position=\"float\" id=\"f6\"><label>Fig. 6</label><caption><p>Comparison between parametric approach using Elastix, non-parametric diffeomorphic Demons algorithm, and the proposed MUVINN-reg. (a) Coronal and axial MIPs of the overlay before co-registration and after co-registration using Elastix, Demons and MUVINN-reg for pair S1 and S3 (normal breast repositioning). The black arrows show regions with misalignment of vascular structures. (b) Coronal and axial MIPs of the overlay before co-registration and after co-registration using Elastix, Demons, and MUVINN-reg for pair S1 and S6 (deliberate breast mispositioning). TRE and computational time () are reported.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"t001\"><label>Table 1</label><caption><p>Overview of the MUVINN-reg registration experiments. For simplicity, the same fixed scan was used within the same experiment.</p></caption><!--OASIS TABLE HERE--><table frame=\"hsides\" rules=\"groups\"><colgroup><col align=\"left\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/></colgroup><thead><tr><th colspan=\"2\" align=\"left\" valign=\"top\"> </th><th align=\"center\" valign=\"top\">Scan</th><th align=\"center\" valign=\"top\">Wavelength (nm)</th><th align=\"center\" valign=\"top\">Cup size</th></tr></thead><tbody><tr><td rowspan=\"3\" align=\"left\">Experiment 1: breast repositioning</td><td align=\"center\">Fixed image</td><td align=\"center\">S1</td><td align=\"center\">720</td><td align=\"center\">7</td></tr><tr><td rowspan=\"2\" align=\"center\">Moving image(s)</td><td align=\"center\">S2 to S5 (normal repositioning)</td><td rowspan=\"2\" align=\"center\">720</td><td rowspan=\"2\" align=\"center\">7</td></tr><tr><td align=\"center\">S6 and S7 (deliberate mispositioning)</td></tr><tr><td rowspan=\"2\" align=\"left\">Experiment 2: breast repositioning with different illumination wavelength</td><td align=\"center\">Fixed image</td><td align=\"center\">S1</td><td align=\"center\">720</td><td align=\"center\">7</td></tr><tr><td align=\"center\">Moving image(s)</td><td align=\"center\">S2 and S3</td><td align=\"center\">870</td><td align=\"center\">7</td></tr><tr><td rowspan=\"4\" align=\"left\">Experiment 3: breast repositioning with different breast-supporting cup</td><td align=\"center\">Fixed image</td><td align=\"center\">S8</td><td align=\"center\">720</td><td align=\"center\">7</td></tr><tr><td rowspan=\"3\" align=\"center\">Moving image(s)</td><td align=\"center\">S10<xref rid=\"t001fn1\" ref-type=\"table-fn\"><sup>a</sup></xref> and S11</td><td align=\"center\">720</td><td align=\"center\">5</td></tr><tr><td align=\"center\">S12 and S13</td><td align=\"center\">720</td><td align=\"center\">4</td></tr><tr><td align=\"center\">S14 and S15</td><td align=\"center\">720</td><td align=\"center\">8</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"t002\"><label>Table 2</label><caption><p>Quantitative results for each experiment.</p></caption><!--OASIS TABLE HERE--><table frame=\"hsides\" rules=\"groups\"><colgroup><col align=\"left\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/><col align=\"center\"/></colgroup><thead><tr><th align=\"left\" valign=\"top\"> </th><th colspan=\"4\" align=\"center\" valign=\"top\">Before co-registration</th><th colspan=\"4\" align=\"center\" valign=\"top\">After co-registration</th></tr><tr><th align=\"left\" valign=\"top\">Pair</th><th align=\"center\" valign=\"top\">PSNR (dB)</th><th align=\"center\" valign=\"top\">NCC</th><th align=\"center\" valign=\"top\">DSC</th><th align=\"center\" valign=\"top\">TRE (mm)</th><th align=\"center\" valign=\"top\">PSNR (dB)</th><th align=\"center\" valign=\"top\">NCC</th><th align=\"center\" valign=\"top\">DSC</th><th align=\"center\" valign=\"top\">TRE (mm)</th></tr></thead><tbody><tr><td align=\"left\">S1 and S2 (both 720 nm)</td><td align=\"center\">39.95</td><td align=\"center\">0.36</td><td align=\"center\">0.33</td><td align=\"center\">10.97 ± 3.78</td><td align=\"center\">44.01</td><td align=\"center\">0.73</td><td align=\"center\">0.63</td><td align=\"center\">0.89 ± 0.48</td></tr><tr><td align=\"left\">S1 and S3 (both 720 nm)</td><td align=\"center\">38.75</td><td align=\"center\">0.14</td><td align=\"center\">0.12</td><td align=\"center\">6.99 ± 2.34</td><td align=\"center\">44.40</td><td align=\"center\">0.75</td><td align=\"center\">0.69</td><td align=\"center\">0.90 ± 0.40</td></tr><tr><td align=\"left\">S1 and S4 (both 720 nm)</td><td align=\"center\">38.79</td><td align=\"center\">0.14</td><td align=\"center\">0.13</td><td align=\"center\">6.22 ± 1.79</td><td align=\"center\">44.80</td><td align=\"center\">0.78</td><td align=\"center\">0.69</td><td align=\"center\">0.64 ± 0.33</td></tr><tr><td align=\"left\">S1 and S5 (both 720 nm)</td><td align=\"center\">38.55</td><td align=\"center\">0.13</td><td align=\"center\">0.10</td><td align=\"center\">5.99 ± 1.23</td><td align=\"center\">44.85</td><td align=\"center\">0.78</td><td align=\"center\">0.69</td><td align=\"center\">0.63 ± 0.26</td></tr><tr><td align=\"left\">S1 and S6 (both 720 nm)</td><td align=\"center\">37.96</td><td align=\"center\">0.12</td><td align=\"center\">0.09</td><td align=\"center\">24.98 ± 6.75</td><td align=\"center\">41.84</td><td align=\"center\">0.58</td><td align=\"center\">0.48</td><td align=\"center\">1.97 ± 2.62</td></tr><tr><td align=\"left\">S1 and S7 (both 720 nm)</td><td align=\"center\">38.27</td><td align=\"center\">0.12</td><td align=\"center\">0.08</td><td align=\"center\">33.31 ± 5.16</td><td align=\"center\">41.43</td><td align=\"center\">0.51</td><td align=\"center\">0.43</td><td align=\"center\">2.33 ± 2.25</td></tr><tr><td align=\"left\">S1 (720 nm) and S2 (870 nm)</td><td align=\"center\">39.95</td><td align=\"center\">0.36</td><td align=\"center\">0.39</td><td align=\"center\">10.97 ± 3.78</td><td align=\"center\">43.57</td><td align=\"center\">0.69</td><td align=\"center\">0.64</td><td align=\"center\">0.75 ± 0.29</td></tr><tr><td align=\"left\">S1 (720 nm) and S3 (870 nm)</td><td align=\"center\">38.75</td><td align=\"center\">0.14</td><td align=\"center\">0.12</td><td align=\"center\">6.99 ± 2.34</td><td align=\"center\">43.80</td><td align=\"center\">0.71</td><td align=\"center\">0.66</td><td align=\"center\">0.92 ± 0.47</td></tr><tr><td align=\"left\">S8 (cup 7) and S11 (cup 5)</td><td align=\"center\">37.69</td><td align=\"center\">0.07</td><td align=\"center\">0.05</td><td align=\"center\">17.3 ± 3.71</td><td align=\"center\">42.82</td><td align=\"center\">0.68</td><td align=\"center\">0.49</td><td align=\"center\">0.82 ± 0.56</td></tr><tr><td align=\"left\">S8 (cup 7) and S12 (cup 4)</td><td align=\"center\">37.21</td><td align=\"center\">0.05</td><td align=\"center\">0.04</td><td align=\"center\">20.77 ± 2.78</td><td align=\"center\">42.45</td><td align=\"center\">0.67</td><td align=\"center\">0.43</td><td align=\"center\">1.32 ± 1.02</td></tr><tr><td align=\"left\">S8 (cup 7) and S13 (cup 4)</td><td align=\"center\">37.40</td><td align=\"center\">0.05</td><td align=\"center\">0.04</td><td align=\"center\">28.11 ± 5.25</td><td align=\"center\">41.79</td><td align=\"center\">0.60</td><td align=\"center\">0.41</td><td align=\"center\">1.45 ± 0.9</td></tr><tr><td align=\"left\">S8 (cup 7) and S14 (cup 8)</td><td align=\"center\">38.15</td><td align=\"center\">0.10</td><td align=\"center\">0.04</td><td align=\"center\">9.19 ± 2.26</td><td align=\"center\">43.42</td><td align=\"center\">0.71</td><td align=\"center\">0.59</td><td align=\"center\">1.30 ± 0.44</td></tr><tr><td align=\"left\">S8 (cup 7) and S15 (cup 8)</td><td align=\"center\">37.96</td><td align=\"center\">0.09</td><td align=\"center\">0.05</td><td align=\"center\">15.17 ± 3.23</td><td align=\"center\">42.27</td><td align=\"center\">0.62</td><td align=\"center\">0.54</td><td align=\"center\">0.81 ± 0.42</td></tr></tbody></table></table-wrap>" ]
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overflow=\"scroll\"><mml:mrow><mml:mn>2.5</mml:mn><mml:mo>·</mml:mo><mml:msub><mml:mrow><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">/</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math30\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>5</mml:mn><mml:mi mathvariant=\"normal\">e</mml:mi><mml:mo>−</mml:mo><mml:mn>5</mml:mn></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math31\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mi>σ</mml:mi><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">{</mml:mo><mml:mn>12</mml:mn><mml:mo>,</mml:mo><mml:mn>9</mml:mn><mml:mo>,</mml:mo><mml:mn>5</mml:mn><mml:mo>,</mml:mo><mml:mn>3</mml:mn><mml:mo>,</mml:mo><mml:mn>2</mml:mn><mml:mo stretchy=\"false\">}</mml:mo></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math32\" display=\"inline\" 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overflow=\"scroll\"><mml:mrow><mml:mn>10.97</mml:mn><mml:mo>±</mml:mo><mml:mn>3.78</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math37\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>6.99</mml:mn><mml:mo>±</mml:mo><mml:mn>2.34</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math38\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>0.75</mml:mn><mml:mo>±</mml:mo><mml:mn>0.29</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math39\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>0.92</mml:mn><mml:mo>±</mml:mo><mml:mn>0.47</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math40\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>17.3</mml:mn><mml:mo>±</mml:mo><mml:mn>3.71</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math41\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>28.11</mml:mn><mml:mo>±</mml:mo><mml:mn>5.25</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math42\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>15.17</mml:mn><mml:mo>±</mml:mo><mml:mn>3.23</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math43\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>0.82</mml:mn><mml:mo>±</mml:mo><mml:mn>0.56</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math44\" display=\"inline\" 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overflow=\"scroll\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math49\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mi>σ</mml:mi></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"math50\" display=\"inline\" overflow=\"scroll\"><mml:mrow><mml:mn>20.42</mml:mn><mml:mo>±</mml:mo><mml:mn>0.56</mml:mn><mml:mtext>  </mml:mtext><mml:mi>min</mml:mi></mml:mrow></mml:math></inline-formula>" ]
[]
[]
[]
[]
[ "<supplementary-material id=\"s01\" position=\"float\" content-type=\"local-data\"></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"t001fn1\"><label>a</label><p>S10 went missing due to technical issues in the data transfer.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"JBO-029-S11515-g001\" position=\"float\"/>", "<graphic xlink:href=\"JBO-029-S11515-g002\" position=\"float\"/>", "<graphic xlink:href=\"JBO-029-S11515-g003\" position=\"float\"/>", "<graphic xlink:href=\"JBO-029-S11515-g004\" position=\"float\"/>", "<graphic xlink:href=\"JBO-029-S11515-g005\" position=\"float\"/>", "<graphic xlink:href=\"JBO-029-S11515-g006\" position=\"float\"/>" ]
[ "<media xlink:href=\"JBO_029_S11515_SD001.pdf\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["7."], "surname": ["Lin"], "given-names": ["L."], "etal": ["et al."], "article-title": ["Photoacoustic computed tomography of breast cancer in response to neoadjuvant chemotherapy"], "source": ["Adv. Sci."], "volume": ["8"], "issue": ["7"], "fpage": ["2003396"], "year": ["2021"], "pub-id": ["10.1002/advs.202003396"]}, {"label": ["14."], "surname": ["Yu"], "given-names": ["Q."], "etal": ["et al."], "article-title": ["Registration of photoacoustic tomography vascular images: comparison and analysis of automatic registration approaches"], "source": ["Front. Phys."], "volume": ["10"], "fpage": ["1045192"], "year": ["2022"], "pub-id": ["10.3389/fphy.2022.1045192"]}, {"label": ["15."], "surname": ["Bise"], "given-names": ["R."], "etal": ["et al."], "article-title": ["Vascular registration in photoacoustic imaging by low-rank alignment via foreground, background and complement decomposition"], "source": ["Lect. Notes Comput. Sci."], "volume": ["9902"], "fpage": ["326"], "lpage": ["334"], "year": ["2016"], "pub-id": ["LNCSD9", "10.1007/978-3-319-46726-9_38"], "issn": ["0302-9743"]}, {"label": ["16."], "surname": ["Fu"], "given-names": ["Y."], "etal": ["et al."], "article-title": ["Deep learning in medical image registration: a review"], "source": ["Phys. Med. Biol."], "volume": ["65"], "issue": ["20"], "fpage": ["20TR01"], "year": ["2020"], "pub-id": ["PHMBA7", "10.1088/1361-6560/ab843e"], "issn": ["0031-9155"]}, {"label": ["17."], "surname": ["Wolterink", "Zwienenberg", "Brune"], "given-names": ["J. M.", "J. C.", "C."], "article-title": ["Implicit neural representations for deformable image registration"], "conf-name": ["Proc. Mach. Learn. Res."], "year": ["2022"]}, {"label": ["18."], "surname": ["Dantuma"], "given-names": ["M."], "etal": ["et al."], "article-title": ["Fully three-dimensional sound speed-corrected multi-wavelength photoacoustic breast tomography"], "year": ["2023"]}, {"label": ["19."], "surname": ["Leshno"], "given-names": ["M."], "etal": ["et al."], "article-title": ["Multilayer feedforward networks with a nonpolynomial activation function can approximate any function"], "source": ["Neural Netw."], "volume": ["6"], "issue": ["6"], "fpage": ["861"], "lpage": ["867"], "year": ["1993"], "pub-id": ["NNETEB", "10.1016/S0893-6080(05)80131-5"], "issn": ["0893-6080"]}, {"label": ["20."], "surname": ["Mildenhall"], "given-names": ["B."], "etal": ["et al."], "article-title": ["NeRF: representing scenes as neural radiance fields for view synthesis"], "source": ["Lect. Notes Comput. Sci."], "volume": ["12346"], "fpage": ["405"], "lpage": ["421"], "year": ["2020"], "pub-id": ["LNCSD9", "10.1007/978-3-030-58452-8_24"], "issn": ["0302-9743"]}, {"label": ["21."], "surname": ["Lozenski", "Anastasio", "Villa"], "given-names": ["L.", "M. A.", "U."], "article-title": ["A memory-efficient self-supervised dynamic image reconstruction method using neural fields"], "source": ["IEEE Trans. Comput. Imaging"], "volume": ["8"], "fpage": ["879"], "lpage": ["892"], "year": ["2022"], "pub-id": ["10.1109/TCI.2022.3208511"]}, {"label": ["22."], "surname": ["Weitz"], "given-names": ["P."], "etal": ["et al."], "article-title": ["The ACROBAT 2022 challenge: automatic registration of breast cancer tissue"], "year": ["2023"]}, {"label": ["26."], "surname": ["Fischer", "Modersitzki"], "given-names": ["B.", "J."], "article-title": ["Ill-posed medicine\u2014an introduction to image registration"], "source": ["Inverse Probl."], "volume": ["24"], "issue": ["3"], "fpage": ["034008"], "year": ["2008"], "pub-id": ["INPEEY", "10.1088/0266-5611/24/3/034008"], "issn": ["0266-5611"]}, {"label": ["27."], "surname": ["Sitzmann"], "given-names": ["V."], "etal": ["et al."], "article-title": ["Implicit neural representations with periodic activation functions"], "conf-name": ["Proc. 34th Int. Confe. Neural Information Process. Syst."], "publisher-name": ["Curran Associates Inc."], "publisher-loc": ["Red Hook, New York"], "fpage": ["12"], "year": ["2020"]}, {"label": ["30."], "surname": ["Mitchell"], "given-names": ["H. B."], "article-title": ["Image similarity measures"], "source": ["Image Fusion: Theories, Techniques and Applications"], "fpage": ["167"], "lpage": ["185"], "publisher-name": ["Springer"], "publisher-loc": ["Berlin, Heidelberg"], "year": ["2010"]}]
{ "acronym": [], "definition": [] }
31
CC BY
no
2024-01-14 23:43:49
J Biomed Opt. 2024 Jan 13; 29(Suppl 1):S11515
oa_package/e7/5c/PMC10787589.tar.gz
PMC10787591
38206278
[ "<title>Introduction</title>", "<p>When touching a slanted surface for a period of time, it will feel less slanted. This mechanism is called adaptation. When next presented with a physical surface that is level, this nonslanted surface will feel as though it was slanted in the opposite direction (adaptation aftereffect). In the past, slant adaptation aftereffects were mainly investigated by adapting some environmental property in a given sense, for instance, the haptic sense in isolation (##REF##27698392##van Dam, Plaisier, Glowania, &amp; Ernst, 2016##; ##REF##32735569##Glowania, Plaisier, Ernst, &amp; Van Dam, 2020##), or the visual sense in isolation (e.g., ##UREF##1##Bergman &amp; Gibson 1959##; ##UREF##0##Balch, Milewski, &amp; Yonas, 1977##; ##REF##15831068##Berends, Liu, &amp; Schor, 2005##; ##REF##16698056##Knapen &amp; van Ee, 2006##). The present study instead focuses on how slant adaptation in one sense (here the haptic sense) can be influenced or interfered with by the availability of information of another sense (here vision) without that information necessarily directly being informative about the slant.</p>", "<p>Previous studies have gained insights into the processing level at which haptic adaptation occurs by investigating to which conditions the adaptation aftereffect transfers. For instance, studies have found that haptic adaptation using static contact with a surface (e.g., by placing two fingers or a hand on a surface to estimate the slant or curvature) is largely posture based (##REF##8861174##Vogels, Kappers, &amp; Koenderink 1996##; ##REF##27698392##van Dam et al., 2016##) and does not transfer to exploration modes that have a dynamic component such as when moving the finger across the surface (##REF##27698392##van Dam et al., 2016##). This indicates that at least part of the adaptation occurs at the level of the proprioceptors. Such specificity of the exploration mode used for haptic adaptation has been shown for both unimanual (##REF##27698392##van Dam et al., 2016##) and bimanual adaptation when the index fingers of both the left and right hand are used instead of multiple fingers of the same hand (##REF##32735569##Glowania et al., 2020##). Moreover, ##REF##32735569##Glowania et al. (2020)## found that these aftereffects were independent of whether an actual object was touched during adaptation or not. That is, slant adaptation aftereffects were observed when simply holding the index fingers of the left and right hand at different prespecified heights in midair during the adaptation phase, before touching a test surface to measure the aftereffect. These findings suggest that we must be adapting constantly to posture rather than haptic shape, regardless of whether we touch something or not. This posture adaptation then influences the perceived shape of subsequently haptically explored surfaces. In our daily life, however, we are hardly aware of the effects of haptic adaptation. This is very surprising, because haptic adaptation aftereffects can already be measured after only two seconds of touching a surface (##REF##8861174##Vogels et al., 1996##). So, either we are not aware of haptic adaptation aftereffects, which is plausible because we also constantly adapt visually without hardly ever noticing it, or we do not adapt in daily life circumstances, perhaps due to additional information from other sensory modalities.</p>", "<p>It is important to note that, in all the studies discussed herein, the participants adapted haptically without being able to see the stimulus. This leads to the question if seeing the object, i.e., in the present case, seeing the slant in combination with our finger positions influences haptic adaptation. From previous studies we know, for instance, that, for spatial dimensions such as slant, the sense of vision under normal viewing conditions is typically dominant to the sense of haptics and proprioception because of its greater reliability for such typical viewing conditions (e.g., ##REF##14080333##Rock &amp; Victor, 1964##; ##REF##11807554##Ernst &amp; Banks, 2002##; ##REF##12015120##van Beers, Wolpert, &amp; Haggard,2002##). A study by ##REF##11516950##Kennett, Taylor-Clarke, &amp; Haggard (2001)## moreover showed that, for a two-point discrimination task, the threshold for detecting being touched by two stimuli instead of one was significantly lower if the forearm was visible just before stimulation. This result shows that vision can enhance touch. On the one hand, this finding could mean that vision might be partially overruling the haptic adaptation aftereffects when the two are integrated, thus leading to a reduced haptic aftereffect. On the other hand, it has to be noted that the visual modality usually also adapts when presented with the same stimulus for a longer period of time, and it has been shown that unimodal sensory adaptation can transfer between vision and touch (##REF##19361996##Konkle et al., 2009##; ##UREF##6##Krystallidou &amp; Thompson, 2016##). This could mean that, when adapting visually and haptically to the same stimulus at the same time, this leads to an enhanced haptic adaptation aftereffect rather than reducing it, when the visual and haptic aftereffects are added together. The latter hypothesized effect would, most likely require visual fixation for several seconds because of the retinotopic nature of many visual shape-related aftereffects (##UREF##5##Knapen et al., 2010##; ##REF##23359857##Mathôt &amp; Theeuwes, 2013##). In general, however, when exploring a visual scene, we move our eyes at a rate of roughly 1 to 2 saccades per second depending also on the task and thus visually we often tend to not fixate long enough for visual aftereffects to reach awareness. That is, in viewing circumstances that don't require strict fixation we can assume vision to adapt very little. Thus, if there is visual dominance when vision and haptics are integrated, this would provide an explanation for the fact that we do not feel any effects of haptic adaptation in our everyday life as vision may be overruling haptic adaptation.</p>", "<p>A recent study by ##REF##21526338##Seizova-Cajic and Azzi (2011)## provides some evidence that it may indeed be the case that vision can overrule or cancel touch, or in their case rather proprioceptive, adaptation aftereffects. Their study investigated the effect vision has on the illusionary movement induced when muscles are stimulated with vibration. In their study, the participants’ biceps were stimulated with a strong vibratory stimulus, resulting in an illusionary movement of the arm. Importantly, when the vibration stops this technique also results in perceptual adaptation aftereffects, i.e., perceived movement of the forearm in the opposite direction. ##REF##21526338##Seizova-Cajic and Azzi (2011)## found that the strength of the perceived movement for the aftereffect depended on whether participants had seen their arm during the adaptation phase or not. When the participants could see their stimulated arm during adaptation, both the perceived illusory movement effect during the adaptation phase as well as the aftereffect after adaptation were considerably reduced. This finding is consistent with previous work by ##REF##6734787##Lackner and Taublieb (1984)##, who proposed that vision reduces the effect of the illusionary limb movement from muscle vibration as soon as it is available. However, in their study they found that just seeing the finger or hand was not as effective as seeing the hand or finger and having external objects as a reference. In their study, they had a condition in which the experiment was performed in the dark and one condition in which the experiment was done with the room lights on. In the dark condition, participants wore gloves that were either completely coated in phosphorescent paint or with just the index finger covered in the paint. In this way, the participants could see either their whole hand or just one finger in otherwise complete darkness. ##REF##6734787##Lackner and Taublieb (1984)## found that the illusionary movement was much more reduced when the room lights were switched on, i.e., when the hand or finger could be seen in relation to external objects when compared with either of the two conditions in the dark. Thus, it seems that vision influences haptic adaptation only when the visual input is informative about one's own position in relation to external objects. However, in their study only the effect of direct muscle-vibration stimulation was measured without looking further into its aftereffects.</p>", "<p>In the present study, we extend these previous findings by investigating in which circumstances and to what extent vision may influence bimanual haptic shape adaptation. In haptic shape adaptation, we can provide visual information not only about our own hand, but also about the touched object and it is not yet clear how these separate elements might each contribute to the visual influence on haptic adaptation. However, given that previous work indicated that haptic slant adaption is largely of a proprioceptive nature (##REF##27698392##van Dam et al., 2016##; ##REF##32735569##Glowania et al., 2020##) we were particularly interested in the role of the visual representation of the fingers. To investigate such potential influences from vision, we used the bimanual haptic slant adaptation paradigm as presented in one of our previous studies (##REF##32735569##Glowania et al., 2020##) in which participants used the index fingers of both hands to adapt to a haptic surface slant. We investigated the influence of vision on haptic adaptation by manipulating the kind and number of visual cues presented to the participants during the adaptation phase (e.g., cues about the space only, fingers only, and various combinations). To measure the size of the resulting haptic aftereffect for each condition, we measured the slant at which the surface felt level in the absence of visual information before and after adaptation. Based on the results by ##REF##21526338##Seizova-Cajic and Azzi (2011)## and ##REF##6734787##Lackner and Taublieb (1984)##, we expected a reduction of haptic adaptation when the visual information participants were presented with was informative about the position of the fingertips. Hence, in this study we investigated in two experiments to what extent and in what way visual information about the touched object may also interfere with the adaptation process.</p>" ]
[ "<title>Methods for experiment 1</title>", "<title>Participants</title>", "<p>Twelve members of the Applied Cognitive Psychology Group of Ulm University, three students of Ulm University, and author C.G. participated in this study (16 participants in total, average age 27.9 ± 5.9 years, 5 male). All participants were self-reported to be right handed. They gave informed consent prior to the start of the experiment and the students received 7€ per hour as compensation for their participation in the experiment. Ethical approval was obtained from the Bielefeld University ethics committee.</p>", "<title>Setup</title>", "<p>Participants were seated in front of a visual-haptic workbench. The workbench consisted of two PHANToM force-feedback devices (PHANToM premium 1.5, SensAble Technologies Inc., Woburn, MA) and a CRT Monitor (Sony CPD G500/G500J, Sony Europe Limited, Weybridge, UK), which was viewed via a mirror in the setup, for visual presentation (##FIG##0##Figure 1##A). The PHANToM force-feedback devices were placed one to each side of the workbench for haptic presentation of the stimulus. The index fingers of the participants were fixed with rubber bands in thimble-like holders at the end of the force feedback devices. These devices were used to create virtual surfaces by generating independent forces to the fingers. This way, the participants could feel and interact with virtual haptic surfaces in an automated setting. Direct vision of the hands was prevented. Visual feedback of the finger position was only available when needed experimentally, in which case it was presented stereoscopically on the CRT monitor. The participants placed their chin on a chinrest to keep their body midline aligned with the center of the workbench and to allow accurate rendering of the visual cues. To present the visual stimulus in three-dimensional (3D) stereoscopic depth, 3D-shutter glasses (RealD Pro CrystalEyes 4S VRLOGIC GmbH, Dieburg, Germany) were used to present the visual scene for the left and right eye separately. The stimuli for this experiment were created using a custom build program in C++, GHOST (for the haptic display) and OpenGL (for the visual display).</p>", "<title>Stimuli</title>", "<p>The stimuli used in this study consisted of several different components, each of which could either be present or absent during the adaptation phase. Note, however, that during test phases only a haptic surface was presented as the test stimulus was kept the same across all adaptation conditions. The haptic surface was rendered by the PHANToM force feedback devices and could be presented at different angles of slant (##FIG##0##Figure 1##). The angle of the surface, if present during adaptation, was kept consistent during the (top-up) adaptation phases at ±10 deg slant, but on test trials could vary according to a 1-up/1-down staircase procedure. The exploration of the haptically rendered surface was limited to 140 mm left to right and 26 mm in depth.</p>", "<p>If the surface was also visually presented during the adaptation phase, the visual surface would have the same surface slant (±10 deg slant). However, visually the surface was presented at a slightly larger scale to avoid potential conflicts at the surface edges. The visual surface extended either 145 mm (when combined with haptic elements) or 175 mm (when presented in isolation) in length and 36 mm in depth. The slightly shorter visual surface length when combined with haptic elements (i.e., 145 mm vs. 175 mm) was chosen to avoid the participants going toward the edges of the haptic space in the bimodal cases.</p>", "<p>For conditions that included cursors indicating the positions of the fingertips, the Visual Cursors were visually rendered spheres (10 mm in diameter). These spheres were used to give feedback about the position of the participants’ fingers, without participants being able to see their own fingers directly.</p>", "<p>In some conditions, a visual spatial reference was provided by adding what we will call Visual Boxes into the visual scene. These Visual Boxes were eight reference boxes (size: 10 mm × 10 mm × 10 mm) positioned 100 mm left and right of the body midline, 25 mm above and below the surface center, and 13 mm to the front and back from the surface midline (##FIG##1##Figure 2##, bottom left). The Visual Boxes were used to provide a visual spatial reference, without necessarily providing direct information about either the slant or the location of the participant's fingertips. Such a stable reference frame can however potentially be relevant to help interpret the visual location of other visual elements (i.e., cursors or a slanted surface) more accurately (see ##REF##6734787##Lackner &amp; Taublieb, 1984##).</p>", "<title>Adaptation conditions</title>", "<p>The elements described above were used in five adaptation conditions, which we explain in this section. The conditions can be divided into unimodal and bimodal adaptation conditions. In the unimodal adaptation conditions the surface was either presented only visually (Vision Only condition) or only haptically (Haptics Only condition) (see ##FIG##1##Figure 2##, top row) and served as baseline conditions to which the bimodal conditions were compared. The bimodal conditions were different combinations of vision and haptics (see ##FIG##1##Figure 2##, bottom row).</p>", "<title>Condition: Haptics Only (unimodal adaptation)</title>", "<p>In the Haptics Only condition (##FIG##1##Figure 2##, top left), only the haptic surface was presented. This means that the screen was black and no visual cues were presented to the participant, but participants were able to touch the slanted surface. Hence, this condition is a unimodal haptic condition and served as the haptic baseline condition to measure the haptic aftereffect without any potential interaction from vision.</p>", "<title>Condition: Vision Only (unimodal condition)</title>", "<p>In the Vision Only condition (##FIG##1##Figure 2##, top right), only the visual surface, was presented. This means that participants viewed the slanted surface during the adaptation phase, but the surface was not rendered haptically and, thus, participants were not able to touch the surface. This condition can be considered as the unimodal visual condition and served as the visual baseline condition to measure to what extent a visual slant aftereffect can transfer to haptics (because the test phase was always haptic only).</p>", "<title>Condition: Boxes (bimodal condition)</title>", "<p>In the Boxes condition (##FIG##1##Figure 2##, bottom left), participants were able to touch the slanted surface haptically and furthermore view eight boxes, rendered always in the same position irrespective of the slant of the haptic surface or the position of the fingers. Thus, these boxes served merely as a visual spatial reference for the general location of the haptic space, without providing any slant or finger information. Since both visual and haptic information was provided, this condition is considered a bimodal condition. Note, however, that, despite both visual and haptic information being available in the general display, no single element within the scene was truly bimodal. This condition, thus, served as a bimodal control for slant adaptation, to verify whether or not the information in the two modalities needs to match.</p>", "<title>Condition: Cursors (bimodal condition)</title>", "<p>In the cursors condition (##FIG##1##Figure 2##, bottom center), the participants could touch the slanted surface haptically and, furthermore, could see two little spheres corresponding in location to the position of their fingertips (i.e., the cursors). These cursor positions also contained some limited visual information about the slant of the surface because participants could visually compare the heights of the fingertips while touching the surface. Note that, in this case, the positions of the fingertips present actual bimodal information, and, using that information, the surface slant can be sensed as well through both proprioception and vision simultaneously. This condition is informative about whether visual information of the fingertips is sufficient to interfere with haptic slant adaptation.</p>", "<title>Condition: Slant and Cursors (bimodal condition)</title>", "<p>In the Slant and Cursors condition (##FIG##1##Figure 2##, bottom right), the participants were able to see as well as touch the slanted surface, and additionally the cursors representing the participant's fingertips were presented. This means that participants could also relate the visual position of the fingertips to the visuohaptic surface. Because visual and haptic information of both the slanted surface and the positions of the fingertips were presented congruently, this condition is considered bimodal and both the slanted surface and the fingers are both bimodal features.</p>", "<title>Procedure</title>", "<p>Each participant conducted each of the five adaptation conditions in a separate session, with a break of at least 10 minutes between sessions. Breaks were introduced to minimize carry-over effects. Because ##UREF##9##Vogels, Kappers, and Koenderink (1995)## showed that the haptic aftereffect of a curved surface decays quickly and is hardly present after 80 Seconds, we inferred that 10 minutes should be more than enough for the haptic aftereffect to vanish. The order of the conditions was randomized across participants.</p>", "<p>For each of the adaptation conditions, we followed a “pre-test–adaptation–post-test” paradigm. Test trials in the pre- and post-tests testing for haptic aftereffects were identical for all five conditions and only contained a haptically rendered surface without visual cues. For test trials, the participant's task was to decide whether the surface was tilted to the left or to the right, that is, to answer the question “Which side of the surface was higher: left or right?” In order to calculate the effect of adaptation on haptic slant perception, we used the participant's answers to these test-trials determining the point at which the participants haptically perceived the surface slant as level—that is, the Point of Subjective Equality (PSE)—before adaptation (in the Pre-Test) and after adaptation (in the Post-Test). To present the trials for the PSE measurements, we used a 1-up/1-down staircase procedure (for more information, see, e.g., ##UREF##2##Gescheider, 1976##), which converges on the point at which participants respond left side higher and right side higher with equal proportions (i.e., the PSE). The step size between test trials started with 8 deg, after two reversals in responses in the staircase procedure, the step size was decreased to 4 deg and after another two reversals to 2 deg. After 12 reversals, the staircase was terminated. To avoid potential hysteresis effects, we presented two intermixed staircases that started at different angles, i.e., one staircase started at –20 deg, while the other started at +20 deg. To calculate the PSE for each condition, we fitted psychometric curves (cumulative Gaussians) to the combined data from the two staircases for each test-phase, resulting in one PSE for the Pre-Test before adaptation and Post-Test after adaptation, respectively. We chose this method for obtaining the PSE, rather than, for example, averaging over reversals points in the staircase, because it allows us to use the data from all trials. For fitting the psychometric curves, we used the psignifit toolbox for MATLAB version 2.5.6 (##REF##11800458##Wichmann &amp; Hill, 2001a##; ##REF##11800459##Wichmann &amp; Hill, 2001b##), with mu (mean) and sigma (standard deviation) as free parameters for the cumulative Gaussian. The lapse-rate was fixed to zero, which given symmetry between left and right side higher stimuli/responses on either side the PSE, should not affect the positioning of the PSE. The PSE on the psychometric curve is the only point of interest in our particular case as 1-up/1-down staircases are not well-suited to determine the slope or the JND because of the sparse sampling away from the PSE. The PSE per test-phase was determined by obtaining the 50% cutoff of the psychometric curve (i.e., equal proportion of left side higher and right side higher responses). The size of the aftereffect was then calculated by subtracting the post-test PSE from the pre-test PSE.</p>", "<p>To familiarize participants with the task, they received as many practice test-trials as they liked at the start of each experimental session. As soon as both participant and experimenter were satisfied that the task was understood, the experimental procedure started.</p>", "<title>Pre-test</title>", "<p>Each trial in the pre-test started by showing the trial number on the screen. The participants lifted both of their index fingers until they reached a predefined threshold (75 mm above the surface, see ##FIG##0##Figure 1##B) and the trial number disappeared. Next, participants lowered their left and right index fingers until they reached the haptic surface. The participants were instructed to keep the fingers in static contact with the surface, that is, without actively moving the fingers over the surface. The surface was haptically rendered for 1 second as soon as one of the fingers touched the surface. After that 1 second for estimating the slant, the surface disappeared. Next, participants responded which side of the previously felt surface appeared higher: left or right. Participants made their response by moving with either the left or right finger into the corresponding response zone located at the top, left (left side higher response) and right (right side higher response) of the exploration space (##FIG##0##Figure 1##C). After providing a response, the next trial number appeared on the screen and the next trial started. Trials continued until the staircases terminated after 12 reversals.</p>", "<title>Adaptation</title>", "<p>Following the pre-test, the main adaptation phase started. Similar to the pre-test, participants started the main adaptation phase by raising their fingers until they crossed the trial-start threshold. During this phase, depending on the adaptation condition, participants could either see only: 1) the slant (Vision Only), 2) nothing (Haptics Only), 3) eight boxes (Boxes), 4) their own fingertips as cursors (Cursors), or 5) a combination of cursors and slant (Slant and Cursors). As before, participants lowered their fingers until they reached the adaptation surface (during adaptation this was always a ±10 deg slant) and held static contact with the surface for the whole adaptation period of 30 seconds. Note that the direction of the adaptation slant was counterbalanced between participants. As soon as one finger touched the surface, the 30 seconds of the adaptation phase started. This was the case for all conditions, except the Vision Only adaptation condition. In the Vision Only condition, since there was no haptic surface rendered, the display time started as soon as the threshold (the 75-mm threshold) (##FIG##0##Figure 1##B) for starting the trial was crossed by the participant's fingers during the downward movement. Furthermore, for this condition the participants were instructed to hold their fingers in the air during the adaptation phases. After the main adaptation phase ended the haptic surface (if rendered) as well as the visual scene (if present) disappeared. For the main adaptation phase, the participants were not asked to judge the surface slant and after the 30 seconds of adaptation, the program automatically proceeded to the post-test phase.</p>", "<title>Post-test</title>", "<p>The procedure of the post-test was the same as in pre-test, with the only difference that a 4 seconds top-up adaptation interval preceded each test trial in the post-test. After the 4 seconds were up, the test trial immediately started. During the top-up adaptation interval, the same visuohaptic condition (Haptics Only, Vision Only, Boxes, Cursors, Slant and Cursors) was presented as in the main adaptation phase. Similar to the main adaptation, the participants did not need to judge the surface slant for top-up adaptation intervals. The task of judging the slant only needed to be performed for the actual test-trials. Note, that for test trials in the post-test the surface slant was again presented only haptically and no visual information was shown. Both staircases were again terminated after 12 reversals each to complete the session.</p>" ]
[ "<title>Results of experiment 1</title>", "<p>The results of five participants (all female, right handed) were removed from the data analysis because their staircases did not converge within 40 trials in at least one of the conditions. Data from the remaining 11 participants were analyzed. To investigate the influence of vision on haptic adaptation, participants adapted to different conditions in which the amount of visual information about the slant varied. Besides two unimodal baseline conditions (Vision Only and Haptics Only rendering of the surface), participants were presented with three bimodal adaptation conditions in which, besides the haptic surface, participants either received only visual information about their fingers position (Cursors), or about the space in which the surface was rendered (Boxes), or about the slant and the fingers position (Slant and Cursors). ##FIG##2##Figure 3## shows the sizes of the aftereffects derived from all five adaptation conditions. First, the two baseline conditions—Haptics Only (the left-most bar) and Vision Only (right-most bar)—reveal very different haptic aftereffects: strong adaptation for the Haptics Only condition and no haptic adaptation for the Vision Only condition. This indicates that presenting only a visual but no haptic surface as an adapter stimulus does not lead to any noticeable haptic slant aftereffect. Note that this does not mean that the visual slant interfered with haptic adaptation in this case, as the Vision Only condition is the only condition in which, during adaptation, no haptic surface was presented and thus no haptic adaptation to interfere with. Rather, this result suggests that adaptation to visual slant does not necessarily transfer into a haptic adaptation aftereffect upon testing. These two baseline conditions furthermore frame the bimodal conditions for comparison.</p>", "<p>To test which conditions resulted in significant haptic slant aftereffects, we performed one sample <italic toggle=\"yes\">t</italic> tests against zero for each condition. A Bonferroni correction was applied by using an alpha of 0.01. The tests revealed that of the two unimodal adaptation conditions only the adaptation condition in which the haptically rendered surface was present as adapter stimulus (Haptics Only) was significantly different from zero (<italic toggle=\"yes\">t</italic>(10) = 4.6, <italic toggle=\"yes\">p</italic> &lt; 0.001), whereas the Vision Only was not (<italic toggle=\"yes\">t</italic>(10) = −0.9, <italic toggle=\"yes\">p</italic> = 0.409). For the bimodal conditions, the <italic toggle=\"yes\">t</italic> tests against zero revealed that the results for the Boxes as well as the Cursors conditions are significantly different form zero (Boxes: <italic toggle=\"yes\">t</italic>(10) = 5.1, <italic toggle=\"yes\">p</italic> &lt; 0.001; Cursors: <italic toggle=\"yes\">t</italic>(10) = 3.7, <italic toggle=\"yes\">p</italic> = 0.004), whereas the condition in which the slant as well as the cursors were visually presented was not (<italic toggle=\"yes\">t</italic>(10) = 1.5, <italic toggle=\"yes\">p</italic> = 0.157).</p>", "<p>To test for significant differences between the conditions, we first performed a one-way repeated measures ANOVA, which was significant (<italic toggle=\"yes\">F</italic>(4,40) = 5.4, <italic toggle=\"yes\">p</italic> = 0.001). We then investigated where the significance came from by performing paired sample <italic toggle=\"yes\">t</italic> tests between the conditions. If vision influences haptic adaptation, we expect a significant reduction of the aftereffect in the conditions containing visual feedback compared with the Haptics Only condition.</p>", "<p>The <italic toggle=\"yes\">t</italic> tests revealed that the two unimodal conditions haptics only and vision only were significantly different from each other (<italic toggle=\"yes\">t</italic>(10) = −4.0, <italic toggle=\"yes\">p</italic> = 0.002). Furthermore, the haptics only condition showed a significant difference when compared with the slant and cursor condition (<italic toggle=\"yes\">t</italic>(10) = −2.4, <italic toggle=\"yes\">p</italic> = 0.040). When compared with the unimodal vision only condition, significant differences were found for the bimodal conditions boxes and cursors, boxes vs. vision only: <italic toggle=\"yes\">t</italic>(10) = −3.9, <italic toggle=\"yes\">p</italic> = 0.003; cursors vs. vision only: <italic toggle=\"yes\">t</italic>(10) = 2.6, <italic toggle=\"yes\">p</italic> = 0.027. Only the bimodal slant and <italic toggle=\"yes\">Cursor</italic> condition was not significantly different from the vision only condition (<italic toggle=\"yes\">t</italic>(10) = 1.8, <italic toggle=\"yes\">p</italic> = 0.110).</p>" ]
[ "<title>Discussion of experiment 1</title>", "<p>In Experiment 1, we tested if visual information impacts the haptic adaptation to slant. To this end, we varied the amount of visual information available to the participant by having conditions in which only one sensory modality was available to participants (Haptics Only, Vision Only) and conditions in which the two modalities were combined. In the conditions with combined modalities, the haptic information was always the same but the type of visual information available was varied (Cursors, Boxes, Slant and Cursors conditions).</p>", "<p>First, the result of the Haptics Only condition is significantly different from zero, showing that adaptation to slant occurs in conditions in which the information from both hands is needed for estimating the slant (bimanual adaptation). This confirms the results from our previous study that focused on the level at which bimanual slant adaptation occurs (##REF##32735569##Glowania et al., 2020##). Furthermore, the Vision Only condition was not significantly different from zero, suggesting that pure visual adaptation does not lead to an aftereffect in a purely haptic test condition (no transfer). That is, when only visual but no haptic slant information is provided during the adaptation phase, there seems to be no measurable effect when using the testing condition in which only a haptic surface was presented.</p>", "<p>Next, we looked at how different visual cues during adaptation affected haptic slant adaptation. We found that the condition in which two visual cues were available, i.e., the Slant and Cursors condition, had a significantly reduced haptic aftereffect compared with the unimodal Haptics Only condition. This result is a strong indicator that vision does reduce haptic/proprioceptive adaptation to slant and extends the findings by ##REF##21526338##Seizova-Cajic and Azzi (2011)## as well as those of ##REF##6734787##Lackner and Taublieb (1984)##, in relation to proprioceptive muscle vibrations, to the case of haptic shape adaptation. In contrast, the two conditions in which only one visual cue was available, that is, the Boxes condition and the Cursors condition, the aftereffect was not significantly different from the condition in which only haptic information was presented. Thus, we did not find a significant reduction of the slant aftereffect in this case. Based on these findings, it seems that the more visual cues are available to the participant during adaptation, the less strong the adaptation aftereffect in the haptic domain is.</p>", "<p>To further test the hypothesis that the combination of visual cues is important for the influence on haptic adaptation, we conducted a second experiment. Note, that in the first experiment the Boxes condition and the Cursors condition alone where not enough to prevent the participants from adapting in the haptic domain. Thus, the question arises as to whether the combination of the two would lead to a reduced aftereffect. Like the visual slant in the Slant and Cursors condition, the boxes could provide a reference cue for the spatial position of the cursors (which in turn can be linked to the proprioceptive estimate of the finger positions). More particularly, however, the Slant and Cursors condition allowed participants to directly link the finger positions to the actual slant of the surface within a single frame of reference across the senses. That is, the visual slant provided both direct visual information about the surface slant, as well as a more general spatial reference in which to interpret the cursors. In a combination of the boxes and the cursors, the link to the surface slant is much less direct, but the boxes could still act as a spatial anchor or reference for the cursors. Hence, if we find a reduced aftereffect when the boxes and the cursors are combined, this would mean that providing a spatial reference to the cursors already reduces the size of the aftereffect, meaning that a direct link between slant and cursors is not needed for a reduction of the aftereffect. If the combination of the boxes and cursors does not result in a reduced aftereffect, however, this would lead to the conclusion that directly seeing and linking the touched surface to the fingers’ position is crucial for reducing the adaptation aftereffect.</p>" ]
[ "<title>Conclusions</title>", "<p>In the present study, we investigated the influence of different types of visual cues on haptic adaptation. Our results indicate that as soon as visual input provides sufficient information about the position of our fingers in space (i.e., vision provides information about both finger positions as well as their spatial context), haptic adaptation no longer occurs. This might explain why we are not constantly affected by haptic adaptation aftereffects when haptically exploring the world around us.</p>", "<p>Our results are of special importance when developing and designing virtual haptic environments. Visual feedback needs to be meaningful and provides a spatial context to the operator to prevent adaptation. Without such a visual reference, adaptation will soon lead to quite substantial inaccuracies in estimated finger/hand positions, which will ultimately prevent operators in such environments from performing their task at an acceptable level.</p>" ]
[ "<p>Adapting to particular features of a haptic shape, for example, the slant of a surface, affects how a subsequently touched shape is perceived (aftereffect). Previous studies showed that this adaptation is largely based on our proprioceptive sense of hand posture, yet the influence of vision on haptic shape adaptation has been relatively unexplored. Here, using a slant-adaptation paradigm, we investigated whether visual information affects haptic adaptation and, if so, how. To this end, we varied the available visual cues during the adaptation period. This process ranged from providing visual information only about the slant of the surface, or the reference frame in which it is presented, to only providing visual information about the location of the fingertips. Additionally, we tested several combinations of these visual cues. We show that, as soon as the visual information can be used as a spatial reference to link the own fingertip position to the surface slant, haptic adaptation is very much reduced. This result means that, under these viewing conditions, vision dominates touch and is one reason why we do not easily adapt to haptic shape in our daily life, because we usually have visual information about both hand and object available simultaneously.</p>" ]
[ "<title>Methods for experiment 2</title>", "<title>Participants</title>", "<p>In total, 11 students of Bielefeld University (average age 24.5 years, 3 male) participated in this study. Ten participants self-reported to be right handed; one was left handed. They gave informed consent before the start of the experiment and received 6€ per hour as compensation for their participation. Ethical approval was obtained from the Bielefeld University ethics committee.</p>", "<title>Setup and procedure</title>", "<p>The experimental setup was the same as for experiment 1. For the procedure, we reduced the number of reversals for the staircase procedure to eight reversals, since the analysis of the results from experiment 1 showed that generally the staircases had already fully converged after this number. Furthermore, we reduced the number of conditions to two: Haptics Only and Boxes and Cursors. The Haptics Only condition was the same as in the previous experiment and the surface could be felt but no visual cues were provided. The condition Boxes and Cursors was a mixture of the Boxes and the Cursors conditions of experiment 1 (##FIG##3##Figure 4##, right). This means that the participants could see the positions of their fingers as cursors but additionally saw the eight boxes providing a spatial reference (see Materials and Methods for Experiment 1 for details). In both conditions, the slanted surface itself was rendered only haptically and thus the participants received haptic feedback when touching the surface without seeing the actual surface.</p>", "<title>Results of experiment 2</title>", "<p>To investigate the influence of vision on haptic adaptation, participants adapted to a slanted surface when vision was not available (Haptics Only) and to a slanted surface when they could see the location of their fingertips represented as spherical cursors as well as eight reference boxes providing a visual reference frame. ##FIG##4##Figure 5## shows a significant aftereffect for the Haptics Only condition (<italic toggle=\"yes\">One-sample t</italic> test: <italic toggle=\"yes\">t</italic>(10) = 7.7, <italic toggle=\"yes\">p</italic> &lt; 0.001) of comparable magnitude to experiment 1. However, there was no significant effect for the Boxes and Cursors condition (one-sample <italic toggle=\"yes\">t</italic> test: <italic toggle=\"yes\">t</italic>(10) = 1.2, <italic toggle=\"yes\">p</italic> = 0.252). Furthermore, there is a significant difference between the Haptics Only condition and the Boxes <italic toggle=\"yes\">and</italic> Cursors condition (<italic toggle=\"yes\">paired t</italic> test: <italic toggle=\"yes\">t</italic>(10) = −4.0, <italic toggle=\"yes\">p</italic> = 0.002).</p>", "<p>In addition, we also statistically compared the results of the two experiments using an independent samples <italic toggle=\"yes\">t</italic> tests. Those confirmed that there are no significant differences between the Haptics Only conditions from experiments 1 and 2 (independent samples <italic toggle=\"yes\">t</italic> test: <italic toggle=\"yes\">t</italic>(20) = 0.96, <italic toggle=\"yes\">p</italic> = 0.351), and between the Boxes and Cursors condition of the second experiment and the Slant and Cursors condition of the first experiment (Indipendent <italic toggle=\"yes\">t</italic> test: <italic toggle=\"yes\">t</italic>(20) = 0.04, <italic toggle=\"yes\">p</italic> = 0.965).</p>", "<title>Discussion of experiment 2</title>", "<p>In the second experiment, we tested whether providing the boxes, instead of the surface slant (the Slant and Cursors condition of experiment 1) as a second visual cue as well as the cursors, would lead to the same reduction of haptic adaptation aftereffects. First, as in experiment 1, we found a significant aftereffect in the Haptics Only condition, showing again that without visual information haptic adaptation to slanted surfaces occurs. Moreover, the results of experiment 2 showed a significant reduction of the aftereffect in the Boxes and Cursors condition when compared with the Haptics Only condition and in this case the aftereffect was also not significantly different from zero.</p>", "<p>When comparing the results of both experiments, no significant differences were found between the two Haptics Only conditions (from experiments 1 and 2) as well as between the two conditions containing visual feedback about the fingertips’ positions (cursors) and a spatial reference (slant for experiment 1 and boxes for experiment 2). This process reveals that a visual spatial reference is relevant in combination with the cursors to see the interference with haptic adaptation, but that the type of reference does not seem to play a greater role.</p>", "<p>The results of experiment 2 indicate that the visual information given by the cursors in combination with reference boxes is enough information to prevent the participants from haptically adapting to the surface slant. Interestingly neither the cursors nor the boxes by themselves provided enough information to reduce haptic adaptation as shown in the results of experiment 1. These findings suggest that spatially linking the visual cursors, that is, giving the visual cursors a spatial context, suffices to reduce the size of the haptic aftereffect. Thus, as the results of experiment 2 show, it is not even necessary to provide visual information about the actual adaptation surface (slant) itself.</p>", "<title>General discussion</title>", "<p>In this study, we investigated how visual information can affect haptic slant adaptation. The results of both experiments show that the availability of visual information does reduce haptic adaptation. However, the results of experiment 1 showed that making just any visual cue available while haptically adapting to slant is not enough to reduce haptic adaptation. This result was most apparent for the condition in which either only the cursors or only the reference boxes were visually presented. In these cases, haptic adaptation was not significantly reduced compared with the Haptics Only condition. It seems that, for vision to affect haptic adaptation, there needs to be a meaningful visual spatial context in which the position of the fingers can be understood. For instance, by the combination of a visual representation of the touched slant and the cursors (Slant and Cursors condition of experiment 1), vision provides information about the position of the fingertips as well as a spatial reference of the space. Interestingly the visual spatial reference did not need to include direct information about the slanted surface, but it sufficed to show reference boxes spanning the area in which the slant was presented as in the Boxes and Cursors condition of Experiment 2.</p>", "<p>Note that our results can be interpreted as being in conflict with a study by ##REF##19361996##Konkle, Wang, Hayward, and Moore, (2009)##. ##REF##19361996##Konkle et al. (2009)## found that motion adaptation aftereffects transfer between vision and touch. Based on this finding, one might expect that, when the surface was seen but not felt (Vision Only condition), any visual adaptation should transfer to the test condition in which only haptic cues were available and no visual information was shown. In the present study we, however, did not find a haptic slant adaptation aftereffect when the surface was presented only visually during adaptation, although it is known that visual slant is subject to visual adaptation (e.g., ##REF##20243615##Köhler &amp; Emery, 1947##; ##UREF##10##Wenderoth, 1970##; ##REF##11584290##Adams, Banks, &amp; van Ee, 2001##). There are several possible explanations for this discrepancy. First, we did not measure the visual aftereffects of adaptation and, therefore, can only speculate about a possible existence of such effects in the present task. On the one hand, visual adaptation to slant has been shown to occur for both stereo disparity and perspective cues (e.g., ##UREF##1##Bergman &amp; Gibson, 1959##; ##REF##16698056##Knapen &amp; van Ee, 2006##). Thus, we have no reason to believe that the visual system did not adapt in the present case. On the other hand, visual adaptation can be retinal location specific and thus needs fixation rather than free eye movements. Even under strict fixation some adaptation effect might not be complete as microsaccades have been shown to prevent certain types of visual adaptation (e.g., ##REF##16423702##Martinez-Conde, Macknik, Troncoso, &amp; Dyar, 2006##; ##REF##28751870##Habtegiorgis, Rifai, Lappe, &amp; Wahl, 2017##). In the present study, however, participants were free to make eye movements and it is likely that the participants moved their eyes across the visual scene. This could potentially lead to less adaptation of the visual system. Second, in the study by ##REF##19361996##Konkle et al. (2009)##, haptic motion judgments were based on cutaneous cues whereas we used PHANToM force-feedback devices in which only proprioceptive cues were available. Because there is evidence that cutaneous and visual receptive fields overlap in the brain (e.g., see ##REF##8131835##Graziano &amp; Gross, 1993##; ##UREF##3##Graziano &amp; Gross, 1995##), the processing of these types of information could potentially be more strongly linked. In our case, however, haptic slant estimates were obtained by comparing the finger positions of the left and right hands (see also ##REF##32735569##Glowania et al., 2020##), and thus the estimate with regard to the seen slant of the actual object is indirect at best and more of proprioceptive nature rather than cutaneous. Since in this case the low-level sensations (haptic finger positions and visual object slant) do not directly correspond to the same estimate, this can explain why we do not see any transfer from visual slant to the haptic domain.</p>", "<p>In light of the above and to interpret the present results, it is meaningful to distinguish between the elements that were truly bimodal (i.e., both visually and haptically represented) and those that were not. Here, it is important to remember that the cursors provide information about the position of the fingers that can also be directly sensed through proprioception. Therefore, it is most likely the cursors, rather than, for instance, the surface slant, that were truly bimodal elements in the present task. Because of the cursors, there is a direct link of the proprioceptive input of the fingertips to the visual input about fingertip position. Furthermore, the seen movement on the screen, especially at the beginning of the trial when the participant lowers the fingers until the haptically rendered surface is reached, is in the temporal alignment of the participants’ finger movement. This temporal alignment does not only lead to optimal integration of the visual and haptic information (##UREF##7##Plaisier, van Dam, Glowania, &amp; Ernst, 2014##), but should also lead to a sense of agency, because what the participants see is in alignment with what they do (##UREF##4##Haggard &amp; Chambon, 2012##). In all the conditions in which the cursors are not present, this direct link between the visual and the haptic input is missing and did not reduce the haptic aftereffect. Still, seeing only the finger positions through the cursors did not result in a significantly lower aftereffect either. This is because the cursors alone do not necessarily provide all that much information about their position in 3D space. Visual depth perception is known to rely more on relative depth cues (e.g., relative disparity) (##REF##4060612##Erkelens &amp; Collewijn, 1985##) rather than absolute depth cues (e.g., convergence of the eyes) (##UREF##8##Richards &amp; Miller, 1969##). When only the cursors are available, there is relatively little relative depth information provided, since, for each cursor, there is only the other cursor (representing the other finger) to compare the visual distance to. Hence, the cursors alone might not give sufficiently reliable visual information to estimate the finger positions visually in 3D space. A second visual cue is needed to provide a relative spatial reference for the cursors, that is, to make the height difference between the visually displayed cursors more obvious. It, however, does not seem to matter whether the reference is itself directly informative about the slant of the surface or not, which is shown in the lack of a significant difference when comparing the Slant and Cursors condition of experiment 1 with the Boxes and Cursors condition in experiment 2. Both, the touched slant as well as simple reference boxes, give the cursors spatial context and result in a significantly reduced haptic adaptation. This is consistent with the results by ##REF##6734787##Lackner and Taublieb (1984)##, who found that the proprioceptive muscle spindle vibration illusion is much weaker when the own body is seen in context with an external reference.</p>", "<p>In short, our results suggest that there may be something special about adding a visual spatial context with respect to which to interpret the positions of the cursors. This addition could be in the form of adding some spatial anchors in the work space that allow to distinguish positions and movements of the cursors more easily and more reliably. If so, it could be of interest to investigate in future work just how the spatial reference might influence the reliability of the visual cursor positions and in turn how this affects haptic slant adaptation. Future studies could, for instance, vary the reliability of the visual reference cues, such as the boxes, and/or the cursors through blurring or similar techniques. If it is the visual relative spacing between cursors and boxes that is relevant for influencing haptic adaptation, then visually degrading either one, thus decreasing the visual reliability of this relative spacing, should lead to haptic aftereffects occurring again to the same extent. Doing the same for a different combination of visual cues without the cursors (e.g., slant and boxes) should not lead to any changes in results. By measuring these reliabilities in separate experiments, these potential relationships could then also be quantified. This was beyond the scope of the present work, however, where we first elucidated the potential relevance of a spatial reference, and we leave the investigation of the relationship between visual reliability of the space and the haptic adaptation aftereffect to future work.</p>" ]
[ "<title>Acknowledgments</title>", "<p>C.G. was supported by the Cluster of Excellence Cognitive Interaction Technology ‘CITEC’ (EXC 277) at Bielefeld University, which is funded by the German Research Foundation (DFG). We acknowledge support for the publication costs by the Open Access Publication Fund of Bielefeld University. This work was partially supported by the European Commission funded project WEARHAP (project 601165).</p>", "<p>Commercial relationships: none.</p>", "<p>Corresponding author: Catharina Glowania.</p>", "<p>Email: [email protected].</p>", "<p>Address: Department of Cognitive Neuroscience, Bielefeld University, Bielefeld, Germany.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1.</label><caption><p>Experimental setup. (<bold>A</bold>) The visuohaptic workbench. The participant was seated in front of the workbench with the body midline aligned with the center of the workbench. Both index fingers were fixed in thimble-like holders attached to the PHANToMs. The visual stimuli were presented on the CRT and viewed via mirror. The system was calibrated to easily present objects both visually and haptically in a spatially aligned way. (<bold>B</bold>) Front view of the virtual space showing the response zones, the threshold for starting a trial and the virtual surface.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2.</label><caption><p>Adaptation conditions. Haptics Only: Subjects could feel the slanted surface but no visual cues were presented. Vision only: Subjects could see the slanted surface but not touch it. Boxes: Subjects could see eight boxes framing the area in which the haptic surface was presented; however, the slanted surface itself could only be felt but not seen. Cursors: Subjects could see the position of their index fingers as cursors, but the slanted surface was only rendered haptically. Slant and cursors: Subjects could see the position of their index fingers as cursors as well as the surface slant, which was also rendered haptically.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3.</label><caption><p>Results of experiment 1. The <italic toggle=\"yes\">y</italic> axis shows the haptic adaptation aftereffects as a result of different visual and haptic adaptation conditions. Each bar represents a different adaptation condition as indicated on the <italic toggle=\"yes\">x</italic> axis. Error bars are standard errors. The dotted line marks the point at which full adaptation would occur. Note that, on the test trials for all adaptation conditions, the surface was rendered only haptically.</p></caption></fig>", "<fig position=\"float\" id=\"fig4\"><label>Figure 4.</label><caption><p>The conditions of experiment 2. Haptics Only: The subjects could only feel the surface, but no visual information was available. Boxes and Cursors: Both the cursors representing the position of the fingertips as well the 8 visual boxes that spanned the available exploration space were presented (for details, see descriptions for experiment 1). The surface itself was not visually represented, but was available for haptic touch.</p></caption></fig>", "<fig position=\"float\" id=\"fig5\"><label>Figure 5.</label><caption><p>Results of experiment 2. The <italic toggle=\"yes\">x</italic> axis shows the labels for each condition, the <italic toggle=\"yes\">y</italic> axis the size of the aftereffect, and error bars are the standard error. The dotted line marks the point at which full adaptation would occur.</p></caption></fig>" ]
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[{"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Balch", "Milewski", "Yonas"], "given-names": ["W.", "A.", "A."], "year": ["1977"], "article-title": ["Mechanisms underlying the slant aftereffect"], "italic": ["Perception and Psychophysics,"], "volume": ["21"], "issue": ["6"], "fpage": ["581"], "lpage": ["585"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Bergman", "Gibson"], "given-names": ["R.", "J."], "year": ["1959"], "article-title": ["The negative aftereffect of a surface slanted in the third dimension"], "italic": ["American Journal of Psychology,"], "volume": ["72"], "fpage": ["364"], "lpage": ["374"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Gescheider"], "given-names": ["G."], "year": ["1976"], "italic": ["Psychophysics: Method and theory"], "publisher-loc": ["Oxford, UK"], "publisher-name": ["Lawrence Erlbaum"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Graziano", "Gross"], "given-names": ["M.", "C."], "year": ["1995"], "article-title": ["The representation of extrapersonal space: A possible role for bimodal, visual-tactile neurons"], "italic": ["Cognitive Neurosciences,"], "fpage": ["1021"], "lpage": ["1034"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Haggard", "Chambon"], "given-names": ["P.", "V."], "year": ["2012"], "article-title": ["Sense of agency"], "italic": ["Current Biology,"], "volume": ["22"], "fpage": ["390"], "lpage": ["392"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Knapen", "Rolfs", "Wexler", "Cavanagh"], "given-names": ["T.", "M.", "M.", "P."], "year": ["2010"], "article-title": ["The reference frame of the tilt aftereffect"], "italic": ["Journal of Vision,"], "volume": ["10"], "issue": ["1"], "fpage": ["1"], "lpage": ["13"], "pub-id": ["10.1167/10.1.8"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Krystallidou", "Thompson"], "given-names": ["D.", "P."], "year": ["2016"], "article-title": ["Cross-modal transfer of the tilt aftereffect from vision to touch"], "italic": ["i-Perception,"], "volume": ["4"], "issue": ["5"], "fpage": ["2014669516668888"], "pub-id": ["10.1177/2041669516668888"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Plaisier", "van Dam", "Glowania", "Ernst"], "given-names": ["M.", "L.", "C.", "M."], "year": ["2014"], "article-title": ["Exploration mode affects visuohaptic integration of surface orientation"], "italic": ["Journal of Vision,"], "volume": ["14"], "issue": ["13"], "fpage": ["22"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Richards", "Miller"], "given-names": ["W.", "J."], "year": ["1969"], "article-title": ["Convergence as a cue to depth"], "italic": ["Perception and Psychophysics,"], "volume": ["5"], "issue": ["5"], "fpage": ["317"], "lpage": ["320"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Vogels", "Kappers", "Koenderink"], "given-names": ["I.", "A.", "J."], "year": ["1995"], "article-title": ["Haptic surface aftereffect is of central not peripheral origin"], "italic": ["Studies in Perception and Action III"], "publisher-loc": ["London"], "publisher-name": ["Routledge"], "fpage": ["319"], "lpage": ["322"]}, {"mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Wenderoth"], "given-names": ["P."], "year": ["1970"], "article-title": ["A visual spatial aftereffect of surface slant"], "italic": [".", "American Journal of Psychology,"], "volume": ["83"], "issue": ["4"], "fpage": ["576"], "lpage": ["590"]}]
{ "acronym": [], "definition": [] }
32
CC BY
no
2024-01-14 23:43:49
J Vis. 2024 Jan 11; 24(1):8
oa_package/4a/dc/PMC10787591.tar.gz
PMC10787594
0
[ "<title>Introduction</title>", "<p>Thoracic procedures involving one-lung ventilation (OLV) present a unique challenge to anesthesiologists. While OLV improves access to the operation field and expedites the surgical process, anesthesiologists must ensure adequate ventilation and oxygenation. To accomplish OLV, anesthesiologists often use a double-lumen endotracheal tube (DLT) [##REF##12411817##1##]. Compared to standard single-lumen endotracheal tubes, the DLT has a larger diameter, a longer length, and a more rigid structure. These factors make intubation with DLT much more difficult in both normal and difficult airways, which could increase the overall morbidity and mortality risk to the patient due to upper airway trauma and post-operative complications [##REF##9583539##2##, ####REF##9539585##3##, ##REF##8910162##4##, ##REF##9583552##5####9583552##5##]. Moreover, DLT use is contraindicated in cases involving a difficult airway, severe tracheal stenosis, or significant distortion of the airway anatomy, all of which may preclude the use of DLT.</p>", "<p>Macintosh blade direct laryngoscopy has been widely used to place both a single-lumen endotracheal tube and DLT. However, an incomplete view of the glottis during direct laryngoscopy due to the challenging airway anatomy often increases the difficulty and duration of achieving endotracheal intubation, hence increasing the risks of hypoxia and trauma to the patient [##REF##9583552##5##,##REF##22540996##6##]. To address the need for easier and safer endotracheal intubation, several different types of video laryngoscopes have been developed. The C-MAC® (Karl Storz GmbH &amp; Co. KG, Tuttlingen, Germany) is a video laryngoscope, and its standard blade has an identical shape to the Macintosh blade; however, its camera attachment allows for both direct and indirect views of the glottis. The C-MAC® has demonstrated higher success rates of tracheal intubation compared with direct laryngoscopy in difficult airway patients [##REF##21156978##7##, ####UREF##0##8##, ##REF##22261795##9####22261795##9##]. GlideScope® (Verathon Inc., Bothell, WA, USA) is another video laryngoscope in which the glottis is indirectly viewed through an optical video apparatus embedded in a 60-degree angulated blade. Studies have also shown that the GlideScope® is more effective than direct laryngoscopes in difficult airway intubations with standard single-lumen tubes by producing a faster intubation time, a higher success rate, and less tissue damage [##REF##21150569##10##,##REF##15960409##11##]. Nevertheless, the effectiveness of these devices when placing a DLT remains unclear. Therefore, this study aims to compare the efficiency of DLT intubation using two different video laryngoscopes, C-MAC® and GlideScope®, and a traditional Macintosh blade direct laryngoscope in a manikin. We utilized a manikin to ensure a standardized comparison and to eliminate patient-related variables, which also enhances safety by avoiding potential airway trauma. We hypothesize that video laryngoscopes will facilitate more efficient intubation compared to the Macintosh blade due to better glottic visualization. Additionally, this study aims to assess if the level of training and experience of the anesthesia provider impacts the efficiency of DLT intubation across these devices. The primary outcome measured is the time to successful intubation, and the secondary outcome is the incidence of failed intubations, specifically esophageal intubations. </p>" ]
[ "<title>Materials and methods</title>", "<p>This is a randomized, crossover manikin study approved by the Institutional Review Board at the University of Iowa (IRB # 201211726). Written consent was obtained from all participants prior to enrollment in the study. Participants were recruited from the University of Iowa directory. A preapproved email was sent to interns, residents, fellows, and attending physicians in the Department of Anesthesia at the University of Iowa Hospitals &amp; Clinics. The inclusion criteria were licensed physicians who worked at the University of Iowa and held either a Doctor of Medicine (MD) or a Doctor of Osteopathic Medicine (DO) degree. Individuals who did not provide study consent were excluded. </p>", "<p>Participants arrived at the simulation suite in the Department of Anesthesia at times that fit their schedules. Each participant was provided with a written description of the study protocol and had the opportunity to ask questions. Additionally, all individuals were asked about their familiarity with all three intubation devices. If they were not familiar with a particular device, they were given time to practice single-lumen intubation on a separate manikin with the unfamiliar devices until they felt comfortable.</p>", "<p>Once each participant felt comfortable with all three devices, they were randomized via a crossover design, with the order of devices determined using a Latin square design. There were six possible permutations for the order of devices used (Figure ##FIG##0##1##). The devices used included the C-MAC® video laryngoscope with a standard size 3 blade, the GlideScope® with a 60-degree angulated size 3 blade (Figure ##FIG##1##2##), and the Macintosh size 3 blade. Every participant intubated an adult manikin (Laerdal, Wappingers Falls, NY, USA), equipped with a head assembly and lungs, using each device, resulting in a total of three timed intubations. In the event of a failed intubation (esophageal intubation), participants were allowed to continue until they successfully intubated the trachea. For all three devices, the same Covidien Mallinckrodt left-sided 37-Fr DLT (Medtronic PLC, Dublin, Ireland) was used with its designated stylet that accompanied the tube. The participants were allowed to pre-shape the DLT using the stylet to match the configuration of the different types of blades as they wished.</p>", "<p>Participants were allowed to set up the room at the simulation suite, such as positioning the device on the table and lubricating the DLT and its stylet, in a manner consistent with their own clinical practice in an operating room. They were instructed to place the DLT through the vocal cords; however, they did not need to achieve or confirm lung isolation. Once the participant believed that the DLT had passed through the vocal cords, the stylet was removed, and the tube was further advanced until resistance was felt. Participants then inflated a tracheal cuff on the DLT and connected the DLT to the Ambu® hyperinflation bag (Ambu Inc., Columbia, MD, USA). Successful intubation was confirmed when bilateral lungs were inflated by squeezing the Ambu® bag. Failed intubation was defined by having the DLT placed in the esophagus. This was confirmed by the failure to inflate the lungs when squeezing the Ambu® bag. This process was repeated with the other two devices. Participants were allowed to have an assistant help with removing the stylet, handing over the Ambu® bag, and applying cricoid pressure if necessary. The primary outcome measure was the time taken to achieve successful intubation, and the secondary outcome measure was the number of failed intubations. </p>", "<p>Data collection</p>", "<p>Intubation time was measured from the first contact with either the DLT, intubation device, or manikin to the first sign of bilateral lung inflation upon squeezing the Ambu® bag. Time spent setting up the room at the simulation suite prior to the actual intubation was not counted as the intubation time. A stopwatch timer was used for measurement and was started/stopped based on visual inspection by a single evaluator. Failed intubation (esophageal intubation) was documented, and the subsequent successful intubations were included in the analysis. When intubation failed, the stopwatch timer was stopped, and then a new timer was started to measure the duration of the subsequent intubation attempt. Only the successful intubation times were included in the primary outcome analysis. The participants' postgraduate years of training were also recorded.</p>", "<p>Data analysis</p>", "<p>To evaluate the effectiveness of the different intubation devices, we formulated a null hypothesis for our statistical analysis. The null hypothesis posited that there would be no significant difference in the intubation time of the C-MAC® video laryngoscope, the GlideScope® video laryngoscope, and the Macintosh blade direct laryngoscope for endotracheal intubation using a DLT in an adult manikin. Our analysis aimed to test this null hypothesis, and we compared the meantime to intubation and the incidence of failed intubations across the three devices. Statistical analyses were performed using Excel’s Analysis ToolPak add-in (Microsoft Corp., Redmond, WA, USA). Mean, median, range, interquartile range, kurtosis, skewness, standard deviation, and standard error were obtained on the duration of successful intubation in all three devices. A Kruskal-Wallis one-way analysis of variance by rank was performed to assess for significance between time to intubation for all three devices and between time to intubation of each device by training level. A subsequent Mann-Whitney U test was used to locate significant differences between devices. Significance was determined by using an alpha set to 0.05. All data are presented as mean ± SD unless otherwise stated.</p>" ]
[ "<title>Results</title>", "<p>Ninety-four anesthesia providers enrolled in and completed the study. The participants were composed of six interns (postgraduate year 1, PGY-1), 49 first-year residents (PGY-2), three second-year residents (PGY-3), nine final-year residents (PGY-4), 15 anesthesia fellows (PGY-5 or more for specialized training), and 12 attending physicians (faculty).</p>", "<p>Each individual attempted intubation with three different devices, totaling 282 intubations. Times to intubation by the devices are illustrated in Figure ##FIG##2##3##. Mean times-to-intubation for the C-MAC®, GlideScope®, and Macintosh blades were 18.57 ± 0.77, 36.26 ± 2.69, and 20.76 ± 0.96 seconds, respectively. There was a significant difference (P&lt;0.001) between the GlideScope® 60-degree angulated blade and the two other laryngoscope standard blades. No significant differences were detected between the C-MAC® and the Macintosh blades (P&gt;0.05). Two individuals failed to place the DLT utilizing the Macintosh blade on their first attempt. The two failed attempts were by anesthesia trainees (PGY-3 and -4). Upon a second attempt, these individuals were able to place the DLT in the trachea rather than the esophagus. One of the successful intubations upon the second attempt is represented by the farthest most outlier data point in the Macintosh data set. The other subject who had a first-attempt failure was able to intubate within a non-outlier timeframe. The outlier data point in the C-MAC data set did not fail any of their first attempts.</p>", "<p>Comparison of the average time to intubation for the C-MAC® standard blade versus the GlideScope® 60-degree angulated blade versus the Macintosh blade by year of training is shown in Table ##TAB##0##1##. Mean times to intubation with the C-MAC® and the Macintosh blades were shorter than with the GlideScope® 60-degree angled blade across all levels of training.</p>" ]
[ "<title>Discussion</title>", "<p>In this manikin study, comparing three different laryngoscopes, we observed that the time required for successful DLT intubation was significantly shorter using both the Macintosh and C-MAC® standard blades compared to the GlideScope® 60-degree angulated blade. There was no significant difference in intubation time between the Macintosh and C-MAC® blades.</p>", "<p>The curvature of the GlideScope® blade differs from that of the standard blades of the C-MAC® and the Macintosh. Specifically, the tip of the GlideScope® blade has a steeper 60-degree angulation compared to the approximately 30-degree angulation of the other two blades. In this study, using this blade with a 60-degree angle, anterior displacement of the tongue and chin was not often necessary during laryngoscopy. This was because the vocal cords were fully visualized on the monitor without much effort. However, the maneuver-creating anterior displacement of the tongue and chin-was essential for achieving optimal alignment of the oral, pharyngeal, and laryngeal axes for DLT intubation. Consequently, most participants initially positioned the DLT too far posteriorly relative to the epiglottis, resulting in a suboptimal trajectory toward the vocal cords. Although all participants eventually manipulated the DLT into the vocal cords, this resulted in a longer intubation time. Additionally, the unique 60-degree angulation of the GlideScope® blade, though advantageous for visualizing the vocal cords, may affect the time required for blade insertion into the mouth. These findings contrast with previous reports where single-lumen tube intubations were studied and the GlideScope® blade had a comparable intubation time to the Macintosh laryngoscope [##REF##21716906##12##,##REF##18434462##13##]. This discrepancy can be attributed to the DLT's larger diameter, its more cumbersome nature, and its reduced flexibility, especially when navigating the curve of the steeply angulated blade.</p>", "<p>There are several studies investigating the effectiveness of various intubation devices for DLT intubation, yielding inconsistent results across different situations [##REF##32434470##14##, ####REF##22324297##15##, ##REF##24219251##16##, ##REF##36620117##17##, ##UREF##1##18####1##18##]. In most past studies, the operators performing the DLT intubation were either not specified or were experienced anesthesia providers, with many studies excluding anesthesia trainees. What distinguishes the current study is the inclusion of anesthesia trainees as operators. As a result, the mean time to successful DLT intubation was consistently the longest with the GlideScope® 60-degree angulated blade across all training levels, as shown in Table ##TAB##0##1##. This suggests that the inexperience in intubation skills could not have fully explained the differences in intubation time, and the results could be generalized regardless of the level of training.</p>", "<p>Notably, of the 282 DLT intubations, two failed (resulting in esophageal intubation) on the first attempt, both using the Macintosh blade. The two failed intubation attempts were by anesthesia trainees (PGY-3 and -4). The fact that PGY-3 and PGY-4 residents were unable to intubate, unlike the PGY-1s, suggests that a lack of experience in intubation skills cannot fully explain the increased rate of failed intubations. This implies that the result could be generalized regardless of the level of training. However, with only two failed intubations out of 282, we do not have a sufficient number of cases to draw any definitive conclusion. While the current study did not ascertain whether participants had an adequate direct view of the vocal cords with the Macintosh laryngoscope, it is possible that inadequate visualization of the vocal cords contributed to the intubation failures. This observation was consistent with previous reports on single-lumen tubes, which revealed that the C-MAC® provided better glottic visualization compared to the direct laryngoscope [##REF##27896321##19##,##REF##22560464##20##], thereby reducing inadvertent esophageal intubations. Hence, the higher success rates and faster intubation times observed with the C-MAC® could hold significant clinical implications.</p>", "<p>In the current study, an adult manikin with normal airway anatomy (Laerdal, Wappingers Falls, NY, USA) was used. This manikin has received a good rating as an airway training device, especially for DLT intubation [##REF##17381577##21##], and was found to be a good alternative airway training device to fresh frozen cadaver models [##REF##20028998##22##]. By using a manikin with consistent airway anatomy, we eliminated the patient’s airway anatomical variation as a variable. This allowed for more controlled experimentation, even with a small sample size when comparing the intubation devices. Subsequently, the results obtained from the current study using a manikin may help with the design of a future study to compare efficacy among intubation devices in a clinical setting.</p>", "<p>There are several limitations to the current study. First, we used a 37 Fr left-sided DLT. Consequently, results might have varied if a different size of DLT had been used. Second, the manikin we used has normal airway anatomy; therefore, outcomes might differ in situations involving difficult airway management. Third, the assumption that the intubation experience increased in the postgraduate years could be inaccurate, as the prior experience of the participants before enrollment in the study with these devices and DLT was not recorded. Thus, it is acknowledged that variations in individual exposure to these specific devices and DLT intubation prior to the study could potentially influence outcomes. Lastly, successful intubation was defined by the placement of the DLT through the vocal cords and the subsequent ability to ventilate the lungs, and the positioning of the tip of the left-sided DLT in the left main bronchus was not confirmed either by fiberoptic bronchoscopy or by assessing one-lung ventilation. Therefore, the findings could be different if the primary outcome were defined as successful one-lung ventilation.</p>" ]
[ "<title>Conclusions</title>", "<p>The current study demonstrated that the C-MAC® and Macintosh standard size 3 blades are more efficient in time to a 37 Fr left-sided DLT placement in the manikin with normal airway anatomy compared to the GlideScope® 60-degree angulated size 3 blade. Intubation failures on the first attempt occurred with the Macintosh blade. These findings suggest that, of the three laryngoscopes studied, the C-MAC® is the most effective for timely and successful DLT intubation in a manikin with normal airway anatomy. Further studies are needed to confirm these results in human subjects.</p>" ]
[ "<p>Background: Macintosh blade direct laryngoscopy is widely used for endotracheal intubation. It may, however, provide an incomplete view of the glottis in patients with challenging airway anatomy. Consequently, various video laryngoscopes have been developed to enhance the visualization of the glottis and facilitate intubation. Yet, the effectiveness of these video laryngoscopes for intubation using a double-lumen endotracheal tube (DLT), which is longer, larger, and more rigid and has a linear configuration as opposed to the naturally semicircular curvature of a single-lumen endotracheal tube, remains uncertain. We hypothesized that video laryngoscopes would be more efficient for DLT intubation compared to the Macintosh blade in an adult manikin.</p>", "<p>Methods: Ninety-four anesthesia providers, comprising 67 residents, 15 fellows, and 12 attendings, attempted to intubate an adult manikin with normal airway anatomy (Laerdal, Wappingers Falls, NY, USA) using a 37 Fr left-sided DLT. Three different intubation devices were used: the C-MAC® video laryngoscope (Karl Storz GmbH &amp; Co. KG, Tuttlingen, Germany), the GlideScope® video laryngoscope (Verathon Inc., Bothell, WA), and the Macintosh blade direct laryngoscope-were used. Each participant intubated a manikin once with each of the three devices. Participants were randomized via a crossover design with the order of devices determined by using a Latin square design. Time to intubation and the number of failed intubations (esophageal intubation) were compared across the three different devices.</p>", "<p>Results: Mean times to intubation for the C-MAC®, GlideScope®, and Macintosh blades were 18.57 ± 0.77, 36.26 ± 2.69, and 20.76 ± 0.96 seconds, respectively. There was a statistically significant difference (P&lt;0.001) between the GlideScope® and the other two laryngoscopes. The times for C-MAC® and Macintosh blades were not significantly different. There were two instances of first-attempt failed intubation with the Macintosh.</p>", "<p>Conclusion: Both the C-MAC® and the Macintosh blades proved more efficient in terms of time to DLT intubation in the manikin with normal airway anatomy, when compared to the GlideScope®. Considering the occurrence of first-attempt failed intubation, the C-MAC® was the most effective device among the three laryngoscopes for timely successful DLT intubation in the adult manikin. Further studies are needed to confirm these results in human subjects.</p>" ]
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[]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Three-way crossover study protocol. </title><p>Each intubation trial was with a different laryngoscope, never repeating a previous laryngoscope. Each laryngoscope was used once for DLT intubation. DLT: double-lumen endotracheal tube.</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Picture of the laryngoscopes used. </title><p>From left to right: Macintosh blade size 3, C-MAC® video laryngoscope with size 3 blade, and GlideScope® with a 60-degree angulated size 3 blade.</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>Comparison of the individual time to intubation for C-MAC® video laryngoscope versus Macintosh direct laryngoscope versus GlideScope® video laryngoscope. </title><p>*P&lt;0.001.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Comparison of average intubation time (seconds) by year of training/practice.</title><p>Values are presented as mean ± SD. PGY: postgraduate year. P values for the ANOVA test.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Subject classification</td><td rowspan=\"1\" colspan=\"1\">C-MAC </td><td rowspan=\"1\" colspan=\"1\">GlideScope</td><td rowspan=\"1\" colspan=\"1\">Macintosh</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PGY-1</td><td rowspan=\"1\" colspan=\"1\">17.3 ± 4.5</td><td rowspan=\"1\" colspan=\"1\">29.0 ± 20.3</td><td rowspan=\"1\" colspan=\"1\">18.7 ± 5.3</td><td rowspan=\"1\" colspan=\"1\">0.021</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">PGY-2</td><td rowspan=\"1\" colspan=\"1\">15.9 ± 4.3</td><td rowspan=\"1\" colspan=\"1\">28.5 ± 22.1</td><td rowspan=\"1\" colspan=\"1\">19.0 ± 7.0</td><td rowspan=\"1\" colspan=\"1\">0.106</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PGY-3</td><td rowspan=\"1\" colspan=\"1\">17.0 ± 8.3</td><td rowspan=\"1\" colspan=\"1\">24.3 ± 13.8</td><td rowspan=\"1\" colspan=\"1\">23.6 ± 19.5</td><td rowspan=\"1\" colspan=\"1\">0.288</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">PGY-4</td><td rowspan=\"1\" colspan=\"1\">14.3 ± 6.8</td><td rowspan=\"1\" colspan=\"1\">29.3 ± 15.4</td><td rowspan=\"1\" colspan=\"1\">19.7 ± 9.3</td><td rowspan=\"1\" colspan=\"1\">0.497</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PGY-5</td><td rowspan=\"1\" colspan=\"1\">20.5 ± 8.1</td><td rowspan=\"1\" colspan=\"1\">43.0 ± 28.4</td><td rowspan=\"1\" colspan=\"1\">21.6 ± 7.4</td><td rowspan=\"1\" colspan=\"1\">0.002</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Faculty</td><td rowspan=\"1\" colspan=\"1\">16.2 ± 6.1</td><td rowspan=\"1\" colspan=\"1\">36.5 ± 25.7</td><td rowspan=\"1\" colspan=\"1\">19.0 ± 7.5</td><td rowspan=\"1\" colspan=\"1\">0.755</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Satoshi Hanada, Srinivasan Rajagopal, Richard N. Gardner, Elizabeth Swanson, Sung Kim, Rakesh Sondekoppam, Kenichi Ueda</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Satoshi Hanada, Srinivasan Rajagopal, Richard N. Gardner, Elizabeth Swanson, Sung Kim, Rakesh Sondekoppam, Kenichi Ueda</p><p><bold>Drafting of the manuscript:</bold>  Satoshi Hanada, Srinivasan Rajagopal, Richard N. Gardner, Elizabeth Swanson, Sung Kim, Rakesh Sondekoppam, Kenichi Ueda</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Satoshi Hanada, Srinivasan Rajagopal, Richard N. Gardner, Elizabeth Swanson, Sung Kim, Rakesh Sondekoppam, Kenichi Ueda</p><p><bold>Supervision:</bold>  Satoshi Hanada, Srinivasan Rajagopal, Sung Kim, Kenichi Ueda</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Institutional Review Board at the University of Iowa issued approval 201211726</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared financial relationships, which are detailed in the next section.</p></fn><fn fn-type=\"financial-disclosure\"><p>Rakesh Sondekoppam declare(s) non-financial support from CIVCO Medical Solutions (<ext-link xlink:href=\"https://www.civco.com\" ext-link-type=\"uri\">https://www.civco.com</ext-link>/). Rakesh Sondekoppam is a consultant for CIVCO Medical Solutions (<ext-link xlink:href=\"https://www.civco.com\" ext-link-type=\"uri\">https://www.civco.com</ext-link>/)</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050523-i01\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050523-i02\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050523-i03\" position=\"float\"/>" ]
[]
[{"label": ["8"], "article-title": ["Endotracheal intubation using the C-MAC\u00ae video laryngoscope or the Macintosh laryngoscope: a prospective, comparative study in the ICU"], "source": ["Crit Care"], "person-group": ["\n"], "surname": ["Noppens", "Geimer", "Eisel", "David", "Piepho"], "given-names": ["RR", "S", "N", "M", "T"], "fpage": ["0"], "volume": ["16"], "year": ["2012"]}, {"label": ["18"], "article-title": ["Comparison of the C-MAC D-blade video laryngoscope and the McCoy laryngoscope for double-lumen endotracheal tube intubation: a prospective randomized controlled study"], "source": ["Medicine (Baltimore)"], "person-group": ["\n"], "surname": ["Kim", "Seo", "Kim", "John", "Moon", "Kim", "Yeon"], "given-names": ["KM", "KH", "YJ", "H", "HS", "N", "N"], "fpage": ["0"], "volume": ["101"], "year": ["2022"]}]
{ "acronym": [], "definition": [] }
22
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50523
oa_package/c5/70/PMC10787594.tar.gz
PMC10787595
0
[ "<title>Introduction</title>", "<p>Non-alcoholic fatty liver disease (NAFLD) is defined as ≥5% hepatic fat content in the absence of excessive consumption of alcohol or use of certain pharmacotherapy that could induce steatosis, viral infection, or other chronic liver diseases [##REF##27188459##1##,##REF##26085906##2##]. NAFLD includes a range of pathological conditions, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma [##REF##26856933##3##].</p>", "<p>A meta-analysis of studies published in 2016 found that NAFLD was the most common cause of chronic liver disease, with a global prevalence of about 25% [##REF##26707365##4##]. However, its prevalence has increased to 30%, as reported in a more recent systemic review; hence, it is considered the leading cause of liver-related morbidity and mortality [##REF##36626630##5##]. In Saudi Arabia, the total NAFLD prevalence was estimated at 8,451,000 cases (25.7%) in 2017, with cases with steatosis only representing 83.6% of all NAFLD cases, and a projected increase of the total to 31.7% by 2030 [##REF##29956688##6##].</p>", "<p>NAFLD is considered the hepatic manifestation of metabolic syndrome (MetS), with obesity, insulin resistance (IR), and some other components of the syndrome among its known risk factors [##UREF##0##7##].</p>", "<p>NAFLD is more common in people with type 2 diabetes mellitus (T2DM) [##REF##26856933##3##,##REF##26005676##8##], increasing their risk of developing cardiovascular disease [##REF##23507799##9##,##UREF##1##10##], as well as the risk of premature mortality [##REF##20145609##11##], and requiring modification to management strategy to avoid or delay these complications. In Saudi Arabia, NAFLD is suspected in patients, especially those with T2DM, presenting with no specific symptoms, accompanied by abnormal liver tests or hepatomegaly, and is usually detected by ultrasound [##REF##30052239##12##]. However, such advanced equipment is not available in public healthcare centers, and those suspected to have NAFLD are usually referred to larger hospitals, which could be in other areas, thus, delaying the diagnosis and management of the disease.</p>", "<p>From the above, it is evident that early diagnosis of NAFLD is needed, especially in people with T2DM. Indeed, there is an urgent need for sensitive biomarkers that can be measured in smaller health centers lacking the more advanced equipment needed for diagnosis.</p>", "<p>In our recent research, we investigated the potential of using specific biological and biochemical markers, along with readily available indices, to identify significant associations with NAFLD in individuals with T2DM to pinpoint those who would benefit from advanced and costly diagnostic tests for confirmation. We employed body mass index (BMI), waist circumference (WC), serum fasting insulin levels, triglycerides, and liver enzymes in various equations to determine the Fatty Liver Index, Hepatic Steatosis Index, NAFLD Liver Fat Score, and the Triglycerides and Glucose Index [##UREF##2##13##]. However, all investigated indices showed low specificity for predicting NAFLD. Therefore, there is still a need to identify novel markers to improve sensitivity and specificity for NAFLD in people with T2DM in primary care.</p>", "<p>Fibroblast growth factor 21 (FGF-21) appears to be a likely candidate for investigation as it has been reported to be engaged in the inter-organ endocrine signaling axes, which are pertinent for the maintenance of the entire-body homeostasis as they govern the metabolism and homeostasis [##REF##26567701##14##,##REF##23217254##15##]. Indeed, increased serum FGF-21 levels have been associated with diabetes, obesity, and MetS [##REF##21987488##16##,##UREF##3##17##]. In addition, FGF-21 is reportedly engaged in the actions of various antidiabetic agents [##REF##27031294##18##,##REF##29989062##19##]. Furthermore, key risk factors for NAFLD, including insulin insensitivity, obesity, and dyslipidemia, are alleviated by FGF-21, and FGF-21 has been reported to reverse liver steatosis while counteracting obesity and enhancing insulin sensitivity [##REF##26441837##20##]. These findings suggest that FGF-21 may be upregulated under the NAFLD condition and may be involved in protecting from the progression of NAFLD by reversing steatosis and enhancing the metabolic energy status.</p>", "<p>Moreover, the accumulation of triglycerides in the liver in people with NAFLD has been reported to be associated with various causes, including increased de novo lipogenesis [##REF##29936596##21##,##REF##24316260##22##], increased lipolysis in the adipocytes, increased delivery of free fatty acids (FFAs) to the liver [##REF##29936596##21##,##REF##23397118##23##], and decreased lipid clearance consequent to impaired fatty acid oxidation and lower lipid secretion [##REF##28585211##24##,##REF##29967350##25##]. Hence, the plasma level of FFAs is a possible marker worthy of investigation as increased lipolysis is suggested as one of the causes of hepatic triglyceride accumulation in NAFLD. This is thought to be a result of increased IR resulting in increased hormone-sensitive lipase (HSL) activity in the adipocytes. This leads to increased release of FFAs and increased FFA flux to the liver, hence, increased synthesis of triglycerides, without an increase in their export [##REF##15254578##26##]. Furthermore, as T2DM is characterized by IR, which is one of the risk factors for NAFLD, investigating the relationship between measures of IR such as homeostatic model assessment for insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) as possible predictors of NAFLD seems logical, especially as it has not been studied before in the Saudi population.</p>", "<p>Therefore, this study measured plasma levels of FGF-21, circulating levels of FFAs, HOMA-IR, and QUICKI in people with T2DM and without NAFLD. The aim was to determine specific and sensitive markers that may be used to predict this condition in Saudi people with T2DM.</p>" ]
[ "<title>Materials and methods</title>", "<p>Participants and study design</p>", "<p>The study design was outlined in detail earlier [##UREF##2##13##], summarized as follows: T2DM patients were recruited from the outpatient endocrine clinics at King Abdulaziz University Hospital in a cross-sectional study design from April 1, 2015, until March 31, 2016. The study was approved by the Committee on the Ethics of Human Research at the Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia (approval number: No-61-15). Written consent was obtained from all participants. Diabetic patients with hemochromatosis, viral hepatitis, Wilson’s disease, primary biliary cirrhosis, autoimmune hepatitis, impaired renal function, sclerosing cholangitis, biliary obstruction, ischemic cardiac or cerebrovascular disease, alpha-1 antitrypsin deficiency, malignancies, or alcohol consumption were excluded from the study. A predesigned questionnaire, including sociodemographic information, medical history, and drugs used, was completed in a face-to-face interview. Blood pressure (BP) was measured using standardized techniques [##REF##14656957##27##]. Anthropometric measurements were taken by following the standard methods and using standardized equipment, and BMI was calculated.</p>", "<p>A sensitive abdominal ultrasound machine (ACUSON X300™ ultrasound system, premium edition (PE) by Siemens, New York, NY, USA) was used to screen for NAFLD. Based on the results, participants were categorized into cases (those with NAFLD) and controls (those without NAFLD).</p>", "<p>Plasma fasting glucose and serum insulin were measured in the Clinical Chemistry Laboratory at the National Guard Hospital, King Abdul-Aziz Medical City in Jeddah. Plasma glucose was measured spectrophotometrically using an Abbott Architect c8000 autoanalyzer (Abbott, Illinois, USA). Serum insulin measurement was performed on an Abbott Architect i2000 autoanalyzer (Abbott, Illinois, USA) using a chemiluminescent microparticle immunoassay method.</p>", "<p>Serum FFA levels were measured using an enzymatic colorimetric method assay for non-esterified fatty acids (Wako Diagnostics USA Corporation). Serum FGF-21 levels were determined using an enzyme-linked immunosorbent assay (FGF-21, UNQ3115/PRO10196; BioVendor LLC, Asheville, North Carolina, USA). Both FFA and FGF-21 were measured manually at the Food, Nutrition, and Lifestyle Research Unit at King Fahd Medical Research Centre.</p>", "<p>The HOMA-IR index was calculated using the following formula: fasting serum insulin (in micro units per milliliter) × fasting serum glucose (in millimoles per liter) divided by the constant 22.5 [##REF##3899825##28##]. The QUICKI was calculated using the following formula: (1/log (fasting insulin) + log (fasting blood glucose) [##REF##10902785##29##].</p>", "<p>Statistical analysis</p>", "<p>The data obtained were analyzed using SPSS version 26 (IBM Corp., Armonk, NY, USA). The independent t-test was used to compare the means of the two groups of people with T2DM with and without NAFLD, and when normality was not confirmed, the Mann-Whitney U test was used. The chi-square test was used to compare the distribution of categorical variables between the two groups.</p>", "<p>The receiver operating characteristics (ROC) curve and the area under the curve (AUC) were used to assess the ability of different indices to identify NAFLD. The optimal cut-off values for the identification of NAFLD were determined from the ROC curve. The association between continuous variables associated with NAFLD was tested with Spearman’s correlation. A p-value &lt;0.05 (two-sided test) was accepted as statistically significant.</p>" ]
[ "<title>Results</title>", "<p>Study participants’ demographic, anthropometric, clinical, and biochemical characteristics</p>", "<p>A total of 67 individuals with T2DM were involved in our study, and 28 were diagnosed with NAFLD through ultrasonography. The average age of those without NAFLD was 57 ± 10.6 years, while for those with NAFLD, the mean age was 57.4 ± 11.9 years. The other characteristics of study participants have been reported in our earlier study [##UREF##2##13##]. Among these participants, there were no significant differences in demographic factors such as age, gender distribution, BMI, and the duration of NAFLD.</p>", "<p>However, the anthropometric measurements WC, HC, and WC to height ratio were significantly higher in women with NAFLD than those without (p &lt; 0.001, at least). Hypertension was also more prevalent among those with NAFLD (p &lt; 0.01). Mean HbA1c% or fasting plasma glucose (FPG) was not significantly different between those with and without NAFLD. However, those with NAFLD had significantly higher serum insulin levels (25.9 ± 27.4 vs. 13.1 ± 9.1, p &lt; 0.01). The means (±SD) of newly measured and calculated biomarkers in participants with and without NAFLD and those with abnormal values are presented in Table ##TAB##0##1##.</p>", "<p>The mean levels of FGF-21 and HOMA-IR were significantly higher, and the mean QUICKI was significantly lower in people with than in those without NAFLD (p &lt; 0.001, p = 0.023, and p = 0.018, respectively (Table ##TAB##0##1##). High FGF-21 was significantly (p &lt; 0.001) more common among participants with NAFLD (92.9%) compared to those without NAFLD (2.6%) (Table ##TAB##0##1##).</p>", "<p>Predictive ability of FGF-21, HOMA-IR, QUICKI, and FFA for NAFLD</p>", "<p>The ROC curves for the four studied indices are shown in Figure ##FIG##0##1##. Among all the four indices, FGF-21 had the highest AUC to predict NAFLD (AUC = 0.981, 95% confidence interval = 0.954-1, p &lt; 0.001) (Figure ##FIG##0##1## and Table ##TAB##1##2##). The AUC for HOMA-IR, QUICKI, and FFA were &lt;0.7 (Figure ##FIG##0##1## and Table ##TAB##1##2##).</p>", "<p>The optimal cut-off values for the four investigated biomarkers and calculated sensitivity, specificity, and positive and negative likelihood ratios (PLR and NLR) for predicting NAFLD in people with T2DM using these calculated cut-off values are presented in Table ##TAB##2##3##. The highest sensitivity, specificity, and PLR combined with the lowest NLR were found when FGF-21 was used to predict NAFLD in people with T2DM (Table ##TAB##2##3##).</p>", "<p>There were no correlations between the level of FGF-21 and the levels of insulin and FPG (data not shown).</p>" ]
[ "<title>Discussion</title>", "<p>Given the high prevalence of T2DM in Saudi Arabia [##UREF##4##30##,##UREF##5##31##], the high incidence of NAFLD among T2DM patients [##REF##26856933##3##], and the absence of robust biomarkers for its prediction, the search for biomarkers for this often-silent complication of diabetes is needed to optimize management. Therefore, we aimed to find specific and sensitive markers that may be used to predict NAFLD in Saudi people with T2DM by assessing the most likely candidates, namely, FGF- 21, FFA, and two measures of IR, i.e., HOMA-IR and QUICKI.</p>", "<p>In our investigation, the mean levels of FGF-21 and HOMA-IR were significantly higher, while those of QUICKI were significantly lower in people with T2DM with NAFLD compared with those without NAFLD. In addition, FGF-21 had the highest AUC to predict NAFLD compared with the other three indices, with a cut-off value of 166 ng/L showing high sensitivity, specificity, and PLR combined with a low NLR.</p>", "<p>We reported earlier [##UREF##2##13##] that the mean levels of HbA1c% or FPG were not significantly different between those with and without NAFLD, but those with NAFLD had significantly higher serum insulin levels. Therefore, the means of the two calculated measures of IR (HOMA-IR and QUICKI) were significantly different in the group of patients with NAFLD compared with the means of the group without the condition, indicating an association between increased IR and NAFLD in people with T2DM.</p>", "<p>IR has long been proposed as the first hit in the two-hit hypothesis [##REF##9547102##32##], which explains the pathogenesis of NASH, with excessive fatty acids in circulation leading to simple hepatic steatosis and IR, thus promoting the progression from simple fatty liver to NASH [##REF##17241878##33##]. Therefore, we studied measures of IR as biomarkers for predicting NAFLD in Saudi people with T2DM. However, following rigorous statistical analysis, the AUC for HOMA-IR and QUICKI were both &lt;0.7, which is reflected in low specificity and unacceptable PLR and NLR for both calculated measures. Therefore, it can be suggested that neither of these measures can be used to predict NAFLD in our T2DM patients.</p>", "<p>The use of HOMA-IR and other IR markers in NAFLD has been investigated by different research groups, particularly among non-diabetic populations. A recent study conducted on 2,148 non-diabetic Chinese adults between 2021 and 2023 found that HOMA-IR along with TyG (triglyceride-glucose index), TyG-BMI (TyG multiplied by body mass index), TG/HDL-c (triglycerides to high-density lipoprotein cholesterol ratio), and METS-IR (metabolic score for insulin resistance) were effective in predicting the risk of NAFLD. However, the study highlighted that the predictive abilities of these IR markers varied between obese and non-obese populations, with HOMA-IR showing significant predictive value in obese populations for NAFLD [##REF##37993481##34##].</p>", "<p>Another published study by the same group included 2,234 participants recruited between 2021 and 22 and investigated the correlation between HOMA-IR and NAFLD in a non-diabetic Chinese population. Similar to our findings, HOMA-IR was significantly higher in the NAFLD group. The efficacy of HOMA-IR for diagnosing NAFLD was substantiated using ROC curves, demonstrating its utility as a predictive tool for NAFLD in lean, non-diabetic Chinese individuals in contrast to our findings [##REF##37862786##35##].</p>", "<p>A prospective, cohort, population-based study was conducted in the northern region of Iran among 2,461 participants without NAFLD (with and without diabetes) between 2009 and 2010, who were recruited using the stratified randomization method based on the sex and age of individuals. Ultrasonographic examination was performed at the baseline and after a seven-year follow-up between 2016 and 2017. Multiple binary regression analysis was applied to evaluate the association between the development of NAFLD and potential risk factors. Based on the numerous binary logistic regression analyses, HOMA-IR had a significant relationship with the incidence of NAFLD in women (odds ratio = 1.164, 95% confidence interval = 1.041-1.301, p = 0.007] but not in men, concluding that HOMA-IR can be considered an independent risk factor for NAFLD in women only [##UREF##6##36##].</p>", "<p>In an earlier published study, the same researchers using the baseline data of the cohort mentioned above and collected from 5,511 participants with and without NAFLD (diabetic and non-diabetic) aged ≥18 years determined the optimal cut-off points for HOMA-IR and QUICKI in the diagnosis of MetS and NAFLD. The optimal cut-off point of HOMA-IR for the diagnosis of NAFLD was 1.79 (sensitivity = 66.2%, specificity = 62.2%) in men and 1.95 (sensitivity = 65.1%, specificity = 54.7%) in women. In addition, the optimal cut-off point of QUICKI for the diagnosis of NAFLD was 0.347 (sensitivity = 62.9%, specificity = 65.0%) in men and 0.333 (sensitivity = 53.2%, specificity = 67.7%) in women, concluding that the optimal cut-off points of HOMA-IR and QUICKI were different for men and women [##REF##26718936##37##]. However, unlike our study, they did not report PLR and NLR, which are necessary to decide whether the investigated biomarker is robust enough to be used to predict a disease.</p>", "<p>Gender differences were not investigated in our study due to the small number of included participants. The noted differences between our findings and the above-mentioned studies could be due to differences in ethnicities, the demographic and clinical characteristics of the studied population, and the differences in lifestyle and dietary practices.</p>", "<p>Another investigated biomarker in our study was the level of circulating FFAs. As mentioned earlier, an increased level of circulating FFAs has been reported to be associated with increased lipolysis and FFA flux to the liver, leading to increased synthesis of triglycerides [##REF##15254578##26##]. We found no difference in the mean levels of FFAs between participants with and without NAFLD, and the calculated AUC was &lt;0.7. However, using the optimal cut-off value of 0.348, the circulating level of FFAs showed a reasonable sensitivity of 0.808 and a moderate specificity of 0.615. Despite this, the calculated PLR and NLR ruled out the possibility of using FFAs as a predictive marker for NAFLD in Saudi people with T2DM.</p>", "<p>A cross-sectional Chinese study, which included 840 participants with NAFLD (114 diabetics and 726 non-diabetics) and 331 healthy controls, investigated the association between fasting serum FFAs and NAFLD. Serum FFA levels were significantly higher in people with NAFLD compared to controls (p &lt; 0.001), and stepwise regression indicated that the level of serum FFAs was an independent factor predicting advanced fibrosis (Fibrosis-4 (FIB-4) ≥1.3) only so that it could be used as an indicator for predicting advanced fibrosis, but not milder fibrosis (FIB-4 &lt;1.3) in NAFLD patients [##REF##25060337##38##]. We did not measure the degree of fibrosis in our study, and all included patients were diabetic, which could explain the difference between our findings and those of the Chinese study.</p>", "<p>Another study evaluated FFA profiles among healthy Chinese individuals and NAFLD patients (lean, overweight, and obese) to identify the most likely FFAs that can be used for the early diagnosis of NAFLD. The serum FFA profiles of NAFLD patients were significantly higher compared to healthy controls. Furthermore, there was no significant difference in total FFA profiles between lean and overweight NAFLD patients. In contrast, the total FFA profiles of obese NAFLD patients were significantly higher compared with the profiles of healthy controls and lean and overweight NAFLD patients. However, following rigorous statistical analysis and adjusting for confounding factors, it was concluded that only myristic acid (14:0) and palmitoleic acid (16:1), and not total FFA, can be considered promising for the early diagnosis of NAFLD, especially among normal-weight individuals [##REF##28870233##39##]. We estimated the total FFA in our study, ruling out its usefulness in predicting NAFLD, and substantiated their findings.</p>", "<p>As mentioned earlier, the development of NAFLD has been reported to be associated with various causes [##REF##29936596##21##, ####REF##24316260##22##, ##REF##23397118##23##, ##REF##28585211##24##, ##REF##29967350##25####29967350##25##]. In addition, dietary energy intake and diet composition have also been shown to play an important role [##REF##30901929##40##]. High-fat diets have been reported to cause fatty liver [##REF##15741262##41##]. Therefore, it can be suggested that an increase in de novo lipogenesis due to dietary intake or other causes can be the leading cause of accumulation of fat in the liver of our studied patients, hence, the lack of difference in the mean levels of FFAs between the two groups of patients with and without NAFLD, especially as both studied groups were T2DM patients with various degrees of IR. This suggestion requires further research which should include dietary intake studies and perhaps the inclusion of non-diabetic individuals with NAFLD to clarify the situation and explore the possibility of utilizing FFAs to diagnose NAFLD in non-diabetic people.</p>", "<p>Our fourth investigated biomarker was the serum level of FGF-21. This showed very promising results for diagnosing NAFLD with very high specificity and sensitivity, giving a high PLR and low NLR. Therefore, serum FGF-21 ≥166 ng/L could be suggested as a good predictor for the diagnoses of NAFLD in Saudi T2DM patients.</p>", "<p>In partial agreement with our study, an earlier study among 179 Chinese NAFLD patients with and without diabetes (68 NASH cases and 111 non-NASH cases) reported that the serum levels of several biomarkers, including FGF-21, were significantly higher in NAFLD patients compared with healthy controls. In addition, these levels positively correlated with NAFLD activity scores (NAS) and pathological characteristics of NAFLD, concluding that these biomarkers could be non-invasive diagnostic markers for NASH, especially if measured in a stepwise combination [##UREF##7##42##]. When measuring FGF-21 alone to diagnose NASH, their reported sensitivity was 79.30% with a specificity of 77.40%, which is lower than our findings. The difference between our results and those of the aforementioned study could be due to differences in the clinical characteristics of included participants (T2DM patients versus mixed population), ethnicity, genetics, and dietary practices, especially as dietary intake and composition have been shown to play a considerable role in the development of NAFLD as mentioned earlier [##REF##15741262##41##], and there have been reports of ethnicity-associated differences in the pathogenesis and development of NAFLD [##REF##34182070##43##]. Furthermore, a study among morbidly obese females undergoing bariatric surgery did not show any association between pathological features of NASH and plasma FGF-21, which suggests that body fat and gender, as well as other comorbidities, may modify this association [##REF##28820393##44##].</p>", "<p>As in most studies, our study has limitations and strengths. The main limitation of our study was the small sample size, which did not allow the investigation of the effect of gender differences, especially as the liver shows a very high degree of sexual dimorphism [##REF##6189449##45##,##REF##16825664##46##]. A second limitation is that this study did not estimate the degree of steatosis, which could affect the level of measured markers. Another limitation was that dietary intake was not studied, even though it might affect the development of the condition and the level of FGF-21. However, as the first study on Saudi T2DM patients, our study shows a strong relationship between NAFLD and the level of FGF-21, suggesting its use as a possible biomarker for predicting NAFLD, thus helping to plan future research. Further studies, including more novel biomarkers on a larger population from different parts of Saudi Arabia, are needed to validate our findings. In addition, studies on non-diabetic individuals are also required and are planned, once funding is available, to explore the possibility of using novel biomarkers to predict NAFLD in these individuals.</p>" ]
[ "<title>Conclusions</title>", "<p>Our research indicates that the serum level of FGF-21 is a highly specific and sensitive biomarker for predicting NAFLD in Saudi patients with T2DM. This biomarker could be utilized for preliminary screening before patients are referred to more advanced healthcare facilities for confirmatory tests such as abdominal ultrasonography. Although HOMA-IR and QUICKI are associated with NAFLD, their predictive power is limited. Additionally, the study found no significant difference in FFA levels between the groups, suggesting that FFAs are not effective in predicting NAFLD.</p>" ]
[ "<p>Background</p>", "<p>Non-alcoholic fatty liver disease (NAFLD) is more prevalent among individuals with type 2 diabetes (T2DM), elevating their risk of cardiovascular diseases (CVDs) and premature mortality. There is a need to modify treatment strategies to prevent or delay these adverse outcomes. Currently, there are no sensitive or specific biomarkers for predicting NAFLD in Saudi T2DM patients. Therefore, we aimed to explore the possibility of using fibroblast growth factor 21 (FGF-21), free fatty acids (FFAs), homeostatic model assessment for insulin resistance (HOMA-IR), and quantitative insulin sensitivity check index (QUICKI) as possible markers.</p>", "<p>Methodology</p>", "<p>In this study, a total of 67 T2DM patients were recruited. NAFLD was detected by ultrasonography in 28 patients. Plasma glucose, FFAs, FGF-21, and serum insulin were measured in fasting blood samples. HOMA-IR and QUICKI were calculated. The means of the two groups with and without NAFLD were statistically compared. The receiver operating characteristics (ROC) curve and the area under the curve (AUC) were used to assess the ability to identify NAFLD.</p>", "<p>Results</p>", "<p>The mean levels of FGF-21 and HOMA-IR were significantly higher and that of QUICKI was significantly lower in patients with NAFLD than in those without (p &lt; 0.001, p = 0.023, and p = 0.018, respectively). FGF-21 had the highest AUC to identify NAFLD (AUC = 0.981, 95% confidence interval = 0.954-1, P &lt; 0.001). The AUCs for HOMA-IR, QUICKI, and FFA were &lt;0.7. The highest sensitivity, specificity, positive likelihood ratio, and the lowest negative likelihood ratio were found when FGF-21 was used to predict NAFLD.</p>", "<p>Conclusions</p>", "<p>FGF-21 may be used as a biomarker to predict NAFLD in people with T2DM due to its high sensitivity and specificity compared to the other markers.</p>" ]
[]
[ "<p>The data used to support the findings of this study have been deposited in the Food, Nutrition and Lifestyle Unit, King Fahd Medical Research Centre, King Abdulaziz University Site. It can be accessed via the following link, http://www.kau.edu.sa/GetFile.aspx?id=313216&amp;fn=NAFLD.rar.</p>" ]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Receiver operating characteristic (ROC) curves.</title><p>ROC curves for free fatty acids (FFAs), fibroblast growth factor 21 (FGF-21), homeostatic model assessment of insulin resistance (HOMA-IR), quantitative insulin sensitivity check index (QUICKI) in patients with type 2 diabetes.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Mean ± SD of fatty liver biomarkers in people with T2DM with and without NAFLD and the number of patients with high biomarker levels.</title><p>^: P-value obtained by Mann-Whitney non-parametric test</p><p><sup>~</sup>: P-value obtained by t-test.</p><p><sup>$</sup>: P-value obtained by the chi-square test.</p><p>Significant p-values are in bold.</p><p>FFA: free fatty acids; FGF-21: fibroblast growth factor 21; HOMA-IR: homeostatic model assessment of insulin resistance; QUICKI: quantitative insulin sensitivity check index; NAFLD: non-alcoholic fatty liver disease; T2DM: type 2 diabetes mellitus</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fatty liver biomarkers</td><td rowspan=\"1\" colspan=\"1\">Participants without NAFLD, N = 39 (men = 16, women = 23)</td><td rowspan=\"1\" colspan=\"1\">Participants with NAFLD, N = 28 (men = 6, women = 22)</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">FGF-21 (ng/L)</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Mean ± SD (actual range)</td><td rowspan=\"1\" colspan=\"1\">113 ± 42 (38–291)</td><td rowspan=\"1\" colspan=\"1\">353 ± 168 (143–767)</td><td rowspan=\"1\" colspan=\"1\">&lt;0.001^</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Number of participants with high FGF-21 (≥166 ng/L) (%)</td><td rowspan=\"1\" colspan=\"1\">1 (2.6%)</td><td rowspan=\"1\" colspan=\"1\">26 (92.9%)</td><td rowspan=\"1\" colspan=\"1\">&lt;0.001<sup>$</sup></td></tr><tr style=\"background-color:#ccc\"><td colspan=\"4\" rowspan=\"1\">HOMA-IR</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mean ± SD (actual range)</td><td rowspan=\"1\" colspan=\"1\">6.18 ± 5.27 (1.16–22.3)</td><td rowspan=\"1\" colspan=\"1\">10.4 ± 10.4 (1.47–45.7)</td><td rowspan=\"1\" colspan=\"1\">0.023^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Number of participants with increased insulin resistance (&gt;1.9) (%)</td><td rowspan=\"1\" colspan=\"1\">33 (84.6%)</td><td rowspan=\"1\" colspan=\"1\">25 (96.2%)</td><td rowspan=\"1\" colspan=\"1\">0.142<sup>$</sup>\n</td></tr><tr><td colspan=\"4\" rowspan=\"1\">QUICKI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Mean ± SD (actual range)</td><td rowspan=\"1\" colspan=\"1\">0.309 ± 0.031 (0.253–0.374)</td><td rowspan=\"1\" colspan=\"1\">0.291 ± 0.029 (0.234–0.361)</td><td rowspan=\"1\" colspan=\"1\">0.018<sup>~</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Number of participants with increased insulin resistance (&lt;0.339) (%)</td><td rowspan=\"1\" colspan=\"1\">31 (79.5%)</td><td rowspan=\"1\" colspan=\"1\">25 (96.2%)</td><td rowspan=\"1\" colspan=\"1\">0.057<sup>$</sup>\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"4\" rowspan=\"1\">FFA</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mean ± SD (actual range)</td><td rowspan=\"1\" colspan=\"1\">0.39 ± 0.18 (0.15–0.85)</td><td rowspan=\"1\" colspan=\"1\">0.44 ± 0.11 (0.24–0.74)</td><td rowspan=\"1\" colspan=\"1\">0.115<sup>~</sup>\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Area under the receiver operating characteristic curve (AUC) and its 95% CI for different biomarkers with NAFLD in patients with type 2 diabetes.</title><p>FFA: free fatty acids; FGF-21: fibroblast growth factor 21; HOMA-IR: homeostatic model assessment of insulin resistance; QUICKI: quantitative insulin sensitivity check index; NAFLD: non-alcoholic fatty liver disease; CI: confidence interval; SE: standard error</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fatty liver biomarkers</td><td rowspan=\"1\" colspan=\"1\">AUC</td><td rowspan=\"1\" colspan=\"1\">SE</td><td rowspan=\"1\" colspan=\"1\">95% CI</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">FGF-21</td><td rowspan=\"1\" colspan=\"1\">0.981</td><td rowspan=\"1\" colspan=\"1\">0.014</td><td rowspan=\"1\" colspan=\"1\">0.954, 1</td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">HOMA-IR</td><td rowspan=\"1\" colspan=\"1\">0.667</td><td rowspan=\"1\" colspan=\"1\">0.067</td><td rowspan=\"1\" colspan=\"1\">0.535, 0.799</td><td rowspan=\"1\" colspan=\"1\">0.023</td></tr><tr><td rowspan=\"1\" colspan=\"1\">QUICKI</td><td rowspan=\"1\" colspan=\"1\">0.333</td><td rowspan=\"1\" colspan=\"1\">0.067</td><td rowspan=\"1\" colspan=\"1\">0.201, 0.465</td><td rowspan=\"1\" colspan=\"1\">0.023</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">FFA</td><td rowspan=\"1\" colspan=\"1\">0.647</td><td rowspan=\"1\" colspan=\"1\">0.069</td><td rowspan=\"1\" colspan=\"1\">0.512, 0.782</td><td rowspan=\"1\" colspan=\"1\">0.046</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>The optimal cut-off values for fatty liver biomarkers and their sensitivity and specificity for the identification of NAFLD in diabetic patients.</title><p>FFA: free fatty acids; FGF-21: fibroblast growth factor 21; HOMA-IR: homeostatic model assessment of insulin resistance; QUICKI: quantitative insulin sensitivity check index; PLR: positive likelihood ratio; NLR: negative likelihood ratio; NAFLD: non-alcoholic fatty liver disease</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nFatty liver biomarkers\n</td><td rowspan=\"1\" colspan=\"1\">\nOptimal cut-off point\n</td><td rowspan=\"1\" colspan=\"1\">\nSensitivity\n</td><td rowspan=\"1\" colspan=\"1\">\nSpecificity\n</td><td rowspan=\"1\" colspan=\"1\">\nPLR\n</td><td rowspan=\"1\" colspan=\"1\">\nNLR\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nFGF-21\n</td><td rowspan=\"1\" colspan=\"1\">\n166\n</td><td rowspan=\"1\" colspan=\"1\">\n0.929\n</td><td rowspan=\"1\" colspan=\"1\">\n0.974\n</td><td rowspan=\"1\" colspan=\"1\">\n35.731\n</td><td rowspan=\"1\" colspan=\"1\">\n0.028\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nHOMA-IR\n</td><td rowspan=\"1\" colspan=\"1\">\n3.8\n</td><td rowspan=\"1\" colspan=\"1\">\n0.846\n</td><td rowspan=\"1\" colspan=\"1\">\n0.487\n</td><td rowspan=\"1\" colspan=\"1\">\n1.649\n</td><td rowspan=\"1\" colspan=\"1\">\n0.606\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nQUICKI\n</td><td rowspan=\"1\" colspan=\"1\">\n0.298\n</td><td rowspan=\"1\" colspan=\"1\">\n0.538\n</td><td rowspan=\"1\" colspan=\"1\">\n0.359\n</td><td rowspan=\"1\" colspan=\"1\">\n0.839\n</td><td rowspan=\"1\" colspan=\"1\">\n1.191\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nFFA\n</td><td rowspan=\"1\" colspan=\"1\">\n0.348\n</td><td rowspan=\"1\" colspan=\"1\">\n0.808\n</td><td rowspan=\"1\" colspan=\"1\">\n0.615\n</td><td rowspan=\"1\" colspan=\"1\">\n0.945\n</td><td rowspan=\"1\" colspan=\"1\">\n0.476\n</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Basmah Eldakhakhny, Suhad Bahijri, Ghada Ajabnoor, Jaakko Tuomilehto</p><p><bold>Drafting of the manuscript:</bold>  Basmah Eldakhakhny, Suhad Bahijri, Ghada Ajabnoor, Aliaa A. Alamoudi, Alaa S. Al-Mowallad, Sumia Enani, Lubna Alsheikh, Amani Alhozali, Anwar Borai</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Basmah Eldakhakhny, Suhad Bahijri, Ghada Ajabnoor, Alaa S. Al-Mowallad, Sumia Enani, Amani Alhozali, Anwar Borai, Jaakko Tuomilehto</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Suhad Bahijri, Ghada Ajabnoor, Aliaa A. Alamoudi, Alaa S. Al-Mowallad, Sumia Enani, Lubna Alsheikh, Amani Alhozali, Anwar Borai, Jaakko Tuomilehto</p><p><bold>Supervision:</bold>  Suhad Bahijri, Ghada Ajabnoor</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. The Committee on the Ethics of Human Research at the Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia issued approval No-61-15</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050524-i01\" position=\"float\"/>" ]
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[{"label": ["7"], "article-title": ["Non-alcoholic fatty liver disease: the mist gradually clears"], "source": ["J Hepatol"], "person-group": ["\n"], "surname": ["de Alwis", "Day"], "given-names": ["NM", "CP"], "fpage": ["0"], "lpage": ["12"], "volume": ["48 Suppl 1"], "year": ["2008"]}, {"label": ["10"], "article-title": ["NAFLD: a multisystem disease"], "source": ["J Hepatol"], "person-group": ["\n"], "surname": ["Byrne", "Targher"], "given-names": ["CD", "G"], "fpage": ["0"], "lpage": ["64"], "volume": ["62"], "year": ["2015"]}, {"label": ["13"], "article-title": ["Exploring the validity of available markers and indices in the diagnosis of nonalcoholic fatty liver disease (NAFLD) in people with type 2 diabetes in Saudi Arabia"], "source": ["Diseases"], "person-group": ["\n"], "surname": ["Ajabnoor", "Bahijri", "Enani"], "given-names": ["GM", "SM", "SM"], "volume": ["11"], "year": ["2023"]}, {"label": ["17"], "article-title": ["Fibroblast growth factor 21 is a metabolic regulator that plays a role in the adaptation to ketosis"], "source": ["Am J Clin Nutr"], "person-group": ["\n"], "surname": ["Domouzoglou", "Maratos-Flier"], "given-names": ["EM", "E"], "fpage": ["901"], "lpage": ["905"], "volume": ["93"], "year": ["2011"]}, {"label": ["30"], "article-title": ["The prevalence of diabetes and prediabetes in the adult population of Jeddah, Saudi Arabia--a community-based survey"], "source": ["PLoS One"], "person-group": ["\n"], "surname": ["Bahijri", "Jambi", "Al Raddadi", "Ferns", "Tuomilehto"], "given-names": ["SM", "HA", "RM", "G", "J"], "fpage": ["0"], "volume": ["11"], "year": ["2016"]}, {"label": ["31"], "article-title": ["IDF Diabetes Atlas"], "date-in-citation": ["\n"], "month": ["11"], "year": ["2023", "2021"], "person-group": ["\n"], "surname": ["Federation. ID:"], "given-names": ["2021"], "uri": ["https://www.diabetesatlas.org"]}, {"label": ["36"], "article-title": ["The incidence and risk factors of non-alcoholic fatty liver disease: a cohort study from Iran"], "source": ["Hepat Mon"], "person-group": ["\n"], "surname": ["Motamed", "Khoonsari", "Panahi"], "given-names": ["N", "M", "M"], "fpage": ["98531"], "volume": ["20"], "year": ["2020"]}, {"label": ["42"], "article-title": ["Combined serum biomarkers in non-invasive diagnosis of non-alcoholic steatohepatitis"], "source": ["PLoS One"], "person-group": ["\n"], "surname": ["Yang", "Xu", "Liu"], "given-names": ["M", "D", "Y"], "fpage": ["0"], "volume": ["10"], "year": ["2015"]}]
{ "acronym": [], "definition": [] }
46
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50524
oa_package/8e/84/PMC10787595.tar.gz
PMC10787596
0
[ "<title>Introduction and background</title>", "<p>According to the World Health Organization (WHO), dementia is classified as a syndrome rather than a disease [##UREF##0##1##]. It is characterized by a progressive decline in the cognitive function of an individual across multiple cognitive domains, consequently impairing functional abilities. The term dementia has been replaced by major neurocognitive disorder (MND) in the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5) [##UREF##1##2##]. However, due to the higher recognition of the term dementia, we will use it in this review. </p>", "<p>The cognitive decline seen in dementia is usually associated with the prior level of cognition of the patient. Furthermore, the decline is often persistent and is not associated with an isolated episode of delirium. The most frequent symptoms of dementia include progressive impairment in thinking, language, memory, learning, and judgment, difficulty in concentrating, being confused about time and place, and mood changes [##REF##33612471##3##,##UREF##2##4##]. According to the DSM-5, there are 13 etiologies that can contribute to dementia. However, Alzheimer's disease (AD) is the underlying etiology in approximately 70% of all cases [##REF##24398425##5##]. Other causes of dementia include Lewy body disease, traumatic brain injury, vascular disease, and frontotemporal lobar degeneration [##UREF##3##6##].</p>", "<p>Several risk factors have been identified for dementia, such as being over the age of 65 and having hypertension, diabetes, smoking, and depression [##UREF##2##4##,##REF##24398425##5##]. These factors make it more likely for older adults to develop dementia. With the growing population of individuals over 65, the number of dementia patients is expected to increase in the future [##REF##24398425##5##]. Additionally, the prevalence of dementia has been linked to socioeconomic status and culture, with a higher occurrence seen in countries with lower and middle incomes [##REF##33612471##3##,##UREF##3##6##].</p>" ]
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[ "<title>Conclusions</title>", "<p>Dementia is a significant contributor to morbidity in elderly individuals. As the number of individuals aged above 65 years is increasing, there is also an increase in dementia cases, and it has emerged as a significant concern for healthcare systems and communities across the globe. However, the latest advances in the diagnosis and treatment of dementia have ushered in a new era of hope and progress in the battle against this debilitating condition. New diagnostic tools have helped early diagnoses of dementia. Currently, research is ongoing both on the pharmacological and non-pharmacological treatment of dementia. In the journey to combat dementia, knowledge is our greatest weapon. As we move forward, collaboration between researchers, healthcare providers, and caregivers is key to managing dementia.</p>" ]
[ "<p>Dementia is a debilitating neurological condition that is characterized by persistent cognitive decline. It is a global health challenge, with a rapidly increasing prevalence due to an increasing aging population. Although definitive diagnosis of various conditions of dementia is only possible by autopsy, clinical diagnosis can be performed by a specialist. The diagnostic process has evolved with recent breakthroughs in diagnostic tools, such as advanced imaging techniques and biomarkers. These tools facilitate early and accurate identification of the condition. Early diagnosis is vital, as it enables timely interventions to improve the quality of life for affected individuals. Treatment strategies for dementia encompass both pharmacological and non-pharmacological approaches. Non-pharmacological treatments include cognitive training and lifestyle modifications. Among pharmacological treatments, acetyl-cholinesterase inhibitors including donepezil, rivastigmine, and galantamine can be used in various doses based on the severity of the disease. Apart from these, N-methyl-D-aspartate receptor antagonists such as memantine can also be used. Furthermore, personalized treatments have also gained significant attention in dementia treatment. Interdisciplinary care, involving healthcare professionals, social workers, and support networks, is crucial for comprehensive and holistic dementia management.</p>" ]
[ "<title>Review</title>", "<p>Prevalence of dementia</p>", "<p>According to WHO, almost 55 million people suffer from dementia across the globe [##UREF##4##7##]. The prevalence of dementia has been associated with socioeconomic level and culture [##REF##25089278##8##]. The majority of dementia patients are from low-and middle-income countries. Furthermore, almost 10 million new cases are being added every year [##UREF##4##7##]. Among various risk factors, age above 65 years, hypertension, diabetes, smoking, and depression are the main contributors. Older adults are particular victims of this condition, as it is the most prevalent condition among neurological diagnoses in this age group [##UREF##2##4##,##UREF##5##9##]. As the number of individuals aged above 65 is increasing, the number of dementia patients is expected to increase to 131.5 million by the year 2050 [##UREF##5##9##]. A meta-analysis by Cao et al. reported that the prevalence of dementia is 244 times higher in individuals aged over 100 years compared to the 50-59 years age group. Furthermore, they revealed that the number of dementia patients doubles every five years [##REF##31884487##10##]. The prevalence of dementia also varies according to the region. For example, a systemic review showed that there was a greater incidence in Latin America (8.5%) and a noticeably lower incidence in the four sub-Saharan African regions (2-4%) [##REF##23305823##11##]. Similarly, a cross-sectional study from Saudi Arabia that included 1613 participants showed that 16.6% of older individuals were suffering from dementia [##UREF##3##6##].</p>", "<p>Pathophysiology of dementia</p>", "<p>The pathophysiology of dementia varies according to the sub-type of dementia. For example, gross examination of the brain of patients with AD indicates lower brain weight, which can be 100-200 g below average or higher depending on the severity of the disease. The temporal, frontal, and parietal regions show cortical atrophy. However, the thalamus, brainstem, cerebellar hemispheres, and basal ganglia typically show normal size and weight. Furthermore, senile plaques and neurofibrillary tangles are visible under a microscope [##REF##11487050##12##]. Extracellular plaques consist of beta-amyloid, whereas intracellular neurofibrillary tangles are composed of hyperphosphorylated tau cytoskeletal filaments. The nucleus basalis, cortex, amygdala, and hippocampus are typically where these changes occur. The degree of clinical illness and cognitive deterioration is inversely correlated with the number of tangles. The damage that beta-amyloid causes to brain cells includes intracellular calcium accumulation, oxygen radical formation, nitric oxide synthesis, and inflammatory processes. The basal forebrain has injured cholinergic neurons, resulting in a diminished level of cholinergic neurotransmission. Further findings include the degeneration of the locus caeruleus and raphe nuclei, which leads to deficits in the neurotransmitters glutamate, noradrenaline (norepinephrine), serotonin, and corticotropin-releasing factor [##REF##11487050##12##, ####REF##11113503##13##, ##REF##28862638##14####28862638##14##].</p>", "<p>Similarly, different types of changes are seen in other types of dementia as well. Lewy body dementia (LBD) is characterized by the accumulation of aggregates of alpha-synuclein protein, called Lewy bodies [##REF##22033743##15##]. HIV-associated dementia can cause neurodegeneration of the brain due to toxic inflammation by HIV [##REF##24156898##16##]. Dementia due to alcohol consumption can be caused by cytotoxic processes [##REF##28950395##17##]. </p>", "<p>Diagnosis of dementia</p>", "<p>The definitive diagnosis of the various types of dementia is only possible by the autopsy of the patient. However, a clinical diagnosis of dementia can be made by a specialist in a primary care setting by clinical examination of the patient. Furthermore, neuroimaging, biomarkers, and digital tools can also be used by the physician. Early diagnosis of dementia is crucial for timely intervention and management of the disease. In recent decades, the diagnostic tools and techniques for dementia have improved significantly.</p>", "<p><italic>Neuroimaging</italic> </p>", "<p>Although diagnostic evaluations are primarily performed based on clinical criteria, the role of neuroimaging has also expanded significantly [##REF##33660199##18##]. Advanced neuroimaging techniques, such as diffusion tensor imaging (DTI), functional MRI (fMRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), have been shown to provide valuable information in the diagnosis of dementia. DTI can detect changes in the structural connectivity of white matter tracts, which can indicate the presence of dementia [##REF##17200485##19##]. The use of fMRI can detect changes in brain activity patterns, which can indicate functional alterations in the brain associated with dementia [##REF##19847046##20##]. PET and SPECT can detect changes in brain metabolism and blood flow, improving the accuracy of the diagnosis of dementia [##REF##15747152##21##] Some studies have investigated the use of novel PET tracers that target specific pathological features of dementia, such as beta-amyloid and tau proteins [##REF##27987560##22##]. Machine learning algorithms that analyze neuroimaging data have also been developed to aid in the diagnosis of dementia [##REF##22019881##23##].</p>", "<p><italic>Biomarkers</italic> </p>", "<p>Recent studies have investigated the use of blood-based biomarkers, such as neurofilament light chain (NfL) and plasma phosphorylated tau (p-tau), for the detection of neurodegeneration in the brain. Elevated levels of these biomarkers indicate the presence of dementia, even before symptoms appear [##REF##31206160##24##]. The use of blood biomarkers is potentially helpful in diagnosing the pathological features of AD. In addition to blood-based biomarkers, CSF biomarkers, such as amyloid beta, tau, and phosphorylated tau, have been shown to improve the accuracy of the diagnosis of AD [##REF##20061647##25##]. Retinal biomarkers, such as retinal nerve fiber layer thickness and macular volume, have also been investigated as potential biomarkers for the early diagnosis of dementia [##REF##25428254##26##]. </p>", "<p>Digital Tools</p>", "<p>The modern healthcare industry has embraced technological advancements to improve the provision of care. Mobile-based applications technology is one such innovative and trending invention that supports increased patient care management and quick diagnostics [##UREF##6##27##]. Digital tools, such as artificial intelligence (AI) machine learning algorithms, smartphone apps, and wearable devices, have gained traction in the diagnosis of dementia. These tools can detect subtle changes in cognitive function and behavior, which can indicate the presence of dementia. AI algorithms can accurately predict the onset of AD even before symptoms appear, by analyzing neuroimaging data [##REF##26927419##28##]. Smartphone apps such as Sea Hero Quest (Glitchers, Edinburgh, Scotland) have been developed to assess spatial navigation skills, which are often impaired in the early stages of dementia [##REF##16829144##29##]. Wearable devices such as smartwatches and fitness trackers have also been used to monitor changes in physical activity and sleep patterns, which can be potential early indicators of dementia. BrainCheck Inc. (Austin, Texas, United States) has developed five distinct games designed based on gold-standard neurocognitive testing. Based on the outcomes of each game, the app shows a graph that indicates the degree of cognitive function relevant to executive function, cognitive processing, immediate memory, visual attention, and delayed recall skills of the user [##UREF##7##30##]. Early diagnosis of dementia is critical for timely intervention and management of the disease [##REF##20838046##31##]. </p>", "<p>Management of dementia</p>", "<p>Due to the neuronal cell loss in dementia, there is no curative treatment available so far. Therefore, symptomatic management remains the mainstay of treatment: treatment of behavioral disturbances, environmental manipulations that support function, and counseling about safety concerns [##REF##32484110##32##]. The main aim of dementia management is to delay cognitive decline and relieve the patient from cognitive suffering. Both non-pharmacologic and pharmacologic approaches are employed, either alone or in combination. The current treatments for dementia vary based on the types of dementia. Two main types of medication have been approved for the treatment of dementia including cholinesterase inhibitors and memantine. AD-related dementia can be treated with acetylcholinesterase inhibitors (AChEIs) including donepezil, rivastigmine, and galantamine in various doses based on the severity of the disease. Apart from these, N-methyl-D-aspartate (NMDA) receptor antagonists such as memantine can also be used. Other types of dementia such as vascular dementia, Parkinson's disease dementia, Down syndrome dementia, LBD, and frontotemporal dementia can also use these pharmacological options. </p>", "<p>AChEIs</p>", "<p>It has been shown that AChEIs such as galantamine, rivastigmine, and donepezil increase cholinergic transmission by halting cholinesterase at the synaptic cleft while providing modest symptomatic relief in certain patients suffering from dementia. It is the mainstay drug used in AD patients as they have lower cerebral content of choline acetyltransferase, resulting in diminished acetylcholine production and poor cortical cholinergic function. Donepezil has been found to significantly improve cognition, clinical assessment, and functional outcomes in the higher-dose group [##REF##22397651##33##, ####REF##33515559##34##, ##REF##33302541##35##, ##UREF##8##36####8##36##]. AChEIs are presently the gold standard for treating LBD-related cognitive and psychiatric symptoms. Rivastigmine is the only one of the three that is FDA-approved for treating LBD [##REF##26515660##37##, ####UREF##9##38##, ##REF##24828899##39##, ##UREF##10##40####10##40##]. The remaining two are used off-label. There is no compelling evidence that one of the three is more effective than the others in treating LBD [##REF##23949147##41##, ####REF##25713599##42##, ##REF##31519472##43####31519472##43##]. There is limited data on the use of AChEIs in treating frontotemporal dementia, and the available ones do not support the use of AChEIs in frontotemporal dementia [##REF##19182475##44##]. The treatment of dementia in Parkinson's disease centers on the use of AChEIs. Most, but not all, trials with AChEIs in Parkinson's disease dementia found a slight to moderate benefit, albeit at the expense of an increased risk of adverse effects such as exacerbated tremors and nausea [##REF##21450164##45##,##REF##18196898##46##].</p>", "<p>Memantine</p>", "<p>Memantine is an NMDA receptor antagonist. In contrast to cholinergic agents, memantine acts in a neuroprotective manner. Cortical and hippocampal neurons' main excitatory amino acid neurotransmitter is glutamate. Moreover, the NMDA receptor, which is involved in memory and learning, is one of the receptors that glutamate activates. It is currently used in treating AD jointly with AChEIs, especially in advanced AD. Agents that block pathologic stimulation of NMDA receptors may also prevent further harm in patients with vascular dementia (VaD), as excessive NMDA stimulation can be generated by ischemia and result in excitotoxicity. The residual neurons' physiological function might also be recovered, improving symptoms [##REF##21482915##47##]. Many clinicians are turning to memantine because of the lack of other established treatments for VaD. Memantine is frequently used in conjunction with AChEIs in people who can afford it. According to the 2020 guidelines for treating LBD in general, AChEIs have the most evidence for usage in cognitive impairment, whereas memantine has mixed evidence [##REF##24828899##39##]. There was no statistically significant difference in the usage of memantine in FTD [##REF##19182475##44##]. Memantine was well-tolerated in some trials in patients with PDD. However, hallucinations and worsening neuropsychiatric symptoms have been recorded using memantine, indicating that it should be used cautiously in PDD [##UREF##11##48##]. </p>", "<p>Antioxidants</p>", "<p>Vitamin E (alpha-tocopherol) and selegiline (a monoamine oxidase inhibitor) have been studied for their potential in treating AD. Research has shown that vitamin E, when administered at a specific dose, may provide a slight reduction in functional decline in patients with mild to moderate AD. However, its impact on cognitive function is not considered significant [##UREF##12##49##,##REF##24381967##50##]. On the other hand, medications targeting serotonergic pathways, such as selective serotonin reuptake inhibitors (SSRIs) and trazodone, as well as atypical antipsychotic treatments, have been found to effectively manage specific behavioral symptoms associated with AD. However, it is important to note that these medications do not improve overall cognitive function. In the case of frontotemporal dementia, SSRIs like sertraline and fluvoxamine have shown positive effects on impulsivity, eating disorders, and anxiety based on case reports and short observational studies [##REF##25238733##51##].</p>", "<p>Most Recent Drugs: Lecanemab and Aducanumab</p>", "<p>Recent advancements in the field of dementia treatment emphasize the significance of focusing on the underlying pathological mechanisms of the disease, like amyloid-beta plaques. By directly addressing these crucial pathophysiological characteristics of AD, both lecanemab and aducanumab have the potential to provide therapeutic benefits by slowing cognitive decline and enhancing measures of cognition and function in affected individuals.</p>", "<p>The FDA has recently approved lecanemab, an anti-amyloid monoclonal antibody, for treating mild cognitive impairment (MCI) and mild dementia caused by AD [##REF##37357276##52##]. This approval represents a significant breakthrough in dementia treatment. Lecanemab specifically targets the underlying causes of AD by binding to amyloid-beta plaques, which are one of the main pathological characteristics of the disease [##UREF##13##53##,##REF##36775284##54##].</p>", "<p>A notable study conducted by van Dyck et al. demonstrated the effectiveness of lecanemab in reducing amyloid markers in individuals with early-stage AD. The study showed that treatment with lecanemab resulted in a slower deterioration in measures of cognition and function over 18 months compared to a placebo [##REF##36449413##55##]. However, it is important to note that adverse events were a major concern following treatment with lecanemab, highlighting the need for careful monitoring and management of potential side effects [##REF##36449413##55##].</p>", "<p>Another study by McDade et al. further supported the efficacy of lecanemab in reducing brain amyloid and slowing cognitive decline in patients with AD. The treatment regimen involved administering lecanemab at a dosage of 10 mg/kg biweekly [##REF##36544184##56##]. These findings provide additional evidence for the potential benefits of lecanemab in managing the progression of AD.</p>", "<p>Similarly, the efficacy of another anti-amyloid-beta monoclonal antibody, aducanumab, has been extensively documented in the literature. A systematic review conducted by Rahman et al. reported that the use of aducanumab led to a reduction of amyloid-beta plaques and a significant decrease in cognitive decline among AD patients [##REF##36775284##54##]. The approval of aducanumab by the FDA in June 2021 further supports its effectiveness as a treatment option for AD.</p>", "<p>Non-Pharmacological Treatments </p>", "<p>Although there is no single dietary intervention that has definitively shown to effectively prevent cognitive degeneration and dementia, it is important to consider various factors that can contribute to maintaining brain health and reducing the risk of AD. Along with maintaining a healthy and balanced diet, regular physical activity, sufficient sleep, and stress management are all important lifestyle factors that have been associated with a decreased risk of cognitive decline [##REF##31374567##57##,##REF##31333085##58##].</p>", "<p>Mediterranean Diet (MedDiet) and Dietary Approaches to Stop Hypertension (DASH):</p>", "<p>In prospective observational studies and trials, the MedDiet has been consistently seen to provide numerous benefits in preventing various non-communicable diseases. This includes cognitive decline and dementia, which are major concerns in aging populations. The MedDiet is characterized by a high consumption of plant-based foods such as fruits, vegetables, whole grains, legumes, nuts, and olive oil. It also involves moderate intake of fish and poultry, and limited consumption of red meat and processed foods. The diet is rich in nutrients such as antioxidants, omega-3 fatty acids, fiber, and vitamins, which have been associated with cognitive health [##REF##28488692##59##,##REF##34204683##60##].</p>", "<p>Several studies have shown that individuals who closely follow the DASH and MedDiet, while also consuming more whole grains, nuts, and legumes, tend to achieve higher scores on the Mini-Mental State Examination (MMSE), a widely used cognitive assessment tool [##REF##28488692##59##, ####REF##34204683##60##, ##REF##24047922##61##, ##REF##28108204##62####28108204##62##]. The MMSE evaluates various cognitive domains such as orientation, memory, attention, and language, with higher scores indicating better cognitive function. These findings suggest a strong association between a healthier dietary pattern and improved cognitive function. Furthermore, a systematic review conducted by Lourida et al. provides additional support for the positive impact of the MedDiet on cognitive health. The review analyzed multiple studies and concluded that adherence to the MedDiet is linked to a slower rate of cognitive decline and a reduced risk of AD [##REF##23680940##63##].</p>", "<p>The potential mechanisms underlying the beneficial effects of the MedDiet on cognitive health are multifactorial. The high intake of fruits, vegetables, and whole grains provides a rich source of antioxidants. These antioxidants help combat oxidative stress and inflammation, which are processes believed to contribute to cognitive decline. The MedDiet is also rich in omega-3 fatty acids, mainly from fish consumption, which have been associated with improved cognitive function and a lower risk of dementia. Additionally, the MedDiet promotes cardiovascular health by reducing the risk of hypertension, diabetes, and obesity. These conditions are known to increase the risk of cognitive impairment [##REF##24047922##61##, ####REF##28108204##62##, ##REF##23680940##63####23680940##63##].</p>", "<p>Ketogenic Diet</p>", "<p>The ketogenic diet has been proposed as a potentially neuroprotective approach against cognitive decline. This diet is characterized by a low carbohydrate composition, moderate protein consumption, and high-fat consumption. In recent years, it has gained attention for its potential in preventing cognitive decline associated with aging. However, it is important to note that there is currently insufficient clinical trial data to draw definitive conclusions about the effectiveness of the ketogenic diet in preventing and treating cognitive decline and AD [##REF##28877457##64##,##REF##28877458##65##]. A systematic review conducted by Devranis et al. included seven studies on the ketogenic diet and reported that it may have the potential to reduce cognitive decline [##REF##36676122##66##]. However, it is crucial to consider the limitations of the existing research. The number of studies available is relatively small, and the quality and design of the studies vary.</p>", "<p>The ketogenic diet is believed to have neuroprotective effects due to several mechanisms. Firstly, the diet's low carbohydrate composition induces ketosis, a state where the body produces ketone bodies as an alternative fuel source for the brain. Ketone bodies, like beta-hydroxybutyrate, are believed to possess neuroprotective properties and offer an alternative energy source for brain cells, potentially enhancing their function [##REF##28877458##65##].</p>", "<p>Additionally, the ketogenic diet has been shown to reduce inflammation and oxidative stress, which are believed to play a role in the development and progression of neurodegenerative diseases. By limiting the intake of carbohydrates and promoting the consumption of healthy fats, the diet may help reduce the production of reactive oxygen species, which can damage brain cells and contribute to cognitive decline [##REF##28877458##65##].</p>", "<p>Furthermore, the ketogenic diet has been found to modulate various signaling pathways and gene expressions that are involved in neuronal health and function. For example, it has been shown to activate pathways that promote the synthesis of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), which supports the growth and survival of neurons [##REF##36676122##66##]. The diet may also enhance mitochondrial function and increase the production of adenosine triphosphate (ATP), the primary energy currency of cells, which is essential for proper brain function [##REF##28877457##64##].</p>", "<p>Physical Activity</p>", "<p>According to recent research, increasing physical activity levels has been found to have a preventive effect on approximately 3% of all dementia cases [##REF##28735855##67##,##REF##32563753##68##]. Additionally, engaging in physical activity and exercise has been shown to improve overall cognitive function in individuals with dementia [##REF##32085951##69##]. This positive impact is likely attributed to multiple underlying processes. Firstly, physical activity and exercise help in managing cardiovascular risk factors that are associated with impaired cognitive performance. By promoting cardiovascular health, physical activity contributes to maintaining optimal brain function [##REF##23243502##70##]. Furthermore, animal studies have demonstrated that physical activity and exercise can stimulate neurogenesis, the generation of new neurons, and synaptic plasticity, the ability of neurons to form connections with one another. These processes are crucial for maintaining brain health and cognitive function [##REF##23243502##70##,##REF##32104516##71##].</p>", "<p>Sleep Patterns</p>", "<p>Sleep disturbance is not only a symptom but also a risk factor for neurocognitive conditions, such as dementia [##REF##32050587##72##]. Adequate sleep is now recognized as crucial for memory consolidation and the removal of excess beta-amyloid and hyperphosphorylated tau, which are characteristic biomarkers of AD. Sleep difficulties often precede the onset of AD pathology and cognitive decline [##REF##32738506##73##]. Non-pharmacological sleep therapies have shown promise in improving sleep quality and may positively impact cognitive function in individuals with dementia. Multidomain approaches that address various aspects of sleep hygiene, including the sleep environment, bedtime routines, and relaxation techniques, have shown particular effectiveness [##REF##32738506##73##,##REF##33871799##74##]. However, it is important to note that the existing research on non-pharmacological sleep therapies for dementia is heterogeneous and limited.</p>", "<p>Challenges of caring for individuals with dementia</p>", "<p>There are multiple challenges in caring for patients with dementia. Behavioral changes such as agitation, aggression, delusions, and hallucinations make it difficult to provide care for patients with dementia. Agitation and aggression are caused by neuroleptic overdose, internal medical conditions, or pain. Cognitive deficit occurring in dementia patients makes it hard for them to express pain and in turn, it manifests as a state of agitation [##REF##21572163##75##]. Sleep disturbance with increased nighttime wakefulness and reduction in the total amount of sleep time is associated with cognitive decline [##REF##24899757##76##]. Another issue is safety, with later stages of dementia patients wandering and becoming lost is one problem that faces caregivers. [##UREF##14##77##] One more thing to consider is eating problems, which are common among demented patients and require nutritional support [##REF##27756815##78##].</p>", "<p>It is important to face the different challenges facing dementia patients' caregivers with a more holistic point of view taken towards dementia with an interdisciplinary team approach. Interventions should be individualized according to the needs of the patients. The effectiveness of psychosocial interventions tailored to the patient's needs for the management of neuropsychiatric symptoms has been demonstrated [##REF##32738937##79##]. An essential aspect of effective management involves rehabilitation, which entails a collaborative effort with healthcare professionals to adopt a patient-centered approach that also incorporates the involvement of caregivers [##REF##37723442##80##]. According to the WHO, rehabilitation is important to meet the needs of the affected people. Older adults and their caregivers have the potential to live an active and social life with interdisciplinary rehabilitation [##REF##37723442##80##]. </p>", "<p>There is a plethora of challenges that caregivers of patients with dementia face. One example is that caregivers constantly need to be alert and involved in the medical treatment of dementia patients due to their cognitive impairment [##REF##31323707##81##]. Care recipients may not value the assistance provided by their caregivers and sometimes even refrain from their medical treatment [##REF##24339089##82##]. Several studies reported that patients sometimes believed that their given medications were poisonous and that they became doubtful and paranoid when given their treatment [##REF##24339089##82##,##UREF##15##83##]. Another challenge is the behavioral changes of elderly patients with dementia that sometimes lead them to refuse care. Some patients lose insight regarding their condition and need for food or medications. As a result, it has been reported in a study that nutritional care may be complicated for patients with dementia as they can accumulate food or become hostile at mealtime [##UREF##16##84##]. Also, caregivers for dementia patients deal with complex treatment regimens. A study reported that care recipients often had around nine comorbidities and received more than 10 medications [##UREF##17##85##]. Lastly, data shows that caregivers did not receive enough information and teaching regarding their medical and nursing tasks. Several studies also showed that the lack of education and training for caregivers was attributed to the limited time available with the healthcare providers [##REF##24339089##82##,##REF##25351043##86##]. </p>", "<p>The treatment approach to people with dementia is complicated as they present with symptoms in multiple domains. These include impaired cognition, neuropsychiatric symptoms, daily activities, and often other medical and comorbidities. Interventions to treat dementia patients must consider their cognitive, physical, emotional, and psychosocial needs [##REF##28735855##67##]. Thus, elderly people with dementia require rehabilitation involving several healthcare professionals to improve their independence to perform their daily activities. One study demonstrated that patients who received interdisciplinary care had stable and slightly improved cognitive status and performance of activities of daily living [##REF##29213915##87##].</p>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Rehab Hafiz, Albatool Ali, Lama Alajlani</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Rehab Hafiz</p><p><bold>Supervision:</bold>  Rehab Hafiz</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Hassan Aljurfi, Albatool Ali, Lama Alajlani, Maria Y. Ashqan, Ghadah A. Algarni , Omar Abdullah M. Alammar, Alanoud Alkhashan</p><p><bold>Drafting of the manuscript:</bold>  Hassan Aljurfi, Albatool Ali, Lama Alajlani, Maria Y. Ashqan, Ghadah A. Algarni , Omar Abdullah M. Alammar, Alanoud Alkhashan</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
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[{"label": ["1"], "article-title": ["Impact of palliative care for dementia patients in tertiary hospitals among Saudi Arabia: a systemic review"], "source": ["Arch Pharm Pract"], "person-group": ["\n"], "surname": ["Al Mani", "Sallam", "Aldosary", "AlGhamdi", "Ghulam", "Shamah"], "given-names": ["TY", "AM", "RA", "JA", "BMI", "WF"], "fpage": ["135"], "volume": ["13"], "year": ["2022"], "uri": ["https://doi.org/10.51847/2GfZKuZB1M"]}, {"label": ["2"], "article-title": ["Major neurocognitive disorder (dementia)"], "source": ["StatPearls [Internet]"], "person-group": ["\n"], "surname": ["Emmady", "Schoo", "Tadi"], "given-names": ["PD", "C", "P"], "publisher-loc": ["Treasure Island (FL)"], "publisher-name": ["StatPearls Publishing"], "year": ["2022"], "uri": ["https://www.ncbi.nlm.nih.gov/books/NBK557444/"]}, {"label": ["4"], "article-title": ["Prevalence of systolic heart failure in patients with dementia in Saudi Arabia: single-center retrospective data review"], "source": ["Middle East J Fam Med"], "person-group": ["\n"], "surname": ["Basheikh", "AlQayidi", "Addas", "Al-Zanbaqi", "Abumelha", "Basheikh"], "given-names": ["M", "A", "M", "M", "A", "M"], "fpage": ["6"], "lpage": ["10"], "volume": ["7"], "year": ["2021"]}, {"label": ["6"], "article-title": ["An assessment of dementia knowledge and its associated factors among health college students in Saudi Arabia"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Aldharman", "Alayed", "Aljohani"], "given-names": ["SS", "FT", "BS"], "fpage": ["0"], "volume": ["15"], "year": ["2023"]}, {"label": ["7"], "article-title": ["Dementia"], "date-in-citation": ["\n"], "month": ["11"], "year": ["2023", "2023"], "uri": ["https://www.who.int/news-room/fact-sheets/detail/dementia"]}, {"label": ["9"], "article-title": ["Prevalence of dementia and its associated risk factors among geriatric patients visiting primary healthcare centers in Riyadh, Saudi Arabia: a cross-sectional study"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Alsebayel", "Alangari", "Almubarak", "Alhamwy"], "given-names": ["FM", "AM", "FH", "R"], "fpage": ["0"], "volume": ["14"], "year": ["2022"]}, {"label": ["27"], "article-title": ["A review of dementia screening tools based on mobile application"], "source": ["Health and Technol"], "person-group": ["\n"], "surname": ["Thabtah", "Peebles", "Retzler", "Hathurusingha"], "given-names": ["F", "D", "J", "C"], "fpage": ["1011"], "lpage": ["1022"], "volume": ["10"], "year": ["2020"], "uri": ["https://doi.org/10.1007/s12553-020-00426-5"]}, {"label": ["30"], "article-title": ["Diagnostic accuracy of tablet-based software for the detection of concussion"], "source": ["PLoS One"], "person-group": ["\n"], "surname": ["Yang", "Flores", "Magal"], "given-names": ["S", "B", "R"], "fpage": ["0"], "volume": ["12"], "year": ["2017"], "uri": ["https://doi.org/10.1371/journal.pone.0179352"]}, {"label": ["36"], "article-title": ["Overlap between pathology of Alzheimer disease and vascular dementia"], "source": ["Alzheimer Dis Assoc Disord"], "person-group": ["\n"], "surname": ["Kalaria", "Ballard"], "given-names": ["RN", "C"], "fpage": ["0"], "lpage": ["23"], "volume": ["13 Suppl 3"], "year": ["1999"]}, {"label": ["38"], "article-title": ["Cholinesterase inhibitors for dementia with Lewy bodies, Parkinson's disease dementia and cognitive impairment in Parkinson's disease"], "source": ["Cochrane Database Syst Rev"], "person-group": ["\n"], "surname": ["Rolinski", "Fox", "Maidment", "McShane"], "given-names": ["M", "C", "I", "R"], "fpage": ["0"], "volume": ["2012"], "year": ["2012"]}, {"label": ["40"], "article-title": ["Pharmacological management of Lewy body dementia: a systematic review and meta-analysis"], "source": ["Essential Reviews in Geriatric Psychiatry"], "person-group": ["\n"], "surname": ["Lane", "Conroy"], "given-names": ["CE", "ML"], "fpage": ["343"], "lpage": ["347"], "publisher-loc": ["Cham, Switzerland"], "publisher-name": ["Springer"], "year": ["2022"], "uri": ["https://link.springer.com/chapter/10.1007/978-3-030-94960-0_60"]}, {"label": ["48"], "article-title": ["Treatment effect of memantine on survival in dementia with Lewy bodies and Parkinson's disease with dementia: a prospective study"], "source": ["BMJ Open"], "person-group": ["\n"], "surname": ["Stubendorff", "Larsson", "Ballard", "Minthon", "Aarsland", "Londos"], "given-names": ["K", "V", "C", "L", "D", "E"], "fpage": ["0"], "volume": ["4"], "year": ["2014"]}, {"label": ["49"], "article-title": ["Vitamin E for Alzheimer's dementia and mild cognitive impairment"], "source": ["Cochrane Database Syst Rev"], "person-group": ["\n"], "surname": ["Farina", "Llewellyn", "Isaac", "Tabet"], "given-names": ["N", "D", "MG", "N"], "fpage": ["0"], "volume": ["1"], "year": ["2017"]}, {"label": ["53"], "article-title": ["Aducanumab"], "source": ["StatPearls [Internet]"], "person-group": ["\n"], "surname": ["Padda", "Parmar"], "given-names": ["IS", "M"], "publisher-loc": ["Treasure Island (FL)"], "publisher-name": ["StatPearls Publishing"], "year": ["2023"], "uri": ["https://www.ncbi.nlm.nih.gov/books/NBK573062/"]}, {"label": ["77"], "article-title": ["People with dementia getting lost in Australia: dementia-related missing person reports in the media"], "source": ["Australas J Ageing"], "person-group": ["\n"], "surname": ["MacAndrew", "Schnitker", "Shepherd", "Beattie"], "given-names": ["M", "L", "N", "E"], "fpage": ["0"], "volume": ["37"], "year": ["2018"]}, {"label": ["83"], "article-title": ["The process of medication management for older adults with dementia"], "source": ["J Nurs Healthc Res"], "person-group": ["\n"], "surname": ["Kaasalainen", "Dolovich", "Papaioannou"], "given-names": ["S", "L", "A"], "fpage": ["407"], "lpage": ["418"], "volume": ["3"], "year": ["2011"]}, {"label": ["84"], "article-title": ["Experiences and nutrition support strategies in dementia care: Lessons from family carers"], "source": ["Nutr Diet"], "person-group": ["\n"], "surname": ["Ball", "Jansen", "Desbrow", "Morgan", "Moyle", "Hughes"], "given-names": ["L", "S", "B", "K", "W", "R"], "fpage": ["22"], "lpage": ["29"], "volume": ["72"], "year": ["2015"]}, {"label": ["85"], "article-title": ["Characterizing caregiver-mediated medication management in patients with memory loss"], "source": ["J Gerontol Nurs"], "person-group": ["\n"], "surname": ["Erlen", "Lingler", "Sereika", "Tamres", "Happ", "Tang"], "given-names": ["JA", "J", "SM", "LK", "MB", "F"], "fpage": ["30"], "lpage": ["39"], "volume": ["39"], "year": ["2013"]}]
{ "acronym": [], "definition": [] }
87
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50522
oa_package/bb/15/PMC10787596.tar.gz
PMC10787602
0
[ "<title>Introduction</title>", "<p>The health benefits of an appropriate level of physical activity (PA) are evident and indisputable (##REF##28708630##1##, ##UREF##0##2##). Recent trends in assessing physical activity involve the development and implementation of national guidelines for physical activity across all age groups, routine surveillance and monitoring of physical activity, policy implementation, and the use of tools such as the Global Physical Activity Questionnaire (GPAQ) and accelerometers for nationwide population monitoring of physical activity in both adults and children (##UREF##1##3##, ##REF##25879680##4##). Studies have also investigated temporal patterns in physical activity levels among different age groups (##REF##18091006##5##, ##REF##33902618##6##), advancements and emerging directions in physical activity surveillance (##REF##34465651##7##), and recent tendencies in adherence to physical activity guidelines (##REF##31348499##8–10##). Moreover, research has examined the close association between physical activity and the prevalence of non-communicable diseases (##UREF##4##11##). Despite numerous studies highlighting the benefits of physical activity, many adults and children fail to meet the recommended levels of physical activity (##REF##25879680##4##, ##REF##30547824##12##). Nurses possess knowledge about healthy behaviors and recognize the importance of physical activity for maintaining health (##REF##28241948##13##, ##REF##26339200##14##). However, research indicates that compared to other occupational groups, nurses often report poorer health and engage in unhealthy behaviors, such as physical inactivity, unhealthy eating habits, and smoking (##REF##31344083##15–17##). These factors, combined with shift work, heavy workloads, and high levels of stress inherent in nursing, contribute to an increased risk of developing various diseases within this profession and a decline in the quality of patient care (##REF##31548933##17–19##).</p>", "<p>The demanding work environment of nurses requires not only comprehensive preparation but also mental resilience and physical fitness (##REF##32901747##20##, ##REF##23527481##21##). Nursing tasks must be performed efficiently and accurately, thus placing high demands on the physical fitness of nurses as it directly impacts the provision of proper patient care (##REF##23527481##21–24##). Nurses experience a higher number of stressful situations compared to other professions, including other healthcare sector employees (##REF##35734805##25##, ##REF##33538823##26##). Regular physical activity improves general physical well-being and is a strategy to reduce work-related tension, stress and protect against professional burnout (##REF##26709860##27–29##).</p>", "<p>Nurses constitute the largest occupational group within the healthcare sector (##UREF##8##30##) and conducting research on this topic offers an excellent opportunity to emphasize the significance of physical activity not only for their personal health but also for the effective functioning of healthcare systems. The objective of this study was to assess physical activity in relation to factors predisposing to the occurrence of specific metabolic disorders.</p>" ]
[ "<title>Materials and methods</title>", "<p>The study was conducted in 2022 among 126 professionally active nurses working at the clinical hospital in the Subcarpathian region.</p>", "<title>Procedures</title>", "<p>The project was approved by the hospital manager and head nurse. In collaboration with hospital authorities, information regarding the dates, hours, and extent of the planned examinations was provided to all nurses working in individual hospital wards. Nurses who wished to participate in the study signed up on the prepared list and selected a convenient date. All measurements (including body composition analysis, glucose level, and lipid profile) were conducted in the morning after a minimum fasting period of 8 h.</p>", "<p>Accelerometers were assigned individual codes for each participant, taking into account their age, weight, and height. They were immediately worn by the participants following the measurements. The inclusion criteria consisted of professionally active nurses without any recent symptoms of infection within the past 2 weeks, no known health issues, and a willingness to participate in the project, which included wearing the accelerometer for seven consecutive days.</p>", "<p>Initially, 154 nurses expressed interest in participating in the physical activity measurement using the accelerometer. However, within 2 days, 7 of them reported being unable to continue due to external circumstances. An additional 11 nurses were excluded from the analysis as it was observed during the data review that the device was not worn according to the established criteria. Ultimately, data from 126 accelerometers were included in the statistical analysis.</p>", "<title>Blood pressure</title>", "<p>Blood pressure was measured three times in the sitting position, after a 10-min rest, following the recommendations of the European Society of Hypertension (##REF##30816983##31##). The measurements were taken using a standardized Welch Allyn 4200B apparatus (Aston Abbotts, UK). The average of the three measurements was calculated for each participant.</p>", "<p>The accepted criteria for blood pressure were as follows:</p>", "<p>normal blood pressure ranged from 120–129 mmHg systolic and 80–84 mmHg diastolic;</p>", "<p>normal high pressure ranged from 130–139 mmHg systolic and 85–89 mmHg diastolic;</p>", "<p>grade 1 hypertension ranged from 140–159 mmHg systolic and 90–99 mmHg diastolic;</p>", "<p>grade 2 hypertension ranged from 160–179 mmHg systolic and 100–109 mmHg diastolic;</p>", "<p>grade 3 hypertension was defined as a systolic blood pressure of ≥180 mmHg and/or a diastolic blood pressure of ≥110 mmHg (##REF##30816983##31##).</p>", "<title>Anthropometry</title>", "<p>Body height was measured to an accuracy of 0.1 cm using a Seca 213 stadiometer in a vertical position, with the participant barefoot (Seca, Hamburg, Germany). Bodyweight was assessed in the early morning after at least 8 h of overnight fasting, in light clothing, in an upright position, with an accuracy of 0.1 kg, using food-to-food bioelectric impedance analysis with a Tanita MC-980 PLUS MA (Tanita, Tokyo, Japan) (##UREF##9##32##).</p>", "<p>Body Mass Index (BMI) was calculated as the participant’s body weight in kilograms divided by their height in meters squared, based on accepted standards:</p>", "<p>17–18.49 = underweight,</p>", "<p>18.5–24.99 = normal body weight,</p>", "<p>25–29.99 = overweight,</p>", "<p>30–34.99 = 1st degree obesity.</p>", "<p>35–39.99 = 2nd degree obesity,</p>", "<p>&gt;40 = 3rd degree obesity (##UREF##10##33##).</p>", "<p>Waist and hip girths were measured with steel anthropometric tape in accordance with the International Society of the Advancement of Kinanthropometry (ISAK) guidelines by an ISAK Level 3 – Instructor anthropometrist.</p>", "<p>Waist-to-hip ratio (WHR) was calculated as the participant’s waist circumference divided by their hip circumference. The following ranges were adopted for the value of the WHR index:</p>", "<p>For men:</p>", "<p>&lt;0.96 WHR within normal limits.</p>", "<p>≥0.96 Abdominal obesity, increased risk of metabolic diseases.</p>", "<p>For women:</p>", "<p>&lt;0.83 WHR within normal limits.</p>", "<p>≥0.83 Abdominal obesity, increased risk of metabolic diseases (##REF##16278693##34##).</p>", "<title>Blood biochemical measurements</title>", "<p>Lipid profile and fasting glucose were assessed using the CardioChek PA (CCPA, PTS Diagnostics, Whitestown, USA) analyzer according to the manufacturer’s instructions. A finger prick capillary blood sample was collected by a registered nurse while observing all rules of asepsis and antisepsis. Blood samples were collected in the morning after an 8-h fast. Total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides (TG), and glucose levels were measured. Quality control testing of the device was done prior to data collection with a Multi-Chemistry Controls set. The CCPA device was checked each time before starting by conducting an Internal Quality Control using a control strip recommended by the manufacturer.</p>", "<p>In accordance with the guidelines of the Polish Cardiological Society, the following lipid profile criteria have been adopted:</p>", "<p>TC 150–190 mg/dL;</p>", "<p>LDL cholesterol less than 115 mg/dL;</p>", "<p>HDL cholesterol above 40 mg/dL for men and over 48 mg/dL for women;</p>", "<p>TG below 150 mg/dL.</p>", "<p>Lipid disorders criteria:</p>", "<p>Hypercholesterolemia was diagnosed when TC was ≥190 mg/dL or LDL &gt; 115 mg/dL;</p>", "<p>Atherogenic dyslipidaemia was determined when TG was ≥150 mg/dL, HDL &lt; 40 mg/dL in males and &lt; 48 mg/dL in females, and elevated LDL fraction (&gt;130 mg/dL);</p>", "<p>Hypertriglyceridemia was determined when TG was &gt;150 mg/dL with normal LDL levels (&lt;115 mg/dL) and severe hypertriglyceridemia – TG ≥ 800 mg/dL;</p>", "<p>Dyslipidaemia was defined as the presence of abnormal concentrations in any component of the lipid profile (##UREF##11##35–37##).</p>", "<p>Fasting glucose criteria:</p>", "<p>less than 70 mg/dL – hypoglycemia;</p>", "<p>70 to 99 mg/dL – normal glucose level;</p>", "<p>100 to 125 mg/dL – elevated glucose levels – pre-diabetes;</p>", "<p>≥ 126 mg/dL at least two measurements – diabetes mellitus (##UREF##11##35–37##).</p>", "<title>Physical activity</title>", "<p>Physical activity parameters were measured using the ActiGraph GT3X-BT triaxial accelerometer (ActiGraph, Pensacola, Florida, USA). Accelerometers have been widely used to assess physical activity and are considered a valid and reliable measure of physical activity in many populations, including adults (##REF##33239356##38##, ##REF##22157770##39##).</p>", "<p>The accelerometer was worn on the subject’s right hip. Subjects were instructed to wear the monitor for seven consecutive days, 24 h a day, and remove it only for water-related activities (e.g., showering or swimming). The sleep-period time was separated from the 24-h activity with the Sadeh algorithm (##REF##7939118##40##). Non-wear time was defined as a period of at least 60 consecutive minutes with zero counts per minute (cpm). The epoch duration was set to 60 s. A minimum wear time of ≥600 min per day was considered a valid day for the analysis. At least 4 days (including at least three valid weekdays and one valid weekend day) were used as the criteria for a valid seven-day period of accumulated data.</p>", "<p>After the recording period ended, the accelerometers were connected to a computer via a mini-USB for data transfer. Data were analyzed using ActiLife software (ActiGraph, software v.6.13, Pensacola, FL, USA). For each participant, time (minutes per week and minutes per day) spent in sedentary, light, moderate, vigorous, moderate-to-vigorous physical activity (MVPA), and the average daily and weekly step counts were calculated. The cut-off points for sedentary (0–99 cpm), light physical activity (100–1,951 cpm), moderate (1,952–5,724 cpm), vigorous (≥5,725 cpm), and MVPA (≥1,952 cpm from all valid days) were applied (##REF##9588623##41##).</p>", "<p>In the present study, the World Health Organization’s (WHO) global recommendations on physical activity for adults were adopted. The cut-off points for meeting the guidelines were set up as 150 min of moderate-intensity aerobic physical activity throughout the week or at least 75 min of vigorous-intensity aerobic physical activity throughout the week or an equivalent combination of moderate- and vigorous-intensity activity (##UREF##1##3##).</p>", "<title>Questionnaire</title>", "<p>The questionnaire developed for this study was prepared in a paper format, which included an envelope for sealing the completed questionnaires to ensure the confidentiality of the responses. The survey consisted of questions regarding the participants’ sociodemographic data (such as age, sex, place of residence, and level of education), work-related questions (such as type of work, work schedule, number of full-time jobs, and years of experience), participation in preventive examinations, smoking habits, and the prevalence of chronic diseases among the surveyed nurses (##SUPPL##0##Appendix 1## – questionnaire template).</p>", "<title>Applied statistical methods</title>", "<p>The analysis was performed using the R program, version 4.2.2 (##UREF##14##42##). The analysis of quantitative variables was performed by calculating the mean, standard deviation, median, and quartiles. The analysis of qualitative variables was performed by calculating the number and percentage of occurrences of each value.</p>", "<p>Multivariate analysis was conducted to assess the influence of multiple variables on the occurrence of individual metabolic disorders. This analysis employed the logistic regression method, which was adjusted to age, shift work, working more than one full-time job, smoking, and chronic diseases.</p>", "<p>The results are presented as Adjusted Odds Ratio (AOR) parameter values along with 95% confidence intervals.</p>", "<p>In this analysis, a significance level of 0.05 was adopted. Therefore, any <italic>p</italic>-values below 0.05 were considered indicative of significant relationships.</p>" ]
[ "<title>Results</title>", "<title>Socio-demographic characteristics of the study group</title>", "<p>Totally, 126 nurses (women) were included to final analysis. The average age of the respondents was approximately 46.5 years (SD ± 9.95), median (quartiles), 50 (39–54), range 23–65. Detailed characteristics of the study group are presented in ##TAB##0##Table 1##.</p>", "<title>Quantity and quality of physical activity undertaken by the surveyed nurses</title>", "<p>Descriptive statistics of accelerometer data, amount, and type of physical activity per day and week are shown in ##TAB##1##Table 2##.</p>", "<p>The results showed the amount and type of physical activity undertaken by nurses, as well as the number of steps taken in relation to the recommended standards (##TAB##2##Table 3##).</p>", "<title>Activity variables influencing the occurrence of metabolic disorders</title>", "<p>Multivariate logistic regression analysis, adjusted to age, shift work and night duty, more than one job, cigarettes smoking and chronic diseases, showed no significant relationships (<italic>p</italic> &gt; 0.05) between physical activity per week and occurrence selected metabolic disorders (##TAB##3##Table 4##).</p>", "<p>Multivariate logistic regression analysis, adjusted to age, shift work and night duty, more than one job, cigarettes smoking and chronic diseases, showed that an inactive lifestyle (7,499–5,000 steps per day) increases the risk of lipid disorders on average 5.712 times (AOR = 5.712) compared to a very active lifestyle (≥10,000) (##TAB##4##Table 5##).</p>", "<p>Multivariate logistic regression analysis, adjusted to age, shift work and night duty, more than one job, cigarettes smoking and chronic diseases, showed no significant relationships (<italic>p</italic> &gt; 0.05) between sedentary (h/day) and occurrence selected metabolic disorders (##TAB##5##Table 6##).</p>" ]
[ "<title>Discussion</title>", "<p>The results demonstrated the impact of objectively measured physical activity, number of steps, sitting time, age, work pattern, and additional employment on the prevalence of obesity, including overweight, abdominal obesity, lipid disorders, elevated blood pressure, and elevated blood glucose levels. Increasing evidence suggests the importance of physical activity for better health and the prevention of non-communicable diseases (##UREF##15##43–47##). This study is one of the first to objectively assess physical activity among nurses in Poland. The aim was to evaluate the physical activity of nurses in relation to factors that predispose them to metabolic disorders. This cross-sectional study found that almost one third (31.75%) of the nurses studied did not meet the criteria for a minimum amount of physical activity of at least moderate intensity. More than half of the nurses surveyed (55.56%) were overweight or obese according to BMI and almost half (42.86%) had abdominal obesity.</p>", "<p>Overweight and obesity have been a recognized health problem for many years, affecting not only Poland but also other countries such as Great Britain, Australia, the United States, and New Zealand (##REF##11978348##48–52##). Other studies have also shown that the prevalence of overweight and obesity among nurses is more frequent than among the general population and other employees in the health sector (##UREF##18##50–53##). For example, Kayaroganam et al. found that one-fifth of the nurses surveyed were overweight, more than a third had abdominal obesity, and more than half were obese, including women over 40 (##UREF##19##54##).</p>", "<p>In our study, lipid disorders were present in 46.83% of the patients, and 38.89% of the nurses had elevated blood pressure. The specificities of the nurses’ work, such as irregular lifestyles, unhealthy eating habits, shift work, and disturbances of the natural rhythm of sleep and rest, may predispose them to many disorders, such as dyslipidemia, hypertension, and elevated glucose levels. Consequently, these factors may lead to the development of cardiovascular diseases, which is a global problem that accounts for the majority of deaths, especially in developed countries. The results of the study conducted by the Cardiovascular Nurses Associates indicate the prevalence of the above relationships among nurses (##UREF##20##55##). Researchers from South India have also shown that more than one-third of nurses had hypercholesterolemia (34.3%) and elevated LDL (41.9%), and two-thirds had low HDL (65.3%) (##UREF##19##54##). Hypertension is another global problem that affects more and more people and leads to morbidity and premature death (##REF##34450083##56##). In the Manakali study, 52% of nurses had hypertension, which is significantly higher than the reported prevalence of hypertension among nurses in South Africa (20%), Brazil (32%), and healthcare professionals in Nigeria (20.1%). This result is also higher than the documented prevalence of hypertension in the general adult population of South Africa (##REF##30551716##57##, ##REF##25363054##58##). Based on the findings of our study, 36.51% participants had elevated glucose levels, which is higher than in the Kayarogana study (11.5%) (##UREF##19##54##). However, a study conducted by Miller et al. on health risk factors of nurses showed that 26% of nurses participating in the measurements were unaware of their diabetes (##REF##18460166##59##). Other studies indicate that elevated glucose levels among nurses may also result from stress experienced during their daily work (##REF##26699764##60##, ##REF##23444388##61##).</p>", "<p>The results in relation to physical activity showed that the nurses participated mainly in light and moderate intensity physical activity and to a lesser extent in vigorous activities. When analyzing the results of the entire study group, the average MVPA was 232 min per week, indicating that the criteria for the recommended amount of physical activity for an adult per week have been met. However, based on the results, 31.75% of nurses do not meet the minimum requirements for a minimum amount of physical activity of at least moderate intensity. The result obtained is higher compared to the data from the Ministry of Sport and Tourism report, which showed that approximately 80% of the Polish society does not engage in moderate or intense physical activity, and about 23% declare moderate activity, mainly related to the need to move (##UREF##21##62##). However, considering the nature of the nursing profession and the need to constantly move while caring for patients (##REF##31427148##63##), the result obtained cannot be considered fully satisfactory. Ottawa Canadian researchers conducted an objective evaluation of physical activity among 364 nurses using accelerometers. Their results showed that only a few nurses (23%) met the WHO physical activity guidelines (≥150 min/week in bouts of ≥10 min) (##REF##29499395##64##). According to a systematic review by Chappel et al. nurses are mostly engaged in light-intensity physical activity (##UREF##22##65##). Other researchers have presented similar results in this regard. According to a survey conducted by Tucker et al. among 3,132 nurses, only 50% of them reported that they met the guidelines for physical activity (##REF##20677722##66##). In a cross-sectional study of 325 British nurses by Blake, Malik, Mo and Pisano, they found that less than half (45.98%) met the government guidelines for physical activity (##REF##21999028##67##).</p>", "<p>In our study, nurses accumulated an average of 232 min per week of moderate to vigorous intensity physical activity. On the other hand, Canadian nurses achieved higher results in this respect, with an average of 288 min per week (##REF##29499395##64##). The number of steps per day is a simple measure of physical activity, which is now easily monitored thanks to various electronic devices that are increasingly available and popular (##UREF##15##43##, ##REF##33239350##44##). Our step count results showed that the average number of steps per day was 9,732. This result is in line with the majority of recommendations on the optimal number of steps per day for good health (##UREF##15##43##). These findings suggest that the nurses who participated in our study are more active in this regard than the general population of Poland, where the average person takes about 5,000 steps per day (##REF##28693034##68##). Data indicate that the highest number of steps per day are taken by people in Switzerland (10,400 for men and 8,900 for women), followed by Belgians (9,500), Japanese (7,200) and then Americans (6,500) (##REF##21798015##69##). When assessing the number of steps per day among nurses, many researchers focus on measuring the number of steps during a 12 and / or 8-h shift (##REF##31427148##63##, ##REF##36209110##70##, ##UREF##23##71##). Croteau et al. showed that nurses take more steps on working days (10,398) compared to non-working days (7,036) (##REF##27758937##72##). There are several recommendations in this regard (##UREF##24##73##), but the goal of achieving 10,000 steps per day for healthy individuals has been widely promoted and advocated by the WHO and various physical activity campaigns (##UREF##15##43##, ##REF##31141585##74##).</p>", "<p>The multivariate logistic regression analysis, adjusted to age, shift work and night duty, more than one job, cigarettes smoking and chronic diseases, showed that an inactive lifestyle (7,499–5,000 steps per day) increases the risk of lipid disorders on average 5.712 times (AOR = 5.712) compared to a very active lifestyle (≥10,000). No significant relationships between physical activity per week, between sedentary (h/day) and occurrence selected metabolic disorders (<italic>p</italic> &gt; 0.05). A meta-analysis of 153,030 nurses from 35 studies demonstrated that shift work, age, night shift, sex, marriage, health status, number of hours worked per week and stress level are positively correlated with overweight and obesity among nurses (##UREF##25##75##). Researchers from Ghana also found that age, sex, and marital status influence the level of obesity and overweight among nurses, with older nurses being more likely to be obese than younger ones (##REF##36209110##70##, ##UREF##26##76##). In Poland, the majority of nurses currently working are 46–60 years old (##UREF##27##77##), which underscores the importance of paying attention to improving the health condition of nurses in the context of an increased risk of developing metabolic disorders and even mortality (##REF##34547483##78##). A meta-analysis conducted by Sheng et al. shows that taking 9,500 steps per day reduces the risk of cardiovascular events by approximately 35% compared to 3,500 steps per day (##REF##34547483##78##). The authors of another meta-analysis showed that among people over 60 years of age, taking 6–9 thousand steps per day reduces cardiovascular risk by up to 40–50% compared to taking approximately 2000 steps per day (##UREF##28##79##). According to Sheng, taking 8,959 steps a day reduces the risk of death by 40.36% compared to taking 4,183 steps a day (##REF##34547483##78##). Garduno et al. showed that every additional 2000 steps per day reduces the risk of diabetes by 12% (##REF##35050362##80##). The benefits of taking the recommended number of steps include weight loss and all the consequences of being overweight and obesity. Additionally, physical activity has a positive effect on stress reduction, which is often present in the work of nurses (##REF##33522994##81##). The results of a meta-analysis published in The Lancet indicate that taking 6–9,500 steps a day is optimal, noting that the more steps taken, the better (##REF##35247352##47##).</p>", "<p>The shortage of nursing staff is a problem currently faced by many countries, including Poland (##UREF##8##30##). The average age of a Polish nurse is currently older than 53 years, meaning that the majority of currently working nurses have reached or will soon reach retirement age. This predisposes them to poor health and absenteeism, which is crucial not only for themselves but also for ensuring high quality healthcare (##UREF##27##77##). Analyses of millions of patient records in the United States and Canada indicate a direct relationship between care satisfaction and the number of adverse events and the health condition of nursing staff (##UREF##29##82##). Nurses are the largest occupational group among health professionals and reach a large proportion of the population, making them an integral part of the health workforce (##UREF##8##30##). According to Fie et al., nurses who have a positive attitude toward physical activity and are physically active are more likely to promote physical activity among their patients compared to inactive nurses (##UREF##30##83##, ##REF##26043015##84##). A very positive solution is the national campaign organized by the American Nurses Association, which supports and promotes pro-health behaviors among nurses (##UREF##31##85##).</p>", "<title>Strengths, limitations, and future research</title>", "<p>To our knowledge, this is the first objective study in Poland that measures physical activity among nurses using certified devices. Most of the research in this area is subjective survey research. The strength of our study is the objective, not the declared assessment of physical activity and the performance of all measurements using standardized high-quality devices. However, several potential study limitations should be considered when interpreting the results. Firstly, since the study is cross-sectional, causality and temporality issues cannot be considered. Second, only women participated in the study, since men make up less than 2% of all nurses, so it was difficult to include them in the study group when randomly selected. Third, the study had a limited geographic scope and should be expanded to more medical facilities in other regions. Fourth, due to limited availability of devices at our disposal (high price), we recruited only some of the nurses. It is possible that those who did not participate in our measurements were more physically active. Future research should include male participation, a larger group of nurses, and consideration of other regions of the country. Our work is a preliminary assessment and provides the basis for implementing multifaceted and supportive activities to improve the general health condition of nurses in Poland by increasing physical activity.</p>", "<p>Future research could include an audit to objectively assess infrastructure and opportunities to increase physical activity.</p>" ]
[ "<title>Conclusion</title>", "<p>The results of this study indicate that the level of physical activity and the presence of factors that contribute to the development of metabolic diseases can have significant implications for the health of nurses, as well as the effectiveness of their work and the quality of patient care. Providing care to those who care for us is necessary, given the requirements imposed by the nurse’s profession and the patient’s right to receive the highest level of care, regardless of age and health condition. Therefore, appropriate interventions should be designed and implemented not only to increase and maintain the level of physical activity of nurses, but also to eliminate barriers that make it difficult to carry out health activities. The activation and support of this professional group is an investment that benefits not only the nurses themselves, but also the health care system and the entire nation.</p>" ]
[ "<p>Edited by: Subhabrata Moitra, University of Alberta, Canada</p>", "<p>Reviewed by: Ruixue Cai, Brigham and Women’s Hospital and Harvard Medical School, United States; Armiya Sultan, Jamia Millia Islamia, India</p>", "<p>Numerous studies demonstrate a relationship between physical activity and the development of non-communicable diseases. Nurses play a crucial role in the healthcare system, and their demanding work can have an impact on their health. The objective of this cross-sectional study was to assess physical activity in relation to factors predisposing to the occurrence of specific metabolic disorders among Polish nurses. The measurements included physical activity level using ActiGraph GT3X, body weight composition using Tanita MC-980, body mass index, waist circumference, blood pressure using Welch Allyn 4200B, lipid profile, and fasting blood glucose using CardioChek PA. The results indicate that nearly one-third (31.75%) of the total sample of studied nurses do not meet the criteria for the minimum amount of physical activity of at least moderate intensity. Furthermore, over half of the surveyed nurses (55.5%) were classified as overweight or obese based on BMI, and almost half (42.86%) had abdominal obesity. The regression model, employing linear regression, revealed that factors predisposing to selected metabolic disorders were age, engaging in multiple jobs, and the number of steps per day. There is a pressing need to implement comprehensive and supportive initiatives to improve the overall health condition of nurses in Poland through increased physical activity. Activating and supporting this professional group is an investment that benefits not only the nurses themselves but also the healthcare system and the entire nation.</p>" ]
[ "<title>Data availability statement</title>", "<p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p>", "<title>Ethics statement</title>", "<p>The study involving humans was approved by the Bioethics Committee of the University of Rzeszów (No. 2022/088 of 05/10/22) and was conducted under the ethical standards stated in the Declaration of Helsinki. The participants provided their written informed consent to participate in this study.</p>", "<title>Author contributions</title>", "<p>AB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review &amp; editing. PM: Data curation, Methodology, Writing – review &amp; editing. JW: Data curation, Investigation, Methodology, Writing – review &amp; editing. EŁ: Data curation, Writing – review &amp; editing. ŁO: Funding acquisition, Writing – review &amp; editing. OA: Funding acquisition, Writing – review &amp; editing. AM-R: Writing – review &amp; editing. AM: Writing – review &amp; editing.</p>" ]
[ "<title>Conflict of interest</title>", "<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>", "<title>Publisher’s note</title>", "<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>", "<title>Supplementary material</title>", "<p>The Supplementary material for this article can be found online at: <ext-link xlink:href=\"https://www.frontiersin.org/articles/10.3389/fpubh.2023.1300662/full#supplementary-material\" ext-link-type=\"uri\">https://www.frontiersin.org/articles/10.3389/fpubh.2023.1300662/full#supplementary-material</ext-link></p>" ]
[]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>General characteristics of the study group.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Variable</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Total (<italic>n</italic> = 126) <italic>n</italic> (%)</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Place of residence*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">City</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">63 (50.0)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Village</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">63 (50.0)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Type of work*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Staff management/administration</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22 (17.5)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Hospital ward</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">104 (82.5)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Work system*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Shift work and night duty (12 h)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">64 (50.8)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">One shift work (8 h)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">62 (49.2)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">More than one job*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">102 (80.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24 (19.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"3\" colspan=\"1\">Education*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Basic nursing education</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20 (15.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Bachelor</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">39 (30.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Master’s degree</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">67 (53.2)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Participation in preventive examinations other than obligatory*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91 (72.2)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">35 (27.8)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Cigarettes smoking*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">110 (87.3)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">16 (12.7)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Chronic diseases*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78 (61.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">48 (38.1)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Anthropometric characteristics</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"5\" colspan=\"1\">BMI* [kg/m<sup>2</sup>]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal body mass</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">56 (44.4)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Overweight</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">40 (31.8)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Class I obesity</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19 (15.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Class II obesity</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9 (7.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Class III obesity</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (1.6)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">WHR*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">72 (57.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Abdominal obesity. Increased risk of metabolic diseases</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">54 (42.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">HDL*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">120 (95.2)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">To low</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6 (4.8)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">TG*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">103 (81.8)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23 (18.2)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">LDL*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">82 (65.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">44 (34.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">TC*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">82 (65.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">44 (34.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">Lipid disorders*</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">67 (53.2)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">59 (46.8)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"4\" colspan=\"1\">BP* [mmHg]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">77 (61.1)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">21 (16.7)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">High blood pressure Stage 1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">26 (20.6)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">High blood pressure Stage 2</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (1.6)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"3\" colspan=\"1\">FG* [mg/dL]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">80 (63.4)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">44 (34.9)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Abnormal glucose – suspicion of diabetes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (1.6)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Parameters of physical activity in the study group (<italic>n</italic> = 126).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Parameter</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SD</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Me</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Min</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Max</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q1</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q3</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sedentary [min per week – sleep]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3976.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1581.1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3802.6</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1282.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13635.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3074.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4373.4</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sedentary [min per day – sleep]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">594.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">134.2</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">574.9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">305.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1050.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">497.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">685.4</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Light activities [min per week]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2657.3</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">805.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2679.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">180.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4456.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2112.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3263.5</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Light activities [min per day]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">397.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">112.3</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">398.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">25.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">674.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">330.1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">479.2</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Moderate activities [min per week]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">225.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">136.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">194.6</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">672</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">127.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">287</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Moderate activities [min per day]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">33.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">28.6</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">101.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19.42</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41.5</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Vigorous activities [min per week]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19.9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">149</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.9</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Vigorous activities [min per day]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.3</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Total MVPA [min per week]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">232</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">141.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">202.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">720</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">127.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">297.2</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Average MVPA [min per day]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31.9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">28.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">102.9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41.6</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Steps counts per week</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">64982.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22658.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">64681.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8,025</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">122,190</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">51232.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">79185.7</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Steps counts per day</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9732.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3282.2</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9773.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1331.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">18,502</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7692.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11758.3</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Level of physical activity and number of steps in relation to the recommendation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" colspan=\"3\" rowspan=\"1\">Parameter</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Total (<italic>n</italic> = 126) n (%)</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"2\" colspan=\"1\">* Physical activity per week (in relation to the recommendation)</td><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">Meet criteria</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">40 (31.7)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">86 (68.3)</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"4\" colspan=\"1\">Steps per day</td><td align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Active lifestyle – meets or exceeds the recommended number of steps per day (≥10,000)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">61 (48.4)</td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Somewhat active lifestyle – meets the recommended number of steps per day (7,500–9,999)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">36 (28.6)</td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Low lifestyle – does not meet the recommended number of steps per day (7,499–5,000)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">21 (16.7)</td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Sedentary lifestyle – does not meet the requirements for the recommended number of steps per day (&lt;5,000)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8 (6.3)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>Physical activity per week and the occurrence of metabolic disorders in the study group.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Physical activity per week</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Overweight</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Obesity</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Abdominal obesity</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipid disorders**</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated blood pressure</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated glucose levels</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Meet criteria</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Do not meet criteria</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.039 (0.472–2.29) <italic>p</italic> = 0.924</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.984 (0.382–2.538) <italic>p</italic> = 0.974</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.58 (0.673–3.707) <italic>p</italic> = 0.293</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.415 (0.638–3.136) <italic>p</italic> = 0.393</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.625 (0.266–1.466) <italic>p</italic> = 0.28</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.632 (0.271–1.471) <italic>p</italic> = 0.287</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab5\"><label>Table 5</label><caption><p>Steps per day and the occurrence of metabolic disorders in the study group.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Steps per day</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Overweight</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Obesity</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Abdominal obesity</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipid disorders**</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated blood pressure</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated glucose levels</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">≥10,000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7,500–9,999</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.833 (0.35–1.985) <italic>p</italic> = 0.68</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.517 (0.542–4.241) <italic>p</italic> = 0.427</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.083 (0.415–2.83) <italic>p</italic> = 0.871</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.534 (0.218–1.306) <italic>p</italic> = 0.169</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.836 (0.326–2.147) <italic>p</italic> = 0.711</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.521 (0.609–3.799) <italic>p</italic> = 0.369</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7,499–5,000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.25 (0.434–3.601) <italic>p</italic> = 0.679</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.177 (0.332–4.176) <italic>p</italic> = 0.801</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 2.251 (0.675–7.502) <italic>p</italic> = 0.187</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 5.712 (1.497–21.798) <italic>p</italic> = 0.011*</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.166 (0.387–3.514) <italic>p</italic> = 0.785</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.43 (0.12–1.538) <italic>p</italic> = 0.194</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">&lt;5,000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 7.296 (0.767–69.433) <italic>p</italic> = 0.084</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.336 (0.03–3.797) <italic>p</italic> = 0.378</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.373 (0.055–2.524) <italic>p</italic> = 0.312</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.601 (0.115–3.146) <italic>p</italic> = 0.547</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0.305 (0.046–2.005) <italic>p</italic> = 0.216</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.622 (0.317–8.298) <italic>p</italic> = 0.561</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab6\"><label>Table 6</label><caption><p>Sedentary and the occurrence of metabolic disorders in the study group.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Sedentary [h/day]</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Overweight</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Obesity</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Abdominal obesity</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipid disorders**</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated blood pressure</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated glucose levels</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[h/day]</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.143 (0.954–1.368) <italic>p</italic> = 0.146</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.007 (0.829–1.223) <italic>p</italic> = 0.942</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 0,892 (0.736–1.081) <italic>p</italic> = 0.243</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.065 (0.894–1.269) <italic>p</italic> = 0.48</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.027 (0.859–1.227) <italic>p</italic> = 0.77</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AOR = 1.007 (0.84–1.207) <italic>p</italic> = 0.941</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"SM1\" position=\"float\" content-type=\"local-data\"></supplementary-material>" ]
[ "<table-wrap-foot><p>* Data presented as: *, n (%); SD, standard deviation; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglycerides; BP, Blood pressure; BMI, Body mass index; WHR, Waist Hip Ratio; FG, Fasting glucose.</p></table-wrap-foot>", "<table-wrap-foot><p>M, arithmetic mean; SD, standard deviation; Me, Median; Min, Minimum; Max, Maximum; Q1, lower quartile; Q3, upper quartile.</p></table-wrap-foot>", "<table-wrap-foot><p>* 150–300 min of moderate-intensity or 75–150 min of vigorous-intensity per week.</p></table-wrap-foot>", "<table-wrap-foot><p>*Statistically significant relationship (<italic>p</italic> &lt; 0.05); AOR (Adjusted Odds Ratio) = odds ratio (OR), adjusted to age, shift work, working more than one full-time job, smoking, and chronic diseases, **lipid disorders – at least one of the parameters is disturbed.</p></table-wrap-foot>", "<table-wrap-foot><p>* Statistically significant relationship (<italic>p</italic> &lt; 0.05); AOR (Adjusted Odds Ratio) = odds ratio (OR), adjusted to age, shift work, working more than one full-time job, smoking, and chronic diseases, ** lipid disorders – at least one of the parameters is disturbed.</p></table-wrap-foot>", "<table-wrap-foot><p>* Statistically significant relationship (<italic>p</italic> &lt; 0.05); AOR (Adjusted Odds Ratio) = odds ratio (OR), adjusted to age, shift work, working more than one full-time job, smoking, and chronic diseases, ** lipid disorders – at least one of the parameters is disturbed.</p></table-wrap-foot>" ]
[]
[ "<media xlink:href=\"Data_Sheet_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["2."], "surname": ["Warburton", "Bredin", "Zibadi", "Watson"], "given-names": ["DER", "SSD", "S", "RR"], "article-title": ["Lost in translation: what does the physical activity and health evidence actually tell us?"], "source": ["Lifestyle in heart health and disease"], "publisher-loc": ["San Diego, CA, USA"], "publisher-name": ["Elsevier"], "year": ["2018"], "fpage": ["175"], "lpage": ["86"]}, {"label": ["3."], "collab": ["World Health Organization"], "year": ["2023"], "ext-link": ["https://www.who.int/publications/i/item/9789240015128"]}, {"label": ["9."], "collab": ["World Health Organization"], "year": ["2010"], "ext-link": ["https://www.who.int/publications/i/item/9789241599979"]}, {"label": ["10."], "collab": ["World Health Organization"], "year": ["2018"], "ext-link": ["https://www.euro.who.int/__data/assets/pdf_file/0005/382334/28fs-physical-activity-euro-rep-eng.pdf"]}, {"label": ["11."], "surname": ["Szychowska", "Zimny-Zaj\u0105c", "Dziankowska-Zaborszczyk", "Grodzicki", "Drygas", "Zdrojewski"], "given-names": ["A", "A", "E", "T", "W", "T"], "article-title": ["Physical activity versus selected health behaviors, subjective physical and psychological health and multimorbidity in a large cohort of Polish seniors during the COVID-19 pandemic (results of the National Test for poles\u2019 health)"], "source": ["Int J Environ Res Public Health"], "year": ["2023"], "volume": ["20"], "fpage": ["556"], "pub-id": ["10.3390/ijerph20010556"]}, {"label": ["18."], "surname": ["Khamisa", "Peltzer", "Ilic", "Oldenburg"], "given-names": ["N", "K", "D", "B"], "article-title": ["Work related stress, burnout, job satisfaction and general health of nurses: a follow-up study"], "source": ["Int J Nursing Practice"], "year": ["2016"], "volume": ["22"], "fpage": ["538"], "pub-id": ["10.1111/ijn.12455"]}, {"label": ["22."], "surname": ["Major"], "given-names": ["D"], "article-title": ["Developing effective nurse leadership skills"], "source": ["Nurs Stand"], "year": ["2019"], "volume": ["34"], "fpage": ["61"], "lpage": ["6"], "pub-id": ["10.7748/ns.2019.e11247"]}, {"label": ["29."], "surname": ["Henwood", "Tuckett", "Bagadi", "Oliffe"], "given-names": ["T", "A", "NE", "J"], "article-title": ["Connecting leisure-time physical activity and quality of sleep to nurse health: data from the e-cohort study of nurses and midwives"], "source": ["J Nurs Care"], "year": ["2015"], "volume": ["4"], "fpage": ["254"], "pub-id": ["10.4172/2167-1168.1000254"]}, {"label": ["30."], "collab": ["World Health Organization"], "source": ["State of world\u2019s nursing 2020: investing in education, jobs and leadership"], "publisher-loc": ["Geneva"], "publisher-name": ["WHO"], "year": ["2020"]}, {"label": ["32."], "collab": ["Tanita"], "ext-link": ["https://tanita.eu/media/wysiwyg/catalogue/tanita_pro_product-guide_april_2017.pdf"]}, {"label": ["33."], "surname": ["Barlow"], "given-names": ["SE"], "collab": ["Expert Committee"], "article-title": ["Expert committee recommendations regarding the prevention, assessment, and treatment of child and adolescent overweight and obesity: summary report"], "source": ["Pediatrics"], "year": ["2007"], "volume": ["120"], "fpage": ["164"], "lpage": ["92"], "pub-id": ["10.1542/peds.2007-2329C"]}, {"label": ["35."], "collab": ["PTS Diagnostics"], "year": ["2018"], "ext-link": ["https://ptsdiagnostics.com/wp-content/uploads/2018/09/ps-002461-en_rev._4_user_guide_cardiochek_pa.pdf"]}, {"label": ["36."], "surname": ["Gao", "Zhu", "Wu", "Guo", "Liu", "Dong"], "given-names": ["Y", "CG", "NQ", "YL", "G", "Q"], "article-title": ["Study on the reliability of CardioChek PA for measuring lipid profile"], "source": ["J Peking Univ Health Sci"], "year": ["2016"], "volume": ["48"], "fpage": ["523"], "lpage": ["8"], "pub-id": ["10.3969/j.issn.1671-167X.2016.03.025"]}, {"label": ["37."], "collab": ["Polskie Towarzystwo Kardiologiczne"], "year": ["2016"], "ext-link": ["https://ptkardio.pl/"]}, {"label": ["42."], "collab": ["R Core Team"], "source": ["R: a language and environment for statistical computing"], "publisher-loc": ["Vienna, Italy"], "publisher-name": ["R Foundation for Statistical Computing"], "year": ["2018"]}, {"label": ["43.", "2018"], "collab": ["Physical Activity Guidelines Advisory Committee"], "source": ["Physical activity guidelines advisory committee scientific report"], "publisher-loc": ["Washington, DC"], "publisher-name": ["US Department of Health and Human Services"], "year": ["2018"]}, {"label": ["46."], "surname": ["Jones", "Davison", "Zoladz"], "given-names": ["AW", "G", "JA"], "article-title": ["Exercise, immunity, and illness"], "source": ["Muscle and exercise physiology"], "publisher-loc": ["London"], "publisher-name": ["Academic Press"], "year": ["2019"], "fpage": ["317"], "lpage": ["44"]}, {"label": ["49."], "surname": ["Finer"], "given-names": ["N"], "article-title": ["Medical consequences of obesity"], "source": ["Medicine"], "year": ["2015"], "volume": ["43"], "fpage": ["88"], "lpage": ["93"], "pub-id": ["10.1016/j.mpmed.2014.11.003"]}, {"label": ["50."], "surname": ["Kyle"], "given-names": ["RG"], "article-title": ["Prevalence of overweight and obesity among nurses in Scotland: a cross-sectional study using the Scottish health survey"], "source": ["BMJ Open"], "year": ["2016"], "volume": ["53"], "fpage": ["126"], "lpage": ["33"], "pub-id": ["10.1016/j.ijnurstu.2015.10.015"]}, {"label": ["54."], "surname": ["Kayaroganam", "Sarkar", "Satheesh", "Tamilmani", "Sivanantham", "Kar"], "given-names": ["R", "S", "S", "S", "P", "SS"], "article-title": ["Profile of non-communicable disease risk factors among nurses in a tertiary care hospital in South India"], "source": ["Asian Nurs Res"], "year": ["2022"], "volume": ["1"], "fpage": ["241"], "lpage": ["8"], "pub-id": ["10.1016/j.anr.2022.07.001"]}, {"label": ["55."], "surname": ["Fair", "Gulanick", "Braun"], "given-names": ["JM", "M", "LT"], "article-title": ["Cardiovascular risk factors and lifestyle habits among preventive cardiovascular nurses"], "source": ["J Cardiovasc Nurs"], "year": ["2009"], "volume": ["240"], "fpage": ["277"], "lpage": ["86"], "pub-id": ["10.1097/JCN.0b013e3181a24375"]}, {"label": ["62."], "collab": ["Ministerstwo Sportu i Turystyki"], "year": ["2018"], "ext-link": ["https://www.gov.pl/web/sport/krajowe-rekomendacje-prozdrowotnej-aktywnosci-fizycznej55"]}, {"label": ["65."], "surname": ["Chappel", "Verswijveren", "Aisbett", "Considine", "Ridgers"], "given-names": ["SE", "SJ", "B", "J", "ND"], "article-title": ["Nurses\u2019 occupational physical activity levels: a systematic review"], "source": ["Int J Nurs Stud"], "year": ["2017"], "volume": ["1"], "fpage": ["52"], "lpage": ["62"], "pub-id": ["10.1016/j.ijnurstu.2017.05.006"]}, {"label": ["71."], "surname": ["Benzo", "Farag", "Whitaker", "Xiao", "Carr"], "given-names": ["R", "A", "KM", "Q", "LJ"], "article-title": ["A comparison of occupational physical activity and sedentary behavior patterns of nurses working 12-h day and night shifts"], "source": ["Int J Nurs Stud Adv"], "year": ["2021"], "volume": ["3"], "fpage": ["100028"], "pub-id": ["10.1016/j.ijnsa.2021.100028"]}, {"label": ["73."], "surname": ["Tudor-Locke", "Hatano", "Pangrazi", "Kang"], "given-names": ["C", "Y", "RP", "M"], "article-title": ["Revisiting \u201chow many steps are enough?\u201d"], "source": ["Med Sci Sports Exerc"], "year": ["2008"], "volume": ["40"], "fpage": ["537"], "lpage": ["43"], "pub-id": ["10.1249/MSS.0b013e31817c7133"]}, {"label": ["75."], "surname": ["Chen", "Lim", "Ivy"], "given-names": ["HC", "T", "N"], "article-title": ["Factors influencing overweight and obesity in nurses: a systematic review and meta-analysis"], "source": ["J Obes Overweight"], "year": ["2021"], "volume": ["7"], "fpage": ["202"], "ext-link": ["https://www.annexpublishers.com/articles/JOO/7203-Factors-Influencing-Overweight.pdf"]}, {"label": ["76."], "surname": ["Aryee", "Helegbe", "Baah", "Sarfo-Asante", "Quist-Therson"], "given-names": ["PA", "GK", "B", "RA", "R"], "article-title": ["Prevalence and risk factors for overweight and obesity among nurses in the tamale metropolis of Ghana"], "source": ["J Med Biomed Sci"], "year": ["2013"], "volume": ["2"], "fpage": ["13"], "lpage": ["23"], "pub-id": ["10.4314/jmbs.v2i4.3"]}, {"label": ["77."], "collab": ["The Supreme Council of Nurses and Midwives"], "year": ["2022"], "ext-link": ["https://nipip.pl/wp-content/uploads/2022/06/2022_Raport-NIPiP-struktura-wiekowa-kadr.pdf"]}, {"label": ["79."], "surname": ["Paluch", "Bajpai", "Ballin", "Bassett", "Buford", "Carnethon"], "given-names": ["AE", "S", "M", "DR", "TW", "MR"], "article-title": ["Prospective association of daily steps with cardiovascular disease: a harmonized meta-analysis"], "source": ["Circulation"], "year": ["2023"], "volume": ["2"], "fpage": ["122"], "lpage": ["31"], "pub-id": ["10.1161/CIRCULATIONAHA.122.061288"]}, {"label": ["82."], "surname": ["Wieczorek-W\u00f3jcik", "Kila\u0144ska"], "given-names": ["B", "D"], "article-title": ["Samotny jak piel\u0119gniarka na oddziale"], "source": ["Mened\u017cer Zdrowia"], "year": ["2017"], "volume": ["2"], "fpage": ["68"], "lpage": ["74"], "ext-link": ["https://www.termedia.pl/Samotny-jak-pielegniarka-na-oddziale,12,29767,1,0.html"]}, {"label": ["83."], "surname": ["Fie", "Norman", "While"], "given-names": ["S", "IJ", "AE"], "article-title": ["The relationship between physicians\u2019 and nurses\u2019 personal physical activity habits and their health-promotion practice: a systematic review"], "source": ["Health Educ J"], "year": ["2013"], "volume": ["72"], "fpage": ["102"], "lpage": ["19"], "pub-id": ["10.1177/0017896911430763"]}, {"label": ["85."], "collab": ["Healthy Nurse Healthy Nation"], "year": ["2021"], "ext-link": ["https://www.healthynursehealthynation.org/"]}]
{ "acronym": [], "definition": [] }
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2024-01-14 23:43:49
Front Public Health. 2023 Dec 29; 11:1300662
oa_package/4d/14/PMC10787602.tar.gz
PMC10787603
37104660
[ "<title><italic toggle=\"yes\">Brucella</italic>: A Brief Introduction</title>", "<p id=\"P4\">The <italic toggle=\"yes\">Alphaproteobacteria</italic> include species that inhabit diverse niches, including the plant rhizosphere and phyllosphere; freshwater, marine, and soil ecosystems; and mammalian and insect hosts (##REF##15550939##11##). <italic toggle=\"yes\">Brucella</italic> is perhaps the most notorious genus in this class. <italic toggle=\"yes\">Brucella abortus</italic> and <italic toggle=\"yes\">Brucella melitensis</italic> were first identified in the late nineteenth century as the etiologic agents of contagious abortion in cows and Malta fever in humans, respectively (##UREF##1##9##, ##UREF##2##18##). These intracellular pathogens cause a disease now known as brucellosis, which remains among the most widespread zoonoses globally (##REF##9204307##32##, ##REF##16439329##96##). The <italic toggle=\"yes\">Brucella</italic> genus is genetically monomorphic, and it has been proposed to be monospecific (##UREF##10##128##). However, <italic toggle=\"yes\">Brucella</italic> species cluster into classifiable phylogenetic groups that align with animal host range and select molecular and physiologic characteristics, including differences in the structure and chemical makeup of the cell envelope (##REF##12414145##90##). In this review we provide an overview of the <italic toggle=\"yes\">Brucella</italic> cell envelope and highlight recent advances in the study of <italic toggle=\"yes\">Brucella</italic> spp. envelope structure, function, and regulation.</p>" ]
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[ "<title>CONCLUSIONS AND OUTLOOK</title>", "<p id=\"P33\"><italic toggle=\"yes\">Brucella</italic> spp. are important animal pathogens that have been studied for over a century. Since their discovery, we have gained significant understanding of animal host responses to infection and the <italic toggle=\"yes\">Brucella</italic> genetic factors that determine infection and pathogenesis. Molecular components of the cell envelope play a crucial role in host interactions, and subtle differences in cell envelope composition or regulation likely contribute to the fascinating animal host preferences exhibited by the highly related <italic toggle=\"yes\">Brucella</italic> species. Deciphering the genetic basis of host preference is an interesting and exciting area of investigation that may be advanced by combining pangenome analyses with omics-based approaches to analyze cell envelope lipids, polysaccharides, and proteins.</p>", "<p id=\"P34\"><italic toggle=\"yes\">Brucella</italic> has emerged as a powerful comparative model for the investigation of cell cycle, polar cell development, and cell division in gram-negative bacteria. The development of fluorescent d-amino acids for study of peptidoglycan synthesis was instrumental in demonstrating that cell growth in <italic toggle=\"yes\">Brucella</italic> is unipolar (##REF##23055266##73##), and this tool will be useful as the community works to decipher the molecular mechanism of polar cell growth in <italic toggle=\"yes\">Brucella</italic> and other <italic toggle=\"yes\">Rhizobiales</italic>. Select genes for other well-studied polar surface structures in <italic toggle=\"yes\">Alphaproteobacteria</italic>, such as unipolar polysaccharide (UPP) (##REF##31209074##63##, ##REF##35191101##93##), are conserved in <italic toggle=\"yes\">Brucella</italic>. It is not known whether <italic toggle=\"yes\">Brucella</italic> spp. elaborate a UPP, but it seems likely that cell cycle–regulated polar envelope structures—perhaps including UPP— contribute to the interesting link between the developmental state of the <italic toggle=\"yes\">Brucella</italic> cell and the process of host infection (##REF##26497941##33##).</p>" ]
[ "<p id=\"P1\">The cell envelope is a multilayered structure that insulates the interior of bacterial cells from an often chaotic outside world. Common features define the envelope across the bacterial kingdom, but the molecular mechanisms by which cells build and regulate this critical barrier are diverse and reflect the evolutionary histories of bacterial lineages. Intracellular pathogens of the genus <italic toggle=\"yes\">Brucella</italic> exhibit marked differences in cell envelope structure, regulation, and biogenesis when compared to more commonly studied gram-negative bacteria and therefore provide an excellent comparative model for study of the gram-negative envelope. We review distinct features of the <italic toggle=\"yes\">Brucella</italic> envelope, highlighting a conserved regulatory system that links cell cycle progression to envelope biogenesis and cell division. We further discuss recently discovered structural features of the <italic toggle=\"yes\">Brucella</italic> envelope that ensure envelope integrity and that facilitate cell survival in the face of host immune stressors.</p>" ]
[ "<title>OVERVIEW</title>", "<title>The Gram-Negative Envelope</title>", "<p id=\"P2\">The cell envelope separates the cytoplasm of a cell from the exterior environment. It determines cell shape; harbors macromolecular complexes that generate usable forms of energy to power motility and growth; and directly interacts with features of the surrounding environment, including plant and animal hosts (##REF##26370932##70##, ##REF##20452953##111##). The envelope of gram-negative bacteria is particularly interesting because it comprises two structurally distinct lipid bilayers with a thin periplasm and peptidoglycan cell wall in the space between them (##REF##20452953##111##). The ability of micron-sized bacterial cells to maintain this structurally complex, didermic envelope while growing and dividing is a remarkable feat.</p>", "<p id=\"P3\">Our understanding of the gram-negative envelope is primarily based on studies of the class <italic toggle=\"yes\">Gammaproteobacteria</italic>, specifically <italic toggle=\"yes\">Enterobacteriaceae</italic> such as <italic toggle=\"yes\">Escherichia coli</italic>. However, gram-negative bacteria are a varied group of organisms that exhibit remarkable diversity in the chemical and structural makeup of their envelopes. In recent years, cellular and molecular studies of the <italic toggle=\"yes\">Alphaproteobacteria</italic> have revealed conserved molecular features that distinguish the envelope of this class. Species in the <italic toggle=\"yes\">Alphaproteobacteria</italic> thus provide useful comparative models when considering which molecular processes of gram-negative envelope biogenesis and homeostasis are general, and which are specific to a particular phylogenetic group (##REF##28697666##38##).</p>", "<title>DEVELOPMENTAL REGULATION OF <italic toggle=\"yes\">BRUCELLA</italic> CELL ENVELOPE BIOGENESIS</title>", "<title>The CtrA Regulatory Network</title>", "<p id=\"P5\">When viewed from afar, <italic toggle=\"yes\">Brucella</italic> looks like any other gram-negative bacteria: it possesses a phospholipid bilayer inner membrane (IM), an outer membrane with a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet, and a peptidoglycan cell wall in the periplasmic space between. Close inspection of single cells has provided strong evidence for an asymmetric growth mechanism. Specifically, peptidoglycan labeling experiments in <italic toggle=\"yes\">B. abortus</italic> demonstrated that new cell envelope material is synthesized strictly from the new cell pole produced immediately after cell division (##REF##22307633##17##, ##REF##27504539##64##). At later stages of the cell cycle, a small growth zone appears at the nascent division site in <italic toggle=\"yes\">B. abortus</italic> (##REF##34662460##101##, ##REF##35748337##103##, ##REF##30635335##127##) (##FIG##0##Figure 1a##). Asymmetry in the biogenesis of new cell material during growth is reflected in asymmetric subcellular organization of key protein regulators of <italic toggle=\"yes\">Brucella</italic> cell cycle and development. For example, the essential polar development sensor histidine kinase, PdhS, colocalizes with the response regulator DivK to the old cell pole during the <italic toggle=\"yes\">B. abortus</italic> cell cycle; DivK localization requires the conserved aspartyl phosphorylation site in its receiver domain. Upon division, polarly localized PdhS is asymmetrically inherited by the mother cell; the newborn cell later acquires PdhS at its old pole, approximately 30 min after division (##REF##17304218##56##) (##FIG##0##Figure 1a##). The developmental processes regulated by PdhS and DivK are integrated into a large regulatory network that is conserved across <italic toggle=\"yes\">Alphaproteobacteria</italic> and that ensures the molecular machinery required to build new cell material is (<italic toggle=\"yes\">a</italic>) localized to the appropriate polar location and (<italic toggle=\"yes\">b</italic>) activated at the appropriate time during the cell cycle (##REF##27517351##77##, ##REF##25793963##95##, ##REF##32970604##125##, ##REF##29783033##136##). This elaborate molecular control system comprises multiple sensor histidine kinases, response regulators, diguanylate cyclases, and phosphodiesterases, all of which influence the expression, phosphorylation, and stability of the essential DNA-binding response regulator, CtrA. It can be reasonably argued that CtrA is the central molecular player of cell cycle and cell development regulation in many <italic toggle=\"yes\">Alphaproteobacteria</italic>, including <italic toggle=\"yes\">Brucella</italic> (##REF##29733367##98##).</p>", "<p id=\"P6\">Like other response regulators, the activity of <italic toggle=\"yes\">Brucella</italic> CtrA as a transcription factor is controlled by its phosphorylation at a single aspartic acid residue. The phosphorylation state of the CtrA protein is directly regulated by a multiprotein phosphorelay comprising the transmembrane sensor histidine kinase CckA and the histidine phosphotransferase ChpT (##REF##26124143##134##) (##FIG##0##Figure 1b##). Chromatin immunoprecipitation sequencing (ChIP-seq) studies of <italic toggle=\"yes\">B. abortus</italic> CtrA have demonstrated that CtrA directly binds the promoter regions of dozens of genes involved in cell division, peptidoglycan cell wall metabolism, outer membrane biogenesis, and biosynthesis of LPS (##REF##27893179##43##). The importance of CtrA as a direct regulator of a diverse set of cell envelope proteins is clearly evident in studies of depletion and temperature-sensitive mutants of <italic toggle=\"yes\">B. abortus</italic> (##REF##27893179##43##, ##REF##26124143##134##). Shifting conditional <italic toggle=\"yes\">ctrA</italic> mutant strains to restrictive conditions resulted in reduced levels of select outer membrane proteins (OMPs) and the formation of filamentous branched cells, consistent with a defect in polar cell development and division. Branched cells were also observable in the mammalian intracellular niche upon depletion of <italic toggle=\"yes\">ctrA</italic> expression; this phenotype was correlated with reduced intracellular viability (##REF##27893179##43##). Attenuation of a conditional <italic toggle=\"yes\">ctrA</italic> mutant in an in vitro infection model is expected, given that <italic toggle=\"yes\">B. abortus</italic> cell cycle progression is intimately tied to intracellular trafficking in the endosomal compartments (##REF##26497941##33##).</p>", "<title>A Role for Cyclic Diguanylate?</title>", "<p id=\"P7\">The concentration and asymmetric localization of the second messenger cyclic-di-GMP (c-di-GMP) directly control the phosphorylation state and stability of CtrA in <italic toggle=\"yes\">Caulobacter crescentus</italic> (##REF##27517351##77##, ##REF##25945741##79##, ##REF##25197043##112##). A direct role for this signaling molecule in regulation of the <italic toggle=\"yes\">Brucella</italic> CtrA pathway has not been shown, but there is evidence that c-di-GMP metabolizing enzymes play a role in <italic toggle=\"yes\">Brucella</italic> envelope biology. A screen of annotated <italic toggle=\"yes\">B. melitensis</italic> diguanylate cyclases and phosphodiesterases in a heterologous <italic toggle=\"yes\">Vibrio</italic> system identified at least three active enzymes, including the phosphodiesterase PdeA (also known as BpdA) (##REF##21856843##97##). <italic toggle=\"yes\">B. melitensis pdeA</italic> mutants were attenuated in vitro and in vivo (##REF##27672085##67##, ##REF##21856843##97##), and deletion of <italic toggle=\"yes\">B. abortus</italic> and <italic toggle=\"yes\">B. melitensis pdeA</italic> resulted in cell rounding, with cells becoming shorter and wider (##REF##27672085##67##, ##REF##34662460##101##). This shape phenotype was independent of any phosphodiesterase activity the enzyme may have in cells, as a catalytically deficient <italic toggle=\"yes\">pdeA</italic><sup>E742A</sup> mutant retained wild-type morphology (##REF##34662460##101##). Peptidoglycan analysis using fluorescent <sc>d</sc>-amino acid (HADA) labeling provided evidence that PdeA is required for localizing the site of peptidoglycan insertion to the pole (##REF##34662460##101##). This result is consistent with studies of a PdeA homolog in <italic toggle=\"yes\">Sinorhizobium meliloti</italic> known as RgsP, which functions at the membrane to control peptidoglycan composition (##REF##30102748##108##).</p>", "<p id=\"P8\">Though PdeA is an important regulator of envelope biology in <italic toggle=\"yes\">Brucella</italic>, a role for the c-di-GMP molecule per se has not been defined. Studies of diverse bacteria have shown that c-di-GMP levels in the cytoplasm control the transition from motile to sessile behavior via a variety of molecular mechanisms (##REF##28171809##31##, ##REF##23471616##105##). <italic toggle=\"yes\">Brucella</italic> spp. contain assorted flagellar genes and pseudogenes (##REF##35056531##26##, ##REF##11756688##36##) but have long been classified as nonmotile bacteria (##UREF##6##109##). A <italic toggle=\"yes\">B. melitensis</italic> strain was reported to elaborate a sheathed flagellum under select growth conditions (##REF##15839898##44##), but efforts to identify actively motile cells among the classical <italic toggle=\"yes\">Brucella</italic> species (<italic toggle=\"yes\">B. abortus</italic>, <italic toggle=\"yes\">B. melitensis</italic>, <italic toggle=\"yes\">B. ovis</italic>, <italic toggle=\"yes\">B. suis</italic>, <italic toggle=\"yes\">B. canis</italic>, and <italic toggle=\"yes\">B. neotomae</italic>) have been unsuccessful to our knowledge. However, several new <italic toggle=\"yes\">Brucella</italic> species have been isolated from a range of animals (##REF##33872784##133##), including amphibian isolates that are clearly motile and elaborate flagella and pilus-like structures from their envelopes (##REF##28300153##3##). Whether c-di-GMP influences motility in these newly identified flagellated and motile <italic toggle=\"yes\">Brucella</italic> species is not known, but it seems likely considering reported connections between <italic toggle=\"yes\">pdeA</italic> and transcription from flagellar promoters in <italic toggle=\"yes\">B. melitensis</italic> (##REF##21856843##97##). Future efforts to define a specific role(s) for c-di-GMP as an effector of envelope processes in <italic toggle=\"yes\">Brucella</italic> may be informed by studies in related free-living rhizobial species (e.g., <italic toggle=\"yes\">S. meliloti</italic>) where c-di-GMP levels are linked to motility, polysaccharide production, and cell wall biosynthesis (##REF##32990642##71##).</p>", "<title>THE <italic toggle=\"yes\">BRUCELLA</italic> OUTER MEMBRANE</title>", "<p id=\"P9\">The outer membrane is the interface between <italic toggle=\"yes\">Brucella</italic> cells and their environment. This asymmetrical lipid bilayer contains primarily LPS in the outer leaflet and phospholipids in the inner leaflet. As an intracellular pathogen, <italic toggle=\"yes\">Brucella</italic> must withstand a variety of host assaults, including compounds that disrupt membranes, such as antimicrobial peptides. Relative to a panel of <italic toggle=\"yes\">Enterobacteriaceae</italic>, including <italic toggle=\"yes\">E. coli</italic>, <italic toggle=\"yes\">Brucella</italic> spp. outer membranes are more resistant to envelope stressors including polymyxin B, melittin, EDTA, and lysozyme (##REF##7622230##84##). This enhanced resistance has been attributed to particular OMPs, distinct chemical features of core lipid A, and the density of O-polysaccharides of the LPS leaflet of the outer membrane, which will be discussed below.</p>", "<title>Outer Membrane Proteins</title>", "<p id=\"P10\">The outer membrane is likely the best-studied of the <italic toggle=\"yes\">Brucella</italic> envelope layers because it contains many antigens, including a diverse array of OMPs, that invoke host immune responses (##REF##15383598##47##). Focused effort to characterize <italic toggle=\"yes\">Brucella</italic> OMPs over the past several decades (##REF##6802764##129##) have been motivated in part by the fact that OMPs can—in some cases—confer protective immunity, facilitate serologic classification of strains, and be developed as vaccines. We now know that <italic toggle=\"yes\">Brucella</italic> spp. encode multiple families of OMPs, including many heat-stable porins and lipoproteins. A large body of literature on <italic toggle=\"yes\">Brucella</italic> OMP content, structure, and immunology has been covered in previous reviews (##REF##12414146##24##, ##REF##26579096##52##, ##REF##33568459##106##), and we encourage readers to consult these for additional information and additional perspectives on this topic.</p>", "<title>Targeting and Assembly: Lol, Bam, and Tam</title>", "<p id=\"P11\">The mechanism by which lipid-modified OMPs (i.e., lipoproteins) become localized to the <italic toggle=\"yes\">Brucella</italic> outer membrane remains an open question. In gram-negative bacteria, the lipoprotein localization (Lol) pathway comprises a set of proteins that traffic outer membrane lipoproteins across the periplasm to their final address (##UREF##4##54##). However, the gene encoding the LolB protein, which is necessary for lipoprotein trafficking in many gram-negative bacteria, is absent in the <italic toggle=\"yes\">Alphaproteobacteria</italic>, including <italic toggle=\"yes\">Brucella</italic> (##REF##26579096##52##, ##REF##21663440##92##). It has been proposed that LolA may serve the functions of both LolA and LolB in clades where LolB is absent (##REF##22410786##118##), though this hypothesis remains untested in <italic toggle=\"yes\">Brucella</italic>. Recent efforts to define the functions of uncharacterized <italic toggle=\"yes\">B. abortus</italic> proteins involved in envelope stress responses identified a periplasmic domain of unknown function protein (DUF1849) conserved in a subset of <italic toggle=\"yes\">Alphaproteobacteria</italic>—primarily <italic toggle=\"yes\">Rhizobiales</italic>—that is now named EipB (##REF##30936371##61##). The <italic toggle=\"yes\">eipB</italic> chromosomal locus exhibits synteny homology across the <italic toggle=\"yes\">Rhizobiales</italic> with genes that function in membrane and cell wall synthesis, LPS synthesis, and outer membrane protein assembly. The overall organization of this genomic region is highly conserved in <italic toggle=\"yes\">Proteobacteria</italic> (##FIG##1##Figure 2a##), and the association of <italic toggle=\"yes\">eipB</italic> with this locus supports a functional role for <italic toggle=\"yes\">eipB</italic> in cell envelope biology. EipB comprises 14 antiparallel β strands, organized in a cylindrical, spiral-like shape, with three α-helical connector segments. Though EipB has no clear structural homologs in the Protein Data Bank, its β-barrel architecture resembles those of <italic toggle=\"yes\">E. coli</italic> LolA and LolB, and the OMP assembly proteins TamA and BamA (##FIG##1##Figure 2b##). Deletion of <italic toggle=\"yes\">eipB</italic> resulted in sensitivity to compounds that disrupt the integrity of the cell envelope and compromised <italic toggle=\"yes\">B. abortus</italic> infection in a murine model of disease (##REF##30936371##61##). These recent results support a functional role for EipB in determining cell envelope integrity, but it is not known whether EipB assumes a LolB-like function in lipoprotein trafficking, whether it functions as an OMP chaperone or assembly factor, or whether it has another role in the <italic toggle=\"yes\">Brucella</italic> cell envelope.</p>", "<p id=\"P12\">In addition to the Lol system, there are other molecular systems that ensure <italic toggle=\"yes\">Brucella</italic> appropriately assembles OMPs into the outer membrane. Among these is the β-barrel assembly machine (BAM) complex, which facilitates protein folding into the outer membrane (##UREF##5##81##). The genus <italic toggle=\"yes\">Brucella</italic> encodes homologs of the BamA, BamD, and BamE proteins and is missing the BamB and BamC proteins present in <italic toggle=\"yes\">E. coli</italic> and other <italic toggle=\"yes\">Gammaproteobacteria</italic>. Our application of Bayesian or HMM gene essentiality algorithms (##REF##26447887##34##, ##REF##26357081##35##) to published transposon sequencing (Tn-Seq) data from <italic toggle=\"yes\">B. abortus</italic> 2308 (##REF##30936371##61##, ##REF##30536925##62##) and <italic toggle=\"yes\">B. ovis</italic> 25840 (##REF##31481543##126##) provides evidence that <italic toggle=\"yes\">bamE</italic>, <italic toggle=\"yes\">bamD</italic>, and <italic toggle=\"yes\">bamA</italic> are essential in both species. Anwari and colleagues (##REF##22524202##4##) identified a BAM complex component, BamF (DUF3035), that is restricted to the <italic toggle=\"yes\">Alphaproteobacteria</italic>, though sequence models in the InterPro-Pfam database (##REF##33156333##15##) indicate that BamF/DUF3035 is absent in the genus <italic toggle=\"yes\">Brucella</italic>. The composition of the BAM complex varies across gram-negative bacteria, and it is not known what proteins—if any—fulfill the functional roles of BamB, BamC, and BamF in <italic toggle=\"yes\">Brucella</italic>. The fact that the periplasmic β-barrel protein, EipB, is encoded from a locus proximal to <italic toggle=\"yes\">bamA</italic> suggests that it could function with the BAM complex in OMP assembly (##FIG##1##Figure 2a##). Notably, the OMP chaperone Skp is encoded adjacent to BamA in many <italic toggle=\"yes\">Proteobacteria</italic>; this gene is absent from <italic toggle=\"yes\">Rhizobiales</italic> cataloged in InterPro. The presence of <italic toggle=\"yes\">eipB</italic> in this conserved cell envelope cluster (instead of <italic toggle=\"yes\">skp</italic>) in <italic toggle=\"yes\">Rhizobiales</italic> raises the possibility that EipB and Skp have analogous chaperoning roles.</p>", "<p id=\"P13\">The outer membrane translocation and assembly module (TAM) is a system that is evolutionarily related to the BAM complex and plays a key role in the assembly of select OMPs (##REF##25994932##58##). The system comprises two protein components: the integral OMP TamA, which is related to BamA, and the inner membrane protein TamB. Studies in <italic toggle=\"yes\">B. suis</italic> have shown that the TamB homolog, known as MapB, controls translocation of a subset of OMPs to the outer membrane (##REF##30770847##13##). A <italic toggle=\"yes\">B. suis mapB</italic> deletion mutant had disrupted localization of select OMPs, a cell division/morphology defect, and was sensitive to multiple envelope-disrupting compounds, including polymyxin B, Triton X-100, and lysozyme (##REF##30770847##13##). <italic toggle=\"yes\">mapB/tamB</italic> deletion in <italic toggle=\"yes\">B. suis</italic> (##REF##30770847##13##) and <italic toggle=\"yes\">B. melitensis</italic> (##REF##17176467##135##) resulted in strain attenuation in infection models, and <italic toggle=\"yes\">B. abortus</italic> strains lacking either <italic toggle=\"yes\">tamA</italic> or <italic toggle=\"yes\">tamB</italic> were attenuated in a mouse macrophage infection model (##REF##29844240##116##). A specific role for the TAM system during infection is supported by proteomic analyses showing that the steady-state level of TamB protein in <italic toggle=\"yes\">B. abortus</italic> (BAB1_0046/Omp160) was enhanced ~20–100-fold just 3 h after macrophage infection and remained high over a two-day infection time course (##REF##19216536##75##).</p>", "<title>The Outer Membrane–Peptidoglycan Connection</title>", "<p id=\"P14\">In <italic toggle=\"yes\">E. coli</italic> and related bacteria, the hyperabundant outer membrane lipoprotein Lpp is covalently linked to the peptidoglycan (##UREF##0##8##, ##REF##4261992##16##, ##REF##4575979##57##), which provides important structural support for the envelope. <italic toggle=\"yes\">Brucella</italic> and other related <italic toggle=\"yes\">Rhizobiales</italic> lack <italic toggle=\"yes\">lpp</italic>, but biochemical evidence for covalent interactions between <italic toggle=\"yes\">Brucella</italic> spp. OMPs and the peptidoglycan cell wall (##REF##1380979##25##, ##REF##3744559##51##) has been long discussed. Recent work by Godessart and colleagues (##REF##33139884##49##) has demonstrated that multiple OMPs in <italic toggle=\"yes\">B. abortus</italic> and the related rhizobial species <italic toggle=\"yes\">Agrobacterium tumefaciens</italic> are covalently linked to the peptidoglycan via a conserved alanyl-aspartyl motif at the protein N terminus (##FIG##2##Figure 3a##). Like the covalent linkage of <italic toggle=\"yes\">E. coli</italic> Lpp to peptidoglycan, this linkage in <italic toggle=\"yes\">Brucella</italic> is catalyzed by <sc>l</sc>,<sc>d</sc>-transpeptidases. This result explains long-noted observations of peptidoglycan linkages to OMPs in <italic toggle=\"yes\">Brucella</italic> and provides new biochemical and structural understanding of <italic toggle=\"yes\">Brucella</italic> envelope integrity. Notably, several other bacteria lacking Lpp have now been shown to anchor the outer membrane to peptidoglycan in a similar fashion to <italic toggle=\"yes\">Brucella</italic> (##REF##33139883##107##).</p>", "<title>LIPOPOLYSACCHARIDE</title>", "<p id=\"P15\">LPS, the primary lipid of the outer leaflet of the outer membrane, is a major pathogen-associated molecular pattern (PAMP) recognized by the innate immune system (##REF##24766885##120##). Accordingly, <italic toggle=\"yes\">Brucella</italic> LPS is a highly studied molecule that has been well-reviewed (##REF##33568459##106##, ##REF##34335551##117##, ##REF##29233768##137##), and we encourage readers to consult these reviews for more information on this topic. Here we provide an abridged overview of distinctive features of <italic toggle=\"yes\">Brucella</italic> LPS structure and biosynthesis.</p>", "<title>LPS Structure</title>", "<p id=\"P16\">The structure of <italic toggle=\"yes\">Brucella</italic> spp. LPS has chemical features that distinguish this envelope component from the highly inflammatory LPS of enteric bacteria (##FIG##2##Figure 3b##) and that enable these pathogens to evade host recognition by the innate immune system (##REF##17637846##10##, ##REF##22589715##28##). Canonically, LPS consists of a hexa-acylated disaccharide called lipid A, which is linked to the core oligosaccharides. In species with smooth LPS, repeating O-polysaccharide units decorate the core polysaccharide (##FIG##2##Figure 3##). The lipid A portion of <italic toggle=\"yes\">Brucella</italic> LPS consists of diaminoglucose sugars linked to C16–C18 fatty acids and very-long-chain fatty acids (VLCFAs), including a significant proportion of 27-OH-28:0 (##REF##2113907##91##) (##FIG##2##Figure 3c##). This unusual feature of <italic toggle=\"yes\">Brucella</italic> lipid A is restricted to the alpha-2 subgroup of <italic toggle=\"yes\">Proteobacteria</italic>, and it may enable lipid A to extend through the outer membrane outer leaflet into the inner leaflet (##FIG##2##Figure 3a##), potentially increasing the structural integrity of the outer membrane (##REF##1854635##12##). The level of VLCFAs on lipid A is controlled in part by the inner membrane–bound BacA protein in both <italic toggle=\"yes\">B. abortus</italic> and the related rhizobial species <italic toggle=\"yes\">S. meliloti</italic> (##REF##15044696##39##), suggesting that this unique LPS feature can be regulated. Relative to other gram-negative bacteria, <italic toggle=\"yes\">Brucella</italic> lipid A is a poor stimulant of the mammalian inflammatory response (##REF##15339879##37##). The core oligosaccharide of <italic toggle=\"yes\">Brucella</italic> spp. lipid A contains two 3-deoxy-<sc>d</sc>-<italic toggle=\"yes\">manno</italic>-2-octulosonic acid (Kdo) sugars (##REF##12761107##89##) linked to diaminoglucose, one of which has a branched linkage to a chain of glucose, mannose, and glucosamine (##REF##24927935##48##) (##FIG##2##Figure 3d##). There is evidence that this branched core structure shields lipid A from recognition by the host innate immune sensor TLR4 (##REF##22589715##28##) and thereby contributes to the reduced inflammatory capacity of <italic toggle=\"yes\">Brucella</italic> LPS.</p>", "<p id=\"P17\">Among the classical <italic toggle=\"yes\">Brucella</italic> species (##REF##19628055##132##), the best-described difference in cell envelope composition is the presence or absence of O-polysaccharide, also known as O-antigen (##REF##34335551##117##). <italic toggle=\"yes\">Brucella</italic> spp. O-antigen consists primarily of N-formylated perosamine (##REF##9784561##50##) (##FIG##2##Figure 3d##) of two major linkage forms: A (from <italic toggle=\"yes\">B. abortus</italic>) and M (from <italic toggle=\"yes\">B. melitensis</italic>) (##REF##23261780##72##, ##REF##2474504##87##). While <italic toggle=\"yes\">Brucella</italic> spp. typically elaborate an O-antigen and are considered to have smooth LPS (S-LPS), <italic toggle=\"yes\">B. ovis</italic> and <italic toggle=\"yes\">B. canis</italic> harbor genetic mutations that result in an inability to synthesize O-antigen and have what is therefore considered naturally rough LPS (R-LPS). O-antigen is an important virulence determinant in <italic toggle=\"yes\">Brucella</italic> (##REF##15694858##76##), yet <italic toggle=\"yes\">B. ovis</italic> and <italic toggle=\"yes\">B. canis</italic> cause disease in their primary hosts, sheep and dogs, respectively. The pathogenicity of these rough strains in their primary hosts is notable because rough mutants of naturally smooth strains have large defects in intracellular growth and replication and can be cytotoxic to host macrophages.</p>", "<p id=\"P18\">The structure of O-antigen from the numerous new <italic toggle=\"yes\">Brucella</italic> species identified in recent years (##REF##33872784##133##) remains largely undefined, but several strains in the recently identified BO2 clade of <italic toggle=\"yes\">Brucella</italic> are missing genes in the <italic toggle=\"yes\">wbk</italic> region that are required for S-LPS synthesis. Experimental analyses of a BO2 strain showed that it produced an S-LPS (##REF##22761298##138##) not composed of <italic toggle=\"yes\">N</italic>-formyl-perosamine (##UREF##11##130##). In the place of the <italic toggle=\"yes\">wbk</italic> genes, this strain contained genes for biosynthesis of a rhamnose-based O-antigen; BO2 S-LPS had biochemical properties that were distinct from those of the classical <italic toggle=\"yes\">Brucella</italic> species (##UREF##11##130##). Future analyses of LPS structure across the expanded <italic toggle=\"yes\">Brucella</italic> clade will likely illuminate new and diverse chemical features of <italic toggle=\"yes\">Brucella</italic> LPS.</p>", "<title>Spatial Control of LPS Biosynthesis in <italic toggle=\"yes\">Brucella</italic></title>", "<p id=\"P19\">An elaborate transport system ensures proper addressing of LPS to the outer membrane in gram-negative bacteria (##UREF##8##115##). In <italic toggle=\"yes\">B. abortus</italic>, the biosynthesis and elaboration of LPS on the surface of the cell is spatially controlled. <italic toggle=\"yes\">B. abortus</italic> has both R-LPS and S-LPS patches on its envelope that can be visualized by immunofluorescence (##REF##30635335##127##). Insertion of new LPS material occurs unipolarly at the new pole of the cell (generated immediately after division), where new cell wall material is also added (##REF##30635335##127##). The inner membrane LPS biosynthesis proteins LptB, LptC, LptF, and LptG are similarly localized to the new pole. There are conflicting reports of the spatial distribution of the outer membrane LPS translocase, LptD. Fluorescent protein fusions to LptD showed a polar localization pattern in wild-type <italic toggle=\"yes\">B. abortus</italic> (##REF##29325043##123##) consistent with the localization of LptBCFG, while staining by immunogold provides evidence that LptD is localized across the cell surface (##UREF##7##110##).</p>", "<title>Periplasmic Factors Linked to LPS</title>", "<p id=\"P20\">Recent studies have identified previously uncharacterized domain of unknown function (DUF) proteins that are secreted to the periplasm and have roles in cell envelope processes. The periplasmic β-barrel protein EipB (##REF##30936371##61##), which may function in OMP targeting, is described above. Studies of <italic toggle=\"yes\">Brucella</italic> RomA and EipA provide evidence that these periplasmic proteins have a functional connection to LPS.</p>", "<p id=\"P21\">The RomA protein is a member of the DUF3126 superfamily, which is restricted to the <italic toggle=\"yes\">Alphaproteobacteria</italic>; in <italic toggle=\"yes\">B. abortus</italic> this protein is secreted to the periplasm (##REF##29325043##123##). A <italic toggle=\"yes\">B. abortus romA</italic> deletion strain (Δ<italic toggle=\"yes\">romA</italic>) had multiple envelope defects including altered LPS composition: Δ<italic toggle=\"yes\">romA</italic> had a higher proportion of S-LPS, with longer O-polysaccharide chains than the wild type (##REF##29325043##123##). This LPS phenotype was correlated with altered localization of a fluorescent protein fusion to the LPS translocase, LptD. Specifically, the Δ<italic toggle=\"yes\">romA</italic> strain showed diffuse localization of LptD across the cell, while LptD was polarly localized in the wild type. Localization of the O-polysaccharide flippase, RfbD (Bab1_0543), was not altered in Δ<italic toggle=\"yes\">romA</italic>. The precise function of RomA remains undefined, but it seems likely that this protein functions in the periplasm to coordinate LPS synthesis.</p>", "<p id=\"P22\">The <italic toggle=\"yes\">Brucella</italic> EipA protein is a member of the DUF1134 protein superfamily, which is largely restricted to the <italic toggle=\"yes\">Alphaproteobacteria</italic> (##FIG##3##Figure 4a##). In the <italic toggle=\"yes\">Rhizobiales</italic>, <italic toggle=\"yes\">eipA</italic> is encoded in the neighborhood of <italic toggle=\"yes\">ctrA</italic> and <italic toggle=\"yes\">chpT</italic> (##FIG##3##Figure 4a##), and its expression is directly controlled by CtrA (##REF##30536925##62##). EipA is secreted to the periplasm, and deletion of <italic toggle=\"yes\">B. abortus eipA</italic> resulted in sensitivity to envelope stressors including EDTA, SDS, and ampicillin (##REF##30536925##62##). As would be expected for a strain with a general envelope defect, Δ<italic toggle=\"yes\">eipA</italic> was attenuated in macrophage and mouse infection models. The molecular function of EipA in the periplasm has not been determined, but a screen for genes that are synthetically lethal with <italic toggle=\"yes\">eipA</italic> deletion in <italic toggle=\"yes\">B. abortus</italic> showed that disruption of O-polysaccharide synthesis genes is not tolerated in the absence of <italic toggle=\"yes\">eipA</italic> (##REF##30536925##62##). Congruent with this genetic analysis in <italic toggle=\"yes\">B. abortus</italic>, <italic toggle=\"yes\">eipA</italic> is essential in the naturally rough species <italic toggle=\"yes\">B. ovis</italic>, which lacks the O-polysaccharide. A high-resolution crystal structure of EipA revealed a novel protein fold (##REF##30536925##62##) composed of 10 antiparallel β strands, with β1–β5 and β8–β10 forming a small β barrel (##FIG##3##Figure 4b##). Though EipA interaction partners have not been defined, a recent solution NMR structure of the lipid/phosphoinositide-binding SYLF domain protein BPSL1445 revealed significant structural similarity to <italic toggle=\"yes\">Brucella</italic> EipA, suggesting that EipA is a lipid-interacting protein (##REF##34968022##99##).</p>", "<title><italic toggle=\"yes\">BRUCELLA</italic> PHOSPHOLIPIDS</title>", "<p id=\"P23\">Phospholipids are a key molecular component of the cell envelope, and <italic toggle=\"yes\">Brucella</italic> has interesting lipid membrane features that merit discussion. Phosphatidylethanolamine (PE) is the dominant phospholipid in most gram-negative bacteria, but <italic toggle=\"yes\">Brucella</italic> membranes contain comparatively low PE and a high proportion of phosphatidylcholine (PC), a phospholipid usually associated with eukaryotic membranes (##REF##3818086##45##, ##REF##12547654##113##, ##REF##4351160##122##). PE synthesis in <italic toggle=\"yes\">B. abortus</italic> is apparently dispensable; deletion of phosphatidylserine synthase (PssA), which catalyzes the first step in PE synthesis, is compensated by increased production of PC and ornithine lipids (OLs) (##REF##18931122##19##). It has been proposed that an elevated PC level may provide a mechanism to mask pathogens, including <italic toggle=\"yes\">Brucella</italic>, from their mammalian hosts (##REF##20656373##2##, ##REF##16484204##27##, ##REF##16882035##30##). This idea is supported by studies of the phospholipase A1, BveA, of <italic toggle=\"yes\">B. melitensis</italic>. Analysis of lipid extracts of a <italic toggle=\"yes\">bveA</italic> mutant revealed elevated levels of PE. Enhanced PE levels were associated with higher susceptibility to polymyxin B and reduced survival in in vitro and in vivo infection models (##REF##26282427##66##).</p>", "<p id=\"P24\">The notable skew in the PC:PE ratio in <italic toggle=\"yes\">Brucella</italic> spp. relative to other gram-negative bacteria has motivated studies of the mechanism of PC biosynthesis. PC can be synthesized through one of two pathways: the methylation pathway and the choline pathway. In the methylation pathway, PmtA mediates successive methylation of phosphatidylethanolamine, using <italic toggle=\"yes\">S</italic>-adenosylmethionine (SAM) as a methyl donor. In the choline pathway, the enzyme Pcs synthesizes PC from choline and cytidine diphosphate-diacylglycerol precursors (##REF##12547654##113##). The <italic toggle=\"yes\">B. abortus</italic> genome contains both <italic toggle=\"yes\">pcs</italic> and <italic toggle=\"yes\">pmtA</italic> homologs, but genetic analysis of Δ<italic toggle=\"yes\">pcs</italic> and Δ<italic toggle=\"yes\">pmtA</italic> mutants showed that only <italic toggle=\"yes\">pcs</italic> was required for normal growth and synthesis of PC in complex medium (##REF##16484204##27##, ##REF##16882035##30##). However, both Δ<italic toggle=\"yes\">pcs</italic> and Δ<italic toggle=\"yes\">pmtA</italic> mutants were attenuated for replication in the mouse spleen at different stages of infection, and a Δ<italic toggle=\"yes\">pcs</italic> Δ<italic toggle=\"yes\">pmtA</italic> double mutant (<italic toggle=\"yes\">a</italic>) had a larger growth defect in defined medium than single mutants and (<italic toggle=\"yes\">b</italic>) was more attenuated in mouse spleen than either single mutant (##REF##16882035##30##). These results provide evidence that both pathways to PC are operational in <italic toggle=\"yes\">B. abortus</italic> and that the nutritional environment influences the requirement for these genes. A related series of experiments on PC synthesis in <italic toggle=\"yes\">B. melitensis</italic> 16M extracts led to the conclusion that Pcs was the sole route to PC production (##REF##14663079##85##), while the conclusion based on experiments in <italic toggle=\"yes\">B. abortus</italic> was that PC was synthesized from host-derived choline exclusively via the choline (Pcs) pathway (##REF##16484204##27##). Acquisition of choline from the host is consistent with a report that <italic toggle=\"yes\">B. abortus</italic> requires the high-affinity choline transporter ChoXWV for PC synthesis when choline concentrations are low (##REF##23161032##59##). Polymorphisms in the SAM-binding site of PmtA across the <italic toggle=\"yes\">Brucella</italic> genus likely influence the enzymatic routes to PC production at the species level and may explain why select <italic toggle=\"yes\">B. abortus</italic>, <italic toggle=\"yes\">B. melitensis</italic>, and <italic toggle=\"yes\">B. suis</italic> strains have differing requirements for <italic toggle=\"yes\">pcs</italic> and <italic toggle=\"yes\">pmtA</italic> in vitro and in vivo (##REF##34421831##5##).</p>", "<p id=\"P25\">In addition to PC and PE, the <italic toggle=\"yes\">Brucella</italic> envelope also contains a significant fraction of OLs (##REF##21249206##94##, ##REF##4351160##122##). Prior to studies in <italic toggle=\"yes\">Brucella</italic>, OL synthesis had been characterized in <italic toggle=\"yes\">S. meliloti</italic>, where two genes, <italic toggle=\"yes\">olsA</italic> and <italic toggle=\"yes\">olsB</italic>, are required for OL synthesis (##REF##15341653##46##, ##REF##12139618##131##). <italic toggle=\"yes\">Brucella</italic> contains homologs of these genes and synthesizes OL through a two-step pathway (##REF##21249206##94##). Deletion of OL synthesis did not impact membrane permeability, susceptibility to antimicrobial peptides, or infection in an in vitro model (##REF##21249206##94##). Moreover, a <italic toggle=\"yes\">B. abortus</italic> strain lacking OLs did not induce an inflammatory response in mice that differed from wild type. These results suggest that <italic toggle=\"yes\">Brucella</italic> OLs have little impact on <italic toggle=\"yes\">B. abortus</italic> envelope stress resistance, infection, or host immunity. Nonetheless, the abundance of OLs in <italic toggle=\"yes\">Brucella</italic> membranes suggests these molecules have an important role in some context.</p>", "<title>THE PEPTIDOGLYCAN CELL WALL</title>", "<p id=\"P26\">As a gram-negative bacterium grows, it builds new peptidoglycan cell wall in the periplasmic space. At the point of division, the cell must cleave and reanneal the peptidoglycan wall that surrounds it. This highly complex process requires exquisite spatiotemporal coordination of a multitude of proteins on a submicron scale and has been the subject of intense study for many years (##REF##34351794##104##). <italic toggle=\"yes\">E. coli</italic>, <italic toggle=\"yes\">C. crescentus</italic>, and many other model bacteria grow laterally and assemble the division site (i.e., the divisome) at the center of the cell after a certain period of growth. The <italic toggle=\"yes\">Rhizobiales</italic> (including <italic toggle=\"yes\">Brucella</italic>) grow by budding, a process in which new peptidoglycan material is added to one cell pole (##REF##22307633##17##) (##FIG##0##Figure 1a##). The mechanism by which <italic toggle=\"yes\">Brucella</italic> and other <italic toggle=\"yes\">Rhizobiales</italic> localize the peptidoglycan biosynthesis machinery to one pole and maintain cell envelope integrity during the process of new cell addition and division remains largely undefined (##UREF##9##124##).</p>", "<p id=\"P27\">Penicillin-binding proteins (PBPs) are critical for peptidoglycan cell wall synthesis, and a transposon screen of <italic toggle=\"yes\">B. abortus</italic> in complex medium uncovered Pbp1a (Bab1_0932) and FtsI as the only two essential PBPs of the seven encoded in the genome (##REF##29844240##116##). As expected, this study also identified numerous divisome (<italic toggle=\"yes\">fts</italic>) and cell division (<italic toggle=\"yes\">tol-pal</italic>) genes as essential for growth. Screening this same transposon library in macrophages identified a set of genes that were conditionally essential in the intracellular niche. Among these was the histidine biosynthesis gene, <italic toggle=\"yes\">hisB</italic>, which exhibited a surprising cell chaining/division defect inside mammalian cells resulting from uncleaved peptidoglycan at the cell division site (##REF##35748337##103##). The <italic toggle=\"yes\">hisB</italic> division phenotype could be suppressed by overexpression of either DipM or CdlP, each of which contains a peptidoglycan-binding LysM domain and is predicted to function as a metallopeptidase. This targeted suppressor approach thus identified two putative peptidoglycan metallopeptidases involved in <italic toggle=\"yes\">Brucella</italic> cell division, though the exact connection between histidine metabolism, peptidoglycan cleavage, and division remains undefined. Cell wall metabolism and cell division are a relatively new area of investigation in <italic toggle=\"yes\">Brucella</italic>, and future studies are certain to elucidate interesting connections between central metabolism and division.</p>", "<title>CONSERVED CELL ENVELOPE REGULATION SYSTEMS</title>", "<p id=\"P28\">When infecting a host, the <italic toggle=\"yes\">Brucella</italic> cell must withstand many host-derived stressors to survive. It is the cell envelope that meets this complex host assault. Accordingly, the composition of the envelope is highly regulated by multiple stress response systems. In this section, we discuss select <italic toggle=\"yes\">Brucella</italic> envelope regulators that are conserved in the <italic toggle=\"yes\">Alphaproteobacteria</italic>.</p>", "<title>The General Stress Response System</title>", "<p id=\"P29\">The general stress response (GSR) system of <italic toggle=\"yes\">Alphaproteobacteria</italic> regulates large-scale changes in gene expression that confer resistance to a range of environmental stressors (##REF##26442844##40##, ##REF##25582885##42##). Activation of the <italic toggle=\"yes\">B. abortus</italic> GSR involves stress-dependent phosphorylation of the anti-anti-sigma protein PhyR (##REF##23546883##68##, ##REF##25257300##69##, ##REF##25993430##119##). PhyR phosphorylation promotes its binding to the anti-σ<sup>E1</sup> protein, NepR (##REF##29052909##80##), thereby releasing the alternative sigma factor σ<sup>E1</sup> (also known as <italic toggle=\"yes\">ecfG</italic>) to regulate transcription of dozens of genes (##FIG##4##Figure 5a##). Studies in <italic toggle=\"yes\">B. abortus</italic> and several other <italic toggle=\"yes\">Alphaproteobacteria</italic> provide evidence that multiple HWE-family sensor kinases (##REF##28193573##60##) coordinately regulate PhyR phosphorylation and GSR activation (##FIG##4##Figure 5a##), which may explain how this regulatory system is able to respond to such a diverse range of stress conditions (##REF##26442844##40##, ##REF##25582885##42##).</p>", "<p id=\"P30\">Deletion of GSR regulatory genes in <italic toggle=\"yes\">B. abortus</italic> and <italic toggle=\"yes\">B. melitensis</italic> results in a variable replication/survival defect in mice depending on the particular gene deletion and the genetic background of the animal host (##UREF##3##53##, ##REF##23546883##68##, ##REF##25257300##69##). The transcription of numerous <italic toggle=\"yes\">B. abortus</italic> membrane transport systems is strongly activated by σ<sup>E1</sup>, including an RND-family efflux system, a CrcB-family transporter, ABC-type transporters, and a major facilitator family transporter (##REF##23546883##68##). Thus <italic toggle=\"yes\">B. abortus</italic> remodels the transport capabilities of its envelope when the GSR system is activated. In addition, transcription of the <italic toggle=\"yes\">cydABX</italic> high-affinity terminal oxidase genes and the O-polysaccharide biosynthesis gene, <italic toggle=\"yes\">pgm</italic>, is activated by σ<sup>E1</sup>, as are multiple hypothetical transmembrane DUF proteins. In <italic toggle=\"yes\">B. melitensis</italic>, deletion of the GSR-activating kinase <italic toggle=\"yes\">lovhK</italic> is reported to activate expression of the type IV secretion system (<italic toggle=\"yes\">virB</italic>) and select flagellar genes (##UREF##3##53##). Together, these studies provide evidence that the GSR system is a major regulator of the <italic toggle=\"yes\">Brucella</italic> cell envelope.</p>", "<title>The BvrR-BvrS Envelope Homeostasis System</title>", "<p id=\"P31\">The BvrR-BvrS two-component regulatory system is conserved in the <italic toggle=\"yes\">Alphaproteobacteria</italic>, where it has important functions in host-bacteria interactions (##REF##8407839##22##, ##REF##8407840##83##). In <italic toggle=\"yes\">Brucella</italic>, this system is a key determinant of infection in in vitro and in vivo models (##REF##12414153##78##, ##REF##9701808##114##). <italic toggle=\"yes\">B. abortus bvrR-bvrS</italic> directly and indirectly regulates the type IV secretion system (##REF##20833814##86##), contributes to homeostasis of membranes, and regulates lipid A acylation and the expression of periplasmic and outer membrane proteins (##REF##12218183##55##, ##REF##17343405##74##, ##REF##16077108##82##) (##FIG##4##Figure 5b##). The general cell envelope defect of strains harboring mutations in this two-component system is evident in the fact that deletion of the envelope integrity protein <italic toggle=\"yes\">eipA</italic> is synthetically lethal with <italic toggle=\"yes\">bvrR</italic> deletion in <italic toggle=\"yes\">B. abortus</italic> (##REF##30536925##62##). Recent ChIP-seq analysis of <italic toggle=\"yes\">B. abortus</italic> BvrR demonstrates that this regulator not only directly binds the promoter regions of type IV secretion genes (<italic toggle=\"yes\">virB</italic>), genes for secreted effectors, and genes that function in multiple aspects of cell envelope homeostasis but also binds promoters of multiple genes with roles in central metabolism (##REF##36129877##102##). This study provides evidence for a broader regulatory role for the BvrR-BvrS system in <italic toggle=\"yes\">Brucella</italic> cell envelope biology and metabolism than has been previously appreciated.</p>", "<title>Ros/MucR</title>", "<p id=\"P32\">Like the BvrR-BvrS system, the zinc finger transcription factor Ros/MucR is conserved in the <italic toggle=\"yes\">Alphaproteobacteria</italic> (##REF##33705385##1##, ##REF##25421297##7##, ##REF##9560269##23##, ##REF##7756693##65##), where it has an established role in virulence gene regulation and plant-rhizobia interactions (##REF##16349248##6##, ##REF##9057324##14##, ##REF##9560269##23##). Deletion of this regulator in multiple <italic toggle=\"yes\">Brucella</italic> species results in alterations in cell envelope properties (##REF##23319565##21##, ##REF##23161025##88##, ##REF##35174240##121##) that, in <italic toggle=\"yes\">B. melitensis</italic>, lead to sensitivity to a range of cell envelope stressors in vitro. Studies in <italic toggle=\"yes\">B. melitensis</italic> further provide evidence that MucR regulates modification of lipid A core and regulates transcription of flagellar genes via the flagellar regulator protein FtcR (##REF##23161025##88##). <italic toggle=\"yes\">Brucella mucR</italic> can functionally complement an <italic toggle=\"yes\">S. meliloti mucR</italic> deletion mutant (##REF##23161025##88##), providing an additional example of how a conserved set of regulatory proteins underpins host-microbe interactions in <italic toggle=\"yes\">Alphaproteobacteria</italic>.</p>" ]
[ "<title>ACKNOWLEDGMENTS</title>", "<p id=\"P35\">Research reported in this publication was supported by the National Institute of General Medical Science of the National Institutes of Health under award number R35GM131762 to S.C.</p>", "<title>DISCLOSURE STATEMENT</title>", "<p id=\"P36\">The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p id=\"P42\">(<italic toggle=\"yes\">a</italic>) <italic toggle=\"yes\">Brucella</italic> spp., like other <italic toggle=\"yes\">Rhizobiales</italic>, exhibit unipolar growth from the new cell pole. As the cell cycle progresses, cell growth shifts from being exclusively polar to being at both the pole and the nascent division site (growth sites colored <italic toggle=\"yes\">orange</italic>). The <italic toggle=\"yes\">Brucella</italic> developmental regulators, PdhS and DivK, exhibit dynamic polar localization to the inner membrane as a function of cell cycle. The new (N) pole and old (O) cell pole are marked. (<italic toggle=\"yes\">b</italic>) Model of the CckA-ChpT-CtrA phosphorelay. The histidine kinase CckA autophosphorylates on a conserved histidine residue and transfers a phosphoryl group to a conserved aspartic acid residue on its C-terminal receiver domain. CckA~P transfers a phosphoryl group to the ChpT phosphotransferase, which can subsequently transfer this phosphoryl group to the receiver domain of CtrA. CtrA~P is a DNA-binding response regulator that modulates transcription of genes controlling cell polarity, division, and intracellular survival.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p id=\"P43\">(<italic toggle=\"yes\">a</italic>) Phylogenetic distribution and synteny of the envelope integrity protein B (<italic toggle=\"yes\">eipB</italic>) genomic region in <italic toggle=\"yes\">Proteobacteria</italic>; gene neighborhood is anchored on <italic toggle=\"yes\">bamA</italic>. <italic toggle=\"yes\">Brucella eipB</italic> is part of a highly conserved cell envelope gene cluster in the <italic toggle=\"yes\">Proteobacteria</italic> that includes genes involved in outer membrane protein assembly (<italic toggle=\"yes\">bamA</italic>), undecaprenyl phosphate biosynthesis, phospholipid synthesis (<italic toggle=\"yes\">cdsA</italic>), lipopolysaccharide synthesis (<italic toggle=\"yes\">lpxDAIB</italic>), and translation (<italic toggle=\"yes\">ef-ts</italic>). <italic toggle=\"yes\">eipB</italic> (<italic toggle=\"yes\">pink</italic>) is conserved in the <italic toggle=\"yes\">Rhizobiales</italic> group of the <italic toggle=\"yes\">Alphaproteobacteria</italic> (phylogenetic classifications on the <italic toggle=\"yes\">right</italic>). Skp is present outside the <italic toggle=\"yes\">Rhizobiales</italic>. Phylogenetic tree is based on BamA protein sequence (<italic toggle=\"yes\">left</italic>). (<italic toggle=\"yes\">b</italic>) Crystal structures of <italic toggle=\"yes\">Brucella</italic> EipB (PDB: 6NTR), <italic toggle=\"yes\">Escherichia coli</italic> LolB (PDB: 1IWM), <italic toggle=\"yes\">E. coli</italic> LolA (PDB: 1IWL), and <italic toggle=\"yes\">E. coli</italic> BamA (PDB: 5OR1). Abbreviation: PDB, Protein Data Bank.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p id=\"P44\">(<italic toggle=\"yes\">a</italic>) Overview of the envelope layers of <italic toggle=\"yes\">Brucella</italic>. Notably, a subset of <italic toggle=\"yes\">Brucella</italic> outer membrane proteins are covalently linked to the peptidoglycan cell wall via a conserved sequence at the protein N terminus. (<italic toggle=\"yes\">b</italic>) Chemical structure of <italic toggle=\"yes\">Escherichia coli</italic> lipid A (##REF##12045108##100##). (<italic toggle=\"yes\">c</italic>) Chemical structure of <italic toggle=\"yes\">Brucella</italic> spp. lipid A showing VLCFA tails and pyrophosphorylethanolamine modification of diaminoglucose backbone (outlined with dashed-line box). Structure adapted from model and data presented in References ##REF##28924631##20## and ##REF##29375522##29##. (<italic toggle=\"yes\">d</italic>) <italic toggle=\"yes\">Brucella</italic> smooth LPS structure showing lipid A, core oligosaccharide, and O-polysaccharide (O-antigen), based on models and data presented in References ##REF##26867577##41##, ##REF##23261780##72##, and ##REF##34335551##117##. Abbreviations: Kdo, 3-deoxy-d-<italic toggle=\"yes\">manno</italic>-2-octulosonic acid; LPS, lipopolysaccharide; OMP, outer membrane protein; PG, peptidoglycan; VLCFA, very-long-chain fatty acid.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p id=\"P45\">(<italic toggle=\"yes\">a</italic>) Phylogenetic distribution and synteny of the envelope integrity protein A (<italic toggle=\"yes\">eipA</italic>) genomic region in <italic toggle=\"yes\">Proteobacteria</italic>; gene neighborhood is anchored on <italic toggle=\"yes\">eipA</italic>. In <italic toggle=\"yes\">Brucella</italic> and other <italic toggle=\"yes\">Rhizobiales</italic>, <italic toggle=\"yes\">eipA</italic> is part of a genetic locus that encodes the essential cell cycle regulatory proteins CtrA and ChpT. (<italic toggle=\"yes\">b</italic>) Crystal structures of <italic toggle=\"yes\">Brucella</italic> EipA (PDB 5UC0) and a <italic toggle=\"yes\">Burkholderia</italic> SYLF domain protein (PDB 7OFN). Abbreviation: PDB, Protein Data Bank.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p id=\"P46\">(<italic toggle=\"yes\">a</italic>) The GSR signaling pathway in <italic toggle=\"yes\">Alphaproteobacteria</italic> is activated by environmental signals and integrates features of two-component and sigma factor–dependent regulation of gene expression. Studies of this pathway in <italic toggle=\"yes\">Brucella abortus</italic> support a model in which phosphorylation of PhyR is controlled by two HWE-family sensor kinases: LovhK and Bab1_1673. PhyR phosphorylation promotes its binding to NepR, which releases σ<sup>E1</sup> to control transcription of select cell envelope genes that influence in vitro stress survival and host infection. The LOV and PAS sensory domains of the cytoplasmic GSR-activating kinase, LovhK, are labeled. The periplasmic CHASE family sensor domain of the transmembrane GSR-repressive kinase, Bab1_1673, is labeled. Solid lines indicate a direct interaction; dashed lines indicate interactions that may be direct or indirect. (<italic toggle=\"yes\">b</italic>) <italic toggle=\"yes\">Brucella</italic> spp. BvrR/BvrS is a conserved, archetypal two-component system that is a critical regulator of infection. In response to detection of intracellular signals during infection, the histidine kinase BvrS phosphorylates BvrR, which then regulates transcription of a suite of cell envelope and metabolic genes important for intracellular survival and replication. Abbreviations: DBD, DNA-binding domain; GSR, general stress response; REC, REC, receiver domain; RNAP, RNA polymerase; OMP, outer membrane protein.</p></caption></fig>" ]
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[{"label": ["8."], "surname": ["Asmar", "Collet"], "given-names": ["AT", "JF"], "year": ["2018"], "article-title": ["Lpp, the Braun lipoprotein, turns 50\u2014major achievements and remaining issues"], "source": ["FEMS Microbiol. Lett"], "volume": ["365"], "issue": ["18"], "fpage": ["fny199"]}, {"label": ["9."], "surname": ["Bang"], "given-names": ["B"], "year": ["1897"], "article-title": ["Die Aetiologie des seuchenhaften (\u201cinfecti\u00f6sen\u201d) Verwerfens"], "source": ["Z. Tiermedizin"], "volume": ["1"], "fpage": ["241"], "lpage": ["78"]}, {"label": ["18."], "surname": ["Bruce"], "given-names": ["D"], "year": ["1887"], "article-title": ["Note on the discovery of a microorganism in Malta fever"], "source": ["Practitioner"], "volume": ["39"], "fpage": ["161"], "lpage": ["70"]}, {"label": ["53."], "surname": ["Gourley", "Petersen", "Harms", "Splitter"], "given-names": ["CR", "E", "J", "G"], "year": ["2015"], "article-title": ["Decreased in vivo virulence and altered gene expression by a "], "italic": ["Brucella melitensis"], "source": ["Pathog. Dis"], "volume": ["73"], "fpage": ["1"], "lpage": ["8"]}, {"label": ["54."], "surname": ["Grabowicz"], "given-names": ["M"], "year": ["2019"], "article-title": ["Lipoproteins and their trafficking to the outer membrane"], "source": ["EcoSal Plus"], "volume": ["8"], "pub-id": ["10.1128/ecosalplus.ESP-0038-2018"]}, {"label": ["81."], "surname": ["Malinverni", "Silhavy"], "given-names": ["JC", "TJ"], "year": ["2011"], "article-title": ["Assembly of outer membrane b-barrel proteins: the Bam complex"], "source": ["EcoSal Plus"], "volume": ["4"], "pub-id": ["10.1128/ecosalplus.4.3.8"]}, {"label": ["109."], "surname": ["Scholz", "Banai", "Cloeckaert", "Kampfer", "Whatmore", "Trujillo", "Dedysh", "DeVos", "Hedlund", "K\u00e4mpfer"], "given-names": ["H", "M", "A", "P", "A", "ME", "S", "P", "B", "P"], "year": ["2018"], "part-title": ["Brucella"], "source": ["Bergey\u2019s Manual of Systematics of Archaea and Bacteria"], "publisher-loc": ["Hoboken, NJ"], "publisher-name": ["John Wiley"], "pub-id": ["10.1002/9781118960608.gbm00807.pub2"]}, {"label": ["110."], "surname": ["Servais", "Vassen", "Verhaeghe", "Kuster", "Carlier"], "given-names": ["C", "V", "A", "NS", "E"], "year": ["2022"], "article-title": ["Lipopolysaccharide synthesis and traffic in the envelope of the pathogen "], "italic": ["Brucella abortus"], "source": ["bioRxiv"], "comment": ["2022.05.19.492625"], "month": ["May"], "day": ["19"]}, {"label": ["115."], "surname": ["Sperandeo", "Deho", "Polissi"], "given-names": ["P", "G", "A"], "year": ["2009"], "article-title": ["The lipopolysaccharide transport system of Gram-negative bacteria"], "source": ["Biochim. Biophys. Acta Mol. Cell Biol. Lipids"], "volume": ["1791"], "fpage": ["594"], "lpage": ["602"]}, {"label": ["124."], "surname": ["Van der Henst", "De Bolle", "Biondi"], "given-names": ["M", "X", "EG"], "year": ["2022"], "italic": ["Brucella abortus"], "source": ["Cell Cycle Regulation and Development in Alphaproteobacteria"], "fpage": ["287"], "lpage": ["301"], "publisher-loc": ["Cham, Switz."], "publisher-name": ["Springer"]}, {"label": ["128."], "surname": ["Verger", "Grimont", "Grimont", "Grayon"], "given-names": ["JM", "F", "PAD", "M"], "year": ["1985"], "article-title": ["Brucella, a monospecific genus as shown by deoxyribonucleic-acid hybridization"], "source": ["Int. J. Syst. Bacteriol"], "volume": ["35"], "fpage": ["292"], "lpage": ["95"]}, {"label": ["130."], "surname": ["Wattam", "Inzana", "Williams", "Mane", "Shukla"], "given-names": ["AR", "TJ", "KP", "SP", "M"], "year": ["2012"], "article-title": ["Comparative genomics of early-diverging "], "italic": ["Brucella"], "source": ["mBio"], "volume": ["3"], "fpage": ["e00246"], "lpage": ["12"]}]
{ "acronym": [ "LPS", "S-LPS", "R-LPS", "PE", "PC" ], "definition": [ "lipopolysaccharide", "smooth lipopolysaccharide", "rough lipopolysaccharide", "phosphatidylethanolamine", "phosphatidylcholine" ] }
138
CC BY
no
2024-01-14 23:43:49
Annu Rev Microbiol. 2023 Sep 15; 77:233-253
oa_package/35/3d/PMC10787603.tar.gz
PMC10787605
0
[ "<title>Introduction</title>", "<p>Over the last decade, evaluation for systemic inflammatory response syndrome (SIRS) and sepsis has been a top priority for hospitals. Ongoing research has shown that early recognition of sepsis and treatments improve the nearly 30% mortality [##REF##11794169##1##, ####REF##20048677##2##, ##REF##28117397##3####28117397##3##]. The most recent Surviving Sepsis Campaign in 2021 recommends using the Sequential Organ Failure Assessment (SOFA) and SIRS instead of Quick Sepsis-related Organ Failure Assessment (qSOFA) for the detection of sepsis [##REF##34599691##4##]. These tools integrate the patient's physical exam findings and laboratory data to identify serious infections. Unfortunately, there are inconsistencies between the utilization of SIRS and SOFA in the literature and clinical practice, as Medicare has failed to adopt the SOFA system. There has been no single laboratory that has been able to predict sepsis.</p>", "<p>The current COVID-19 pandemic has put increased strain on the healthcare system. Research is underway on biomarkers, calculators, and now cytomarkers for evaluating sepsis and the risk of severe disease to aid in the triage of patients. In Surviving Sepsis, the ultimate driving force for early detection of infection is timing to antibiotics, as numerous studies demonstrate that time to antibiotics impacts survival [##REF##11794169##1##,##REF##20048677##2##]. Despite its initial promise, there is significant variability in predicting infection, which prevented the biomarker procalcitonin from being widely accepted [##REF##26903335##5##]. The 2021 Surviving Sepsis Campaign has recommended against the routine use of procalcitonin due to poor sensitivity [##REF##26903335##5##,##REF##33675202##6##]. Traditionally, the white blood cell (WBC) count is among the first laboratory tests available to clinicians in the ED and is used in the SIRS determination. Additionally, other components of the CBC such as bands, or immature white blood cells, are also used to clue physicians into possible infections [##REF##34599691##4##]. Unfortunately, the WBC count elevation can also be nonspecific for sepsis as patients can have low, normal, or high counts and still have a serious life-threatening infection. Seymour et al. in the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) identified that once patients are admitted to the hospital, the detection of infection is typically delayed by &gt; four hours after admission [##REF##26903335##5##]. For patients presenting to the hospital, delays can be &gt; eight hours in approximately 30% of cases presenting to the ED [##REF##34599691##4##,##REF##26903335##5##,##REF##24201179##7##]. These delays are associated with worse outcomes; therefore, there is an ongoing need for additional biomarkers that would be routinely available and reliable to help guide clinicians in evaluating patients for sepsis in the emergency room setting [##REF##24201179##7##].</p>", "<p>Beckman Coulter developed an FDA-approved cytomarker for the detection of sepsis. This cytomarker is a calculation based on monocyte distribution width (MDW) and WBCs [##UREF##0##8##,##REF##31107278##9##]. It is different from biomarkers as it is based on cell size and not specific protein. The calculation uses WBC measurements known as cell morphometric parameters to characterize the cell volume variability and distribution [##UREF##0##8##,##REF##31107278##9##]. The MDW calculation is as follows: MDW + WBC = area under the curve (AUC) of 0.85 [##UREF##0##8##]. This cytomarker was validated by Elliott D. Crouser et al., who conducted a multicenter, blinded, observational, prospective cohort study at three academic centers using this new biomarker to determine the diagnostic accuracy of peripheral blood MDW alone and in combination with WBC count for early sepsis detection in the emergency department [##REF##31107278##9##]. They were able to identify that the MDW value of greater than 20.0 U was effective for sepsis detection [##REF##31107278##9##]. Crouser et al. also found that when used in tandem with WBC, MDW was able to enhance early sepsis detection [##REF##31107278##9##].</p>", "<p>Monocytes are white blood cells that play a crucial role in the innate immune response to inflammation and infection [##REF##19132917##10##]. They have the ability to differentiate into macrophages, including alveolar macrophages and dendritic cells [##UREF##1##11##]. Both differentiated cells can present antigens from digested viruses, bacteria, or other microorganisms to stimulate the adaptive immune response [##UREF##1##11##]. They have a significant role in cytokine production and may have unique responses to COVID-19. The monocyte life is about three days. A recent publication in the Lancet using this knowledge found that trending MDW can be helpful in determining clinical outcomes [##REF##33675202##6##,##UREF##1##11##]. A cartooned description of the proposed pathophysiology of monocyte enlargement and cytokine production in COVID-19 is demonstrated in the Appendix.</p>", "<p>In this retrospective study, we evaluate the new cytomarker, MDW, in the determination of sepsis in COVID-19 patients and the correlation of clinical outcomes, including hypoxia or respiratory failure and all causes of mortality.</p>" ]
[ "<title>Materials and methods</title>", "<p>This is a retrospective, single-center study that was conducted in a Community Hospital in Southern California. This study was approved by our Institutional Review Board (IRB). This study did not contain any personal patient information. The study population consisted of adult patients who presented to the emergency room, with positive polymerase chain reaction testing for COVID-19 and MDW measurement that required admission between September 1, 2020, and November 30, 2021. The calculation uses WBC measurements known as cell morphometric parameters to characterize the cell volume variability and distribution [##UREF##0##8##,##REF##31107278##9##]. The MDW calculation is as follows: MDW + WBC = AUC of 0.85 [##UREF##0##8##]. A total of 331 were included for evaluation. No repeat values were included. Data extraction utilized the electronic medical record system using the International Classification of Diseases, Tenth Revision, Clinical Modification for the determination of SIRS, sepsis, severe sepsis, septic shock, hypoxia, and respiratory failure.</p>", "<p>For statistical analyses, general descriptive statistics and box plots were calculated for cell population distribution parameters. The diagnostic capability was evaluated in terms of the AUC, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The scoring approach was utilized to be used to calculate confidence intervals (CIs) for sensitivity, specificity, PPV, and NPV. The Youden index will be used for the cutoff. Three approaches will be used to demonstrate the added value of MDW in comparison with WBC count. The first was the differences in AUCs. The AUC was calculated using a one-predictor variable logistic model with WBC count as the predictor and sepsis status as the response. In addition, the AUC was calculated for the two predictor variables logistic model with both WBC count and MDW as predictors and sepsis as a response. The comparison between the AUC from the two models (WBC count vs. WBC count þ MDW) along with their CIs were calculated. The second was the Cochran-Mantel-Haenszel (CMH) approach. Both WBC count and MDW were dichotomized to 0 and 1 based on their values falling into the normal or abnormal category. Comparing the binary threshold variable indicator indicating MDW values greater than or less than 24.9 against survival status, a chi-square test was performed, which detected a significant association between MDW values and final clinical outcomes of COVID-19. The following statistical analyses were applied through the use of open-source R statistical software packages (https://www.r-<ext-link xlink:href=\"https://project.org\" ext-link-type=\"uri\">project.org</ext-link>) to correlate MDW values with routine laboratory parameters and final outcomes.</p>" ]
[ "<title>Results</title>", "<p>The mean age of this population (N = 331) was 64.25 ± 16.78 years old. There were 168 (50.76%) male and 163 (49.24%) female patients. Table ##TAB##0##1## summarizes the demographics of the study population.</p>", "<p>MDW as well as inflammatory biomarker values, such as ferritin, D-dimer, procalcitonin, and lactic acid, were collected and compared against any level of sepsis (SIRS, sepsis, severe sepsis, or septic shock). Average MDW was found to be significantly higher in patients with sepsis (25.50 ± 5.93) vs. patients without (23.13 ± 4.46) (p &lt; 0.01); average D-dimer values were found to be significantly higher in patients with sepsis (7560.11 ± 17698.53) vs. patients without (1933.25 ± 3725.41) (p = 0.02); average procalcitonin values were found to be significantly higher in patients with sepsis (2.91 ± 6.10) vs. patients without (0.95 ± 4.47) (p = 0.02); average lactic acid values were found to be significantly higher in patients with sepsis (2.33 ± 1.88) vs. patients without (1.64 ± 0.84) (p &lt; 0.01) (Table ##TAB##1##2##).</p>", "<p>Average MDW was found to be significantly higher in patients with sepsis (25.50 ± 5.93) vs. patients without (23.13 ± 4.46) (p &lt; 0.01).</p>", "<p>Results demonstrating average MDW were found to be significantly higher in patients with sepsis (25.50 ± 5.93) vs. patients without (23.13 ± 4.46) (p &lt; 0.01).</p>", "<p>In this study cohort, there were 284 (85.54%) favorable cases where patients were discharged to home or other care settings, and 47 (14.16%) had fatal outcomes. Applying the Mann-Whitney test for independent values, we found a significant correlation (p &lt; 0.001) between the MDW detected in each patient (n = 331), and the final clinical outcome (survival/discharge vs. expiration). The median MDW value was about 24.9 between the 47 patients with death, which could be indicative of a prognostic threshold. Receiver operating characteristic (ROC) curve analysis is a statistical tool based on the notions of specificity and sensitivity, used for assessing diagnostic tests and predictive models. We utilized this approach to determine the best MDW cut-off to assess the probability of fatal outcomes. We did not have repeat MDW variables and only presenting MDW was utilized. We identified an MDW value of 24.9 as the best cut-off to assess the probability of fatal outcomes during the disease course in our cohort of COVID-19 patients. This analysis provided an AUC value of 0.69 (95% CI: 0.55-0.71; sensitivity: 0.83; specificity: 0.71). Among 118 patients with an MDW value of &gt;24.9, 29 cases had fatal outcomes, meaning that high MDW values are associated with a mortality rate or absolute risk of about 25%. A total of 18 out of 213 patients (9%) died while presenting MDW values lower than 24.9 (NPV = 0.91). Comparing the binary threshold variable indicator indicating MDW values greater than or less than 24.9 against survival status, a chi-square test was performed, which detected a significant association between MDW values and final clinical outcomes of the COVID-19 patient (OR = 3.52, 95% CI: 1.78-7.11, p &lt; 0.001), which indicates that MDW values are associated with clinical outcomes in COVID-19 patients.</p>", "<p>Respiratory failure and hypoxia were determined on ICD billing codes. A two-tailed t-test was completed comparing the average MDW between COVID-19 patients with respiratory failure. The MDW was statistically higher in patients with respiratory failure (24.9 vs. 21.9; p &lt; 0.01).</p>" ]
[ "<title>Discussion</title>", "<p>This research demonstrates that MDW is a reliable and easily exploitable cytomarker that can be utilized in patients with COVID-19 to detect sepsis and correlate with clinical outcomes of hypoxia and respiratory failure. Given much is still being discovered about this pandemic and the triage of patients, it is important to recognize the niche of this novel marker's potential in evaluating patients. MDW is a marker of the immune response to inflammation and unlike other biomarkers, it is calculable from the WBC count [##UREF##0##8##]. This is unique since patients with COVID-19 can present with relative leukopenia; it is important to recognize that the monocyte count distribution width was still able to identify ill patients and correlate with clinical outcomes in this study. Dedicated research on MDW in patients with specific illnesses that can affect monocyte production and maturation will need to be completed to determine utilization.</p>", "<p>MDW correlates with clinical outcomes of survival</p>", "<p>The biggest discovery identified is that an MDW value of 24.9 (25) is able to correlate with fatal outcomes. In fact, an MDW of greater than 24.9 had an absolute risk of 25%. Patients with a lower MDW had a negative predictive value of 0.91. When we compared the binary thresholds of MDW, they correlated to final outcomes with an odds ratio of 3.5. This is similar to Riva et al., except we are able to demonstrate a slightly lower MDW value of 25 (24.9) as a powerful predictor [##REF##32887652##12##,##REF##34135448##13##]. Our research also supports Lippi et al.’s pooled analysis that illustrated that MDW at hospital admission is higher in subjects with COVID-19 [##UREF##2##14##]. Furthermore, our research adds diagnostics values to clinical correlations using a larger population.</p>", "<p>MDW is able to detect hypoxia and respiratory failure</p>", "<p>COVID-19 patients with hypoxia and respiratory failure had a significantly higher average MDW value. The MDW value for respiratory failure matched the MDW prediction for death. Since respiratory failure is one of the biggest clinical outcomes for COVID-19, it is not surprising that these are correlated. It is important to note that the MDW was helpful for risk stratification. Our data support Hossain et al., who found that there is a meaningful difference in MDW in patients with hypoxic respiratory failure [##UREF##3##15##]. Our data utilized the International Classification of Diseases (ICD) codes in the determination of hypoxia and respiratory failure, and we were able to demonstrate that patients with respiratory problems had a significantly higher MDW of 24.9 compared to those without respiratory failure (24.9 vs. 21.9; p &lt; 0.01).</p>", "<p>MDW is able to detect sepsis in COVID-19</p>", "<p>We are also able to illustrate that MDW was able to detect sepsis in patients with COVID-19. We compared MDW as well as the inflammatory biomarker ferritin, D-dimer, procalcitonin, and lactic acid and compared against whether the patient had any level of sepsis (SIRS, sepsis, severe sepsis, or septic shock). The average MDW was found to be significantly higher in patients with sepsis (25.50 ± 5.93) vs. patients without (23.13 ± 4.46) (p &lt; 0.01).</p>", "<p>Surprisingly, MDW did not correlate with inflammatory markers, D-dimer, procalcitonin, lactic acid, or ferritin. This is dissimilar to Riva et al., where MDW correlated with laboratory values of inflammation, including C-reactive protein (CRP), ferritin, and fibrinogen [##REF##32887652##12##,##REF##34135448##13##]. These are not routinely utilized inflammatory markers and may represent an area of further research. It is possible that CRP, ferritin, and fibrinogen may be more specific to COVID-19. These markers were not available for comparison in our study. However, procalcitonin, lactic acid, and D-dimer are routinely used in the emergency department for the determination of inflammatory response to COVID-19. It is possible that this is directly linked to the pathophysiology of monocytes and their role in COVID-19 and cytokine production [##REF##32861199##16##]. MDW may have a specific niche for COVID-19, especially since MDW did not correlate with other biomarkers. Our study differs from Ennio Polilli et al., as we found the MDW did not correlate with procalcitonin in the detection of sepsis [##UREF##4##17##]. It is possible that MDW is able to predict a new kind of sepsis or viral sepsis. This is also similar to Piva et al., who found MDW increase is higher in patients with sepsis, and the MDW increase was not affected by the etiology of sepsis even in patients with COVID-19 [##REF##33675202##6##].</p>", "<p>Strengths and weakness</p>", "<p>Due to the retrospective nature of this study design, we are unable to control for confounding variables. To mitigate this, we created a very specific and homogenous population in hopes of making the evidence meaningful and applicable. We hope to repeat this study and compare MDW's ability to predict sepsis in patients with and without COVID-19. Additionally, our homogenous population of admitted patients may have allowed for selection bias for sicker patients with increased risk for sepsis or death. Currently, our hospital and staff primarily use Sepsis-2 criteria for the identification of sepsis because it correlates to ICD coding and billing. However, future studies can evaluate MDW with SOFA criteria from Sepsis-3. Our methods are similar to the recent study published in Nature by Riva et al. for mortality prediction using MDW [##REF##32887652##12##,##REF##34135448##13##]. This study is expanded to compare MDW against existing biomarkers to compare sepsis, respiratory failure, and death, and also has a larger sample size.</p>", "<p>This study could have been stronger if we had been able to further differentiate COVID-19 from secondary bacterial infections using microbiotic data from sputum cultures and blood cultures. In this study, the MDW was not evaluated to determine if sepsis was related to viral infection with COVID-19 or other co-occurring bacterial infections. In the setting of COVID-19, having a marker for bacterial infections would be clinically useful for antimicrobial stewardship.</p>" ]
[ "<title>Conclusions</title>", "<p>MDW is a novel and reliable cytomarker for identifying sepsis in patients with COVID-19 infection. High MDW values are associated with clinical outcomes of respiratory failure and death with a mortality rate or absolute risk of 25%. MDW is easily obtained from routine laboratory evaluation in the emergency room and has the potential to be a useful tool in the triage of COVID-19 patients.</p>" ]
[ "<p>Introduction</p>", "<p>Sepsis is the leading cause of hospital mortality nationwide. Early recognition has been shown to improve outcomes. This research investigates the use of monocyte distribution width’s (MDW) ability to detect sepsis and clinically correlate to outcomes in COVID-19 infection.</p>", "<p>Methods</p>", "<p>This is a retrospective, single-center cohort study of adult patients with confirmed COVID-19 requiring hospital admission over a 14-month period (September 2020 to November 2021). MDW was evaluated as a cytomarker to predict disease severity, mortality, and determination of sepsis in patients with COVID-19. Additionally, MDW was compared to existing inflammatory markers, including procalcitonin, D-dimer, ferritin, and lactic acid.</p>", "<p>Results</p>", "<p>MDW was able to predict sepsis in patients with COVID-19. The average MDW was found to be significantly higher in the detection of sepsis (25.50 ± 5.93) vs. patients without (23.13 ± 4.46) (p &lt; 0.01). MDW was able to correlate with clinical outcomes or respiratory failure/hypoxia and death. An MDW value of 24.9 was shown to be the best cut-off value in determining fatal outcomes; receiver operating characteristic curve analysis revealed an area under the curve value of 0.69 (95% CI: 0.55-0.71) with a sensitivity of 83% and specificity of 71%. A chi-square test was performed, which detected a significant association between MDW values and the final clinical outcome of COVID-19 (OR = 3.52, 95% CI: 1.78-7.11, p &lt; 0.001). Additionally, the mean MDW of patients with hypoxia or respiratory failure was significantly higher (22 vs. 25, p &lt; 0.1). MDW did not correlate with any of the existing inflammatory markers.</p>", "<p>Conclusion</p>", "<p>MDW is a novel and reliable cytomarker for identifying sepsis in patients with COVID-19 infection. High MDW values are associated with clinical outcomes of respiratory failure and death with a mortality rate or absolute risk of 25%. MDW is easily obtained from routine laboratory evaluation in the emergency room and has the potential to be a useful tool in the triage of COVID-19 patients.</p>" ]
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[ "<title>Appendices</title>" ]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Cartooned description of proposed pathophysiology of monocyte enlargement and subsequent cytokine production in COVID-19</title><p>Image made with the use of BioRender. Permission and publication licensing obtained for use.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Descriptive characteristics of the population</title><p>ICU: intensive care unit; MDW: monocyte distribution width.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Variable</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Percent (%)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age group</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">18-44</td><td rowspan=\"1\" colspan=\"1\">48</td><td rowspan=\"1\" colspan=\"1\">14.50%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">45-54</td><td rowspan=\"1\" colspan=\"1\">40</td><td rowspan=\"1\" colspan=\"1\">12.08%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">55-64</td><td rowspan=\"1\" colspan=\"1\">65</td><td rowspan=\"1\" colspan=\"1\">19.64%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">65-74</td><td rowspan=\"1\" colspan=\"1\">69</td><td rowspan=\"1\" colspan=\"1\">20.85%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">75+</td><td rowspan=\"1\" colspan=\"1\">109</td><td rowspan=\"1\" colspan=\"1\">32.93%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Gender</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">168</td><td rowspan=\"1\" colspan=\"1\">50.76%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ethnicity</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">White</td><td rowspan=\"1\" colspan=\"1\">126</td><td rowspan=\"1\" colspan=\"1\">38.07%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Asian</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">2.72%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Black/African American</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">1.51%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hispanic/Latino</td><td rowspan=\"1\" colspan=\"1\">191</td><td rowspan=\"1\" colspan=\"1\">57.70%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">MDW</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Greater than 20 (&gt;20)</td><td rowspan=\"1\" colspan=\"1\">247</td><td rowspan=\"1\" colspan=\"1\">74.62%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Less than 20 (&lt;20)</td><td rowspan=\"1\" colspan=\"1\">84</td><td rowspan=\"1\" colspan=\"1\">25.38%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">ICU hospitalization</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">91</td><td rowspan=\"1\" colspan=\"1\">27.49%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">240</td><td rowspan=\"1\" colspan=\"1\">72.51%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Outcomes</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Discharged (survivors)</td><td rowspan=\"1\" colspan=\"1\">284</td><td rowspan=\"1\" colspan=\"1\">85.80%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Expired (death)</td><td rowspan=\"1\" colspan=\"1\">47</td><td rowspan=\"1\" colspan=\"1\">14.20%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sepsis</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">71</td><td rowspan=\"1\" colspan=\"1\">21.45%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">260</td><td rowspan=\"1\" colspan=\"1\">78.55%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Sepsis severity</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">No sepsis</td><td rowspan=\"1\" colspan=\"1\">260</td><td rowspan=\"1\" colspan=\"1\">78.55%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">SIRS (systemic inflammatory response syndrome)</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">0.91%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sepsis</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">2.42%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Severe sepsis</td><td rowspan=\"1\" colspan=\"1\">36</td><td rowspan=\"1\" colspan=\"1\">10.88%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Septic shock</td><td rowspan=\"1\" colspan=\"1\">24</td><td rowspan=\"1\" colspan=\"1\">7.25%</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Comparison table of inflammatory markers including MDW in the detection of sepsis</title><p>Average MDW was found to be significantly higher in patients with sepsis (25.50 ± 5.93) vs. patients without (23.13 ± 4.46) (p &lt; 0.01).</p><p>MDW: monocyte distribution width.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Sepsis</td><td rowspan=\"1\" colspan=\"1\">No sepsis</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Variable</td><td rowspan=\"1\" colspan=\"1\">Mean +/- SD</td><td rowspan=\"1\" colspan=\"1\"> Mean +/- SD</td><td rowspan=\"1\" colspan=\"1\">t (95% CI)</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">MDW</td><td rowspan=\"1\" colspan=\"1\">25.50 +/- 5.93</td><td rowspan=\"1\" colspan=\"1\">23.13 +/- 4.46</td><td rowspan=\"1\" colspan=\"1\">-3.13 (-3.87, -0.86)</td><td rowspan=\"1\" colspan=\"1\">&lt;0.01</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ferritin</td><td rowspan=\"1\" colspan=\"1\">858.47 +/- 1164.07</td><td rowspan=\"1\" colspan=\"1\">638.80 +/- 826.44</td><td rowspan=\"1\" colspan=\"1\">-1.17 (-539.62, 154.29)</td><td rowspan=\"1\" colspan=\"1\">0.25</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">D-dimer</td><td rowspan=\"1\" colspan=\"1\">7560.11 +/- 17698.53</td><td rowspan=\"1\" colspan=\"1\">1933.25 +/- 3725.41</td><td rowspan=\"1\" colspan=\"1\">-2.38 (-10353.05, -900.67)</td><td rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Procalcitonin</td><td rowspan=\"1\" colspan=\"1\">2.91 +/- 6.10</td><td rowspan=\"1\" colspan=\"1\">0.95 +/- 4.41</td><td rowspan=\"1\" colspan=\"1\">-2.28 (-3.68, -0.25)</td><td rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Lactic acid</td><td rowspan=\"1\" colspan=\"1\">2.33 +/- 1.88</td><td rowspan=\"1\" colspan=\"1\">1.64 +/- 0.84</td><td rowspan=\"1\" colspan=\"1\">-2.91 (-1.16, -0.22)</td><td rowspan=\"1\" colspan=\"1\">&lt;0.01</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Amanda Frugoli, Johnson Ong, Bashar Khiatah, Graal Diaz</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Amanda Frugoli, Johnson Ong, Brittany Meyer, Bashar Khiatah, Robert Bernstein, Anthony Hernandez, Graal Diaz</p><p><bold>Drafting of the manuscript:</bold>  Amanda Frugoli, Johnson Ong, Brittany Meyer, Robert Bernstein, Anthony Hernandez, Graal Diaz</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Amanda Frugoli, Bashar Khiatah, Robert Bernstein, Anthony Hernandez, Graal Diaz</p><p><bold>Supervision:</bold>  Amanda Frugoli, Graal Diaz</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Community Memorial Health System IRB issued approval 2021-HSR010. Exempt Category 4.</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050525-i01\" position=\"float\"/>" ]
[]
[{"label": ["8"], "article-title": ["What is monocyte distribution width (MDW) and what role does it play in the early detection of sepsis?"], "person-group": ["\n"], "surname": ["Bentahar", "A"], "given-names": ["Bentahar", "A"], "year": ["2022"], "uri": ["https://www.beckmancoulter.com/en/blog/diagnostics/monocyte-distribution-width"]}, {"label": ["11"], "article-title": ["Histology, Monocytes"], "person-group": ["\n"], "surname": ["Espinoza", "Emmady"], "given-names": ["VE", "PD"], "publisher-loc": ["Treasure Island, FL"], "publisher-name": ["StatPearls Publishing"], "year": ["2023"], "uri": ["https://www.ncbi.nlm.nih.gov/books/NBK557618/"]}, {"label": ["14"], "article-title": ["Pooled analysis of monocyte distribution width in subjects with SARS\u2010CoV\u20102 infection"], "source": ["Int J Lab Hematol"], "person-group": ["\n"], "surname": ["Lippi", "Sanchis\u2010Gomar", "Henry"], "given-names": ["G", "F", "BM"], "fpage": ["0"], "lpage": ["3"], "volume": ["43"], "year": ["2021"], "uri": ["https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013932/"]}, {"label": ["15"], "article-title": ["Monocyte distribution width adds prognostic value in detection of COVID\u201019 respiratory failure"], "source": ["Int J Lab Hematol"], "person-group": ["\n"], "surname": ["Hossain", "Ayub", "Tarabichi"], "given-names": ["R", "S", "Y"], "fpage": ["0"], "lpage": ["6"], "volume": ["44"], "year": ["2021"], "uri": ["https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8652943/"]}, {"label": ["17"], "article-title": ["Comparison of monocyte distribution width (MDW) and procalcitonin for early recognition of sepsis"], "source": ["PLoS One"], "person-group": ["\n"], "surname": ["Polilli", "Sozio", "Frattari"], "given-names": ["E", "F", "A"], "fpage": ["0"], "volume": ["15"], "year": ["2020"], "uri": ["https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227300"]}]
{ "acronym": [], "definition": [] }
17
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50525
oa_package/2e/49/PMC10787605.tar.gz
PMC10787606
0
[ "<title>Introduction</title>", "<p>Immunoglobulin G4-related disease (IgG4-RD) is a rare immune-mediated fibro-inflammatory condition with a wide range of organs involved [##REF##21881964##1##,##REF##25865251##2##]. Progressive destruction of involved structures results from a growing deposition of IgG4-positive plasma cells within their tissues, leading to chronic inflammation and fibrosis [##REF##21881964##1##]. Even though physio-pathogenic and immunological mechanisms behind this disease are yet to be totally understood, histopathological patterns are well established and very similar through all the organs possibly involved [##REF##20733352##3##]. The mesenteric manifestation of IgG4-RD referred to as immunoglobulin G4-related sclerosing mesenteritis (IgG4-RSM) is an uncommon expression of IgG4-RD.</p>" ]
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[ "<title>Discussion</title>", "<p>Sclerosing mesenteritis (SM) is a rare idiopathic inflammatory disorder with varying degrees of inflammation, fat necrosis and fibrosis [##REF##30829677##4##]. IgG4-RD is a recently studied clinical entity, a systemic disease with a wide range of organs involved, including the mesentery and retroperitoneum. It consists of chronic inflammation with infiltration of IgG4-positive plasma cells within tissues involved. IgG4-RSM is an entity that started to emerge in the literature at the beginning of the 2010s, but remains sparsely reported [##REF##18682417##5##, ####REF##22449233##6##, ##REF##26581887##7##, ##UREF##0##8##, ##UREF##1##9####1##9##].</p>", "<p>Diagnosis criteria rely on clinical features, IgG4 serum levels, IgG4-positive plasma cells and pathological features such as storiform fibrosis, obliterative phlebitis and eosinophilia [##REF##28165852##10##,##REF##22596100##11##]. In our case, diagnosis was not certain after both biopsies; there was storiform fibrosis but no obliterative phlebitis, few IgG4-positive plasma cells and eosinophilia. IgG4 serum levels before treatment were not elevated. However, the multi-organ involvement because of the systemic nature of this disease, even though all imaging characteristics of an IgG4-RAP were not met [##REF##26473450##12##], and the fact that the patient responded clinically and at imaging to corticotherapy, were strong arguments for IG4-RD diagnosis. Indeed glucocorticoid responsiveness is considered as a major diagnosis criterion [##UREF##2##13##]. Additionally, radiological features of the mesenteric lesion were in line with an IgG4-RSM, displaying features such as soft mass appearance and fat preserved ring around mesenteric vessels. We can then claim with a high level of confidence that our multidisciplinary team was facing an IgG4-RSM case with a concomitant IgG4-RAP.</p>", "<p>Our patient had chronic abdominal pain and an abdominal mass feeling. Patients with IgG4-RSM can present diverse symptoms from digestive symptoms such as chronic abdominal pain, abdominal distention, abdominal mass feeling, nausea, vomiting and diarrhea to more systemic symptoms such as fever, anorexia, and weight loss [##UREF##0##8##].</p>", "<p>Usually, diagnosis is made after explorative surgery with pathological examination due to the fact that IgG4-RSM mimic neoplastic masses [##UREF##1##9##].</p>", "<p>The recommended initial systemic treatment for abdominal IgG4-RD is glucocorticoids [##UREF##2##13##]. Immunosuppressive agents can be considered after relapse or to maintain remission. More studies should be conducted to specify treatment modalities and treatment recommendations for IgG4-RSM that is a proper entity. Optimal treatment and prognosis may vary across different abdominal expressions of IgG4-RD.</p>" ]
[ "<title>Conclusions</title>", "<p>Our case displayed an IgG4-related sclerosing mesenteritis with concomitant IgG4-related autoimmune pancreatitis. The multi-organ involvement, radiological, biological, histopathological findings and glucocorticoid responsiveness favored an IgG4-related disease, even though pathological diagnosis elements were not met to assert a definitive diagnosis.</p>" ]
[ "<p>A 63-year-old male presented to our oncological hospital with a one-year evolving abdominal pain, with an abdominal mass feeling. Contrast-enhanced computed tomography displayed two soft tissue masses, one at the mesentery root and the second around the pancreatic tail; at the same time the patient presented with hyperlipasemia. Endoscopic biopsy for the pancreatic mass and surgical biopsy of the mesenteric one were performed in order to narrow diagnosis. No neoplastic cells but only dense fibro-inflammatory changes with immunoglobulin G4 (IgG4)-positive plasma cell inclusions were observed for both biopsies. A diagnostic and therapeutic strategy based on high suspicion of IgG4-related disease was adopted, with good clinical and imaging response to corticotherapy.</p>" ]
[ "<title>Case presentation</title>", "<p>A 63-year-old Caucasian male patient presented to our oncological hospital with a one-year evolving abdominal pain radiating to the back, with an abdominal mass feeling and a recent constipation. The physician only palpated epigastric mass with no peritoneal irritation symptoms at physical examination. The patient had a medical history of gastroduodenal peptic ulcers, a collarbone fracture and a family oncological history (sister breast cancer at 35 years old and father lung cancer at 58 years old). On laboratory examination complete blood count, cytolysis hepatic markers, renal and hepatic function, blood coagulation, ionogram, serum IgG4 concentration and tumor markers were normal. However, lipasemia was at 363 UI/L, above the normal range (60-260 UI/L).</p>", "<p>Contrast-enhanced abdominal computed tomography displayed a mesentery root soft mass with no noticeable mass effect on surrounding structures, with a small amount of fat preserved around some mesenteric vessels (Figure ##FIG##0##1##). A second mass around the pancreatic tail was observed with at the same time an enlargement of the pancreatic tail (Figure ##FIG##1##2##). A small amount of ascites was noticed in the pelvic floor.</p>", "<p>Positron emission tomography/computed tomography (PET-CT) displayed a low to moderate F-fluorodeoxyglucose (FDG) uptake of both masses and the pancreatic tail (Figure ##FIG##2##3##).</p>", "<p>First an echo-endoscopic biopsy of the pancreas and the mass around the pancreatic tail, then a surgical biopsy of the mesenteric mass was performed to narrow the differential diagnosis between a neoplastic cause, more likely a lymphoma due to the soft tissue nature of the different masses and an IgG4-RD with the concomitance of an IgG4-RSM and an IgG4-related autoimmune pancreatitis (IgG4-RAP).</p>", "<p>Histopathology of both biopsy samples showed storiform fibrosis, no obliterative phlebitis but a substantial white cell infiltration of fat tissues (lymphocytes, plasmocytes and neutrophils), some of the plasma cells were IgG4-positive (Figure ##FIG##3##4##), and no neoplastic cells were found.</p>", "<p>Based on the radiological biological and histopathological findings, the multidisciplinary medical team suspected IgG4-RSM with a concomitant IgG4-RAP and opted for a prednisone test and treat strategy. Prior to corticotherapy, IgG4 serum level was at 33.6 mg/dl (normal) and quiescent tuberculosis was assessed with a low dose Chest CT and Quantiferon tuberculosis (TB) serum level. After two weeks of oral prednisone at 0.6 mg/kg per day, patient's symptoms decreased. An abdominal CECT and PET-CT were performed at six weeks and at 18 weeks of treatment showed a size and activity decrease of the mesenteric and peri-pancreatic pseudo-masses with recovery of the normal aspect of the pancreatic tail (Figure ##FIG##4##5##). Prednisone was tapered after six weeks of treatment with a resolution of symptoms in the third month.</p>" ]
[]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Initial contrast-enhanced computed tomography (CECT)</title><p>CECT soft mesenteric root mass (arrowhead) displaying a small amount of preserved fat around a mesenteric vessel called the halo sign (arrow).</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Initial contrast-enhanced computed tomography (CECT)</title><p>Soft mass around the pancreatic tail (arrowhead). We can notice the moderate enlarged pancreatic tail within the mass, compared to its body (arrow).</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>Initial F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET-CT) </title><p>We can observe the FDG uptake on both mesenteric (arrow) on A and pancreatic mass (arrow) on B.</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG4\"><label>Figure 4</label><caption><title>Pathology</title><p>(A) The mesenteric tissue specimen reveals storiform fibrous proliferation (arrowhead) infiltrated by plasma cells and lymphocytes (arrow). (B) Immunostaining of IgG4-positive plasma cells (arrowhead). (C) Scattered eosinophils (arrowhead).</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG5\"><label>Figure 5</label><caption><title>Six-week follow-up</title><p>Contrast-enhanced computed tomography (CECT) (A and B) and F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET-CT) (C and D) after six weeks of treatment. Size and activity decrease of the pancreatic and mesenteric masses (arrows in A, B, C and D); notice the recovery of the normal aspect of the pancreatic tail (arrow on A).</p></caption></fig>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Ilias Bennouna, Maria Antonietta Bali, Ana Veron Sanchez</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Ilias Bennouna, Maria Gomez Galdon</p><p><bold>Drafting of the manuscript:</bold>  Ilias Bennouna, Ana Veron Sanchez</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Ilias Bennouna, Maria Antonietta Bali, Maria Gomez Galdon, Ana Veron Sanchez</p><p><bold>Supervision:</bold>  Ana Veron Sanchez</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
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[]
[{"label": ["8"], "article-title": ["A case of IgG4-related sclerosing mesenteritis"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Butt", "Alam", "Semeniuk", "Singh", "Chhabra", "Tan"], "given-names": ["Z", "SH", "O", "S", "GS", "IJ"], "fpage": ["0"], "volume": ["10"], "year": ["2018"]}, {"label": ["9"], "article-title": ["Sclerosing mesenteritis as an uncommon site of involvement of IgG4-related disease: a case report with an updated review of the literature"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Bertoni", "Giani", "Tozzini", "Di Natale"], "given-names": ["M", "A", "S", "ME"], "fpage": ["0"], "volume": ["14"], "year": ["2022"]}, {"label": ["13"], "article-title": ["European Guideline on IgG4-related digestive disease - UEG and SGF evidence-based recommendations"], "source": ["United European Gastroenterol J"], "person-group": ["\n"], "surname": ["L\u00f6hr", "Beuers", "Vujasinovic"], "given-names": ["JM", "U", "M"], "fpage": ["637"], "lpage": ["666"], "volume": ["8"], "year": ["2020"]}]
{ "acronym": [], "definition": [] }
13
CC BY
no
2024-01-14 23:43:49
Cureus.; 15(12):e50529
oa_package/6c/03/PMC10787606.tar.gz
PMC10787628
0
[ "<title>Introduction and background</title>", "<p>The term tissue dielectric constant (TDC) was originally introduced in 2007 to refer to the permittivity of biological tissue as measured at a frequency of 300 MHz [##REF##17853619##1##]. At that time, the aim was to evaluate the skin-to-fat tissue of the arm to characterize the relative amount of water in localized tissue to assess breast cancer-related lymphedema (BCRL). However, measurements of the dielectric constant of biological materials had long predated that time. Pioneering work in the application of such measurements was done by Schwan et al. [##REF##5970566##2##, ####REF##5798200##3##, ##REF##836937##4##, ##REF##2651001##5####2651001##5##], who built upon and extended the work of Cole et al. related to cell suspensions [##REF##19872447##6##,##REF##13880427##7##]. The use of radio and microwave frequencies combined with transmission line principles and associated mathematics led to the development of methods to measure dielectric constant values of tissues in vitro from various organs [##REF##7295367##8##, ####UREF##0##9##, ##REF##7111397##10##, ##REF##4048279##11##, ##REF##8938025##12####8938025##12##]. Measurements were made in vivo in a single subject [##REF##3399514##13##], but for the most part, these tissue measurements were confined to the laboratory given the equipment needed. However, based on analyses and engineering design efforts [##REF##9775538##14##, ####REF##9533128##15##, ##UREF##1##16####1##16##], a device in the form of a portable open-ended transmission line emerged [##REF##15132310##17##]. A commercialized version of the measurement system was developed that had four different-sized probes with diameters ranging from 10 to 55 mm and connected to a control box that generated a 300 MHz signal, as pictured in Figure ##FIG##0##1##.</p>", "<p>The 300 MHz signal was sent through the probe, which functioned as an open-ended transmission line. As a consequence, the reflected wave could be analyzed within the software of the control box to calculate the effective dielectric constant through well-established methods [##UREF##0##9##,##REF##9775538##14##,##REF##29874833##18##]. With further developments in technology, these processes were all integrated into a compact device, shown in Figure ##FIG##1##2##, that had an effective penetration depth of between 2.0 and 2.5 mm [##REF##26333210##19##].</p>", "<p>These developments in technology and capability allowed the utilization of TDC values for the assessment of skin tissue properties that were revealed by the dielectric constant of the tissue volume interrogated by the incident electromagnetic wave. One of these properties relates to the amount of water within the measured tissue volume since the TDC value is strongly dependent on tissue water content [##REF##32775853##20##]. As a consequence, measurements of TDC provide a convenient method to assess tissue water and its change. Capitalizing on this property, some of the earliest medical applications targeted conditions in which tissue water and its change were of clinical interest, such as cerebral edema [##REF##2089916##21##, ####REF##1355280##22##, ##REF##10493075##23####10493075##23##] or lung edema [##UREF##2##24##] which were studied in experimental animals. Applications that used probes similar in function to those shown in Figure ##FIG##0##1## and applied to humans in 2003 targeted changes in skin-to-fat water associated with cardiac surgery in a group of 29 patients [##REF##12812423##25##]. Subsequently, TDC measurement use has expanded to a variety of medical applications. One of these is related to assessing upper extremity lymphedema, which is a complication of breast cancer treatment referred to as BCRL. This condition has an incidence that ranges from 20 to 60% [##REF##37436762##26##, ####REF##31874124##27##, ##REF##30521080##28##, ##REF##29397555##29##, ##REF##28992556##30####28992556##30##] and is evaluated with various methods to detect its presence and track its progression. These include arm circumference measurements [##UREF##3##31##, ####REF##34176909##32##, ##REF##34121139##33##, ##REF##28749717##34####28749717##34##], arm volume measurements [##REF##33761290##35##, ####REF##33667586##36##, ##REF##23912961##37##, ##REF##15560108##38##, ##REF##12444879##39####12444879##39##], and the determination of the electrical impedance of the arms [##REF##36946918##40##, ####REF##28831632##41##, ##REF##26359689##42##, ##REF##25141461##43##, ##REF##19522677##44####19522677##44##]. This same type of measurement can be applied when the edematous or lymphedematous condition is in the lower extremities [##REF##35860819##45##, ####REF##12570322##46##, ##REF##12669673##47##, ##REF##8277752##48####8277752##48##]. However, when it comes to measuring localized edema or lymphedema, either at specific locations on the limbs or anywhere on the body where lymphedema detection or tracking is indicated, there are limited methods available. One such method, which is the main subject of this paper, is the measurement of TDC. Thus, the specific aim herein was to review and describe the uses and findings of TDC measurements, considering and including their wide array of medical applications.</p>" ]
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[ "<title>Conclusions</title>", "<p>The findings demonstrate multiple research and medical uses and applications of TDC measurements, focusing on detecting and quantifying localized edema and lymphedema in multiple target sites. These include the upper and lower extremities, breasts, and trunk as regions involved in medical conditions causing lymphedema. In addition, the findings suggest that TDC evaluations are a convenient, non-invasive method to study and evaluate other conditions impacting skin, including DM and skin wounds or ulcers. Its ability to detect aspects of tissue changes simply and rapidly at almost any anatomical location makes it a useful tool for investigating multiple dermatological conditions and their treatment as future applications of this method.</p>" ]
[ "<p>Tissue dielectric constant (TDC) values assess certain skin properties that are dependent on multiple factors but mainly on the relative amount of water content within a locally measured tissue volume. Because of the non-invasive nature of these measurements and their ease of use, the method has been widely used in various medically related applications. The goal of this paper was to review and describe the uses and findings of such TDC measurements, considering and including the wide array of medical applications. The review is in part based on information derived from an analysis of published material obtained via literature searches of four major electronic databases and, in part, based on the author’s experience with the TDC measurement methods and their various applications and his professional experiences. The databases searched were PubMed, Web of Science, EMBASE, and CINAHL Complete. Based on the initial search criteria, a total of 1257 titles were identified. After removing duplicates and filtering according to relevancy, 160 remained for detailed further review. In some cases, the bibliography of these retrieved articles provided additional sources. The findings demonstrate multiple research and medical uses and applications of TDC measurements, focusing on detecting and quantifying localized edema and lymphedema in multiple target sites. These include the upper and lower extremities, breasts, and trunk as regions involved in medical conditions causing lymphedema. In addition, the findings suggest that TDC evaluations are a convenient, non-invasive method to study and evaluate other conditions impacting skin, including diabetes mellitus and skin wounds or ulcers. Its ability to detect aspects of tissue changes simply and rapidly at almost any anatomical location makes it a useful tool for investigating multiple dermatological conditions and their treatment as future applications of this method.</p>" ]
[ "<title>Review</title>", "<p>Methods</p>", "<p>This review is in part based on information derived from an analysis of published material obtained via literature searches of four major electronic databases and, in part, based on the author’s experience with the TDC measurement methods and their various applications, his professional experiences, and the original material of the author. The databases searched were PubMed, Web of Science, EMBASE, and CINAHL Complete. The primary search term strategy for each of these was as follows: The term “dielectric constant” was searched in combination with a logical AND for the term’s “skin” or “edema” or “lymphedema” as (dielectric constant) AND (skin OR edema OR lymphedema). Using this strategy, a total of 1257 titles were identified. Duplicate titles from the four databases were eliminated, and the retrieved titles were screened for potential relevance via an abstract review for further clarifications if warranted. This review and filtering process reduced the likely relevant articles to 160. The present author was a primary or co-author on about half of these (59), and the remainder were retrieved if not already available and reviewed. In some cases, the bibliography of the retrieved articles provided additional sources. Supplemental searches were done as needed. The manuscript is organized as follows: The first part describes general aspects of TDC applications by considering many of the main factors that impact their value and interpretation. These include factors such as the number of measurements per site, variations by anatomical location, depth of the measurement, subject age, gender, or race, and even the time of day of the measurement. This is followed by a review of its many medical applications.</p>", "<p>General aspects</p>", "<p>Each available device has pros and cons. The multiprobe set (Figure ##FIG##0##1##) can measure to varying depths (0.5 mm to 5.0 mm) [##REF##18540873##49##] but is less mobile than the compact version (Figure ##FIG##1##2##). The compact device also has a pressure sensing process to help increase repeatability but offers measurements only to a single depth [##REF##32268598##50##].</p>", "<p>Number of TDC Measurements per Site</p>", "<p>The measured TDC value is localized in that it reflects the value of the tissue volume roughly equal to the product of the probed diameter and its effective measurement depth. Because the TDC value largely depends on tissue water content, it is useful in the assessment of edema, lymphedema, and its change. As with any such measurement, there is a need to balance the number of measurements per site with the time required for multiple measurements. This aspect has been studied [##REF##19306665##51##,##REF##19076729##52##].</p>", "<p>In one study, similar TDC values were found based on a single measurement or the average of triplicate measurements in healthy and lymphedematous arms. In that study, ventral forearms were measured bilaterally to a depth of 2.5 mm in 10 women with unilateral arm lymphedema. The 95% confidence interval for differences between single and averaged values was found to be less than ± 1 TDC unit for both arms [##REF##19306665##51##]. Another study measured the anterior forearms of 20 healthy women to 0.5, 1.5, 2.5, and 5.0 mm depths and also measured the lateral thorax bilaterally on 10 women with BCRL to a depth of 2.5 mm. The 95% confidence interval for differences between single and averaged TDC values was less than ± 1 TDC unit, and the limits of agreement between methods were less than ± 2.5 TDC units (± 6.5%) for each condition, site, and depth measured. If this level of agreement is acceptable, suitable clinical assessments can be made using a single TDC measurement.</p>", "<p>Extensions to other anatomical locations were carried out on a group of 60 female subjects [##REF##30339476##53##]. TDC was measured bilaterally in triplicate at the forearm, hand palm, lateral calf, medial calf, and foot dorsum. Single, duplicate, and triplicate values were compared for absolute TDC values and inter-side ratios. Differences between single and multiple measurement averages were anatomical site-dependent, with the smallest coefficient of variation (2.19%) at the forearm and the largest at the lateral calf (4.59%). Thus, if clinical time is of major concern, useful TDC data may be obtained in upper limbs by a single TDC measurement per site, but lower extremity skin assessments are best with duplicate and preferably triplicate measurements.</p>", "<p>Variations in TDC Values by Anatomical Location</p>", "<p>This issue has been studied at various sites on the forearm, different upper arm sites, and lower extremities [##REF##18540873##49##,##REF##20923455##54##, ####REF##23046199##55##, ##REF##30427746##56####30427746##56##]. In a study of 30 healthy women, TDC was measured on the anterior forearm midline and also 1.2 cm medial and lateral to the midline at sites 4, 8, and 12 cm distal to the antecubital crease [##REF##20923455##54##]. The midline and medial TDC values increased progressively from 4 to 8 to 12 cm sites (p&lt;0.001). At a depth of 2.5mm, TDC values increased, with a maximum difference of 8.2 ± 10.6%. For all sites, TDC values were lower (p&lt;0.001) with increasing depths. Thus, TDC increases from proximal to distal sites and should be considered since such differences are important when evaluating patients with arm lymphedema [##REF##30427746##56##]. Handedness does not appear to have an impact on measured TDC values [##REF##28795491##57##].</p>", "<p>Other information on the variability of TDC by location emerged from a study of 36 women who were followed for 78 weeks after their breast cancer surgery [##REF##30427746##56##]. TDC and arm girth were measured bilaterally at multiple arm sites on multiple occasions. An inverse correlation was found between TDC and girth (p&lt;0.001), but TDC values decreased with distance from the wrist (p&lt;0.001). Differences in TDC values between anterior and medial sites at the same longitudinal position have also been investigated [##REF##31525830##58##]. Forearm TDC was measured to multiple depths in 40 women, with no statistical differences between anterior and medial sites found. In that study, a moderate correlation was found between the total body water percentage and the TDC value, which was greatest when measured to a depth of 5 mm.</p>", "<p>Variations in TDC Values by Measurement Depth</p>", "<p>TDC values depend on free and bound water in the measured tissue volume; thus, tissues with more fat hold less water, and the TDC value is lower. Thus, in anatomical areas with more subcutaneous fat, TDC values tend to be lower, especially when measurements are made at deeper levels. This finding has been experimentally demonstrated [##UREF##4##59##, ####REF##26040759##60##, ##REF##25525747##61####25525747##61##]. In one of these studies, the anterior forearm TDC was determined bilaterally at depths of 0.5, 1.5, 2.5, and 5.0 mm in 40 healthy women with BMI values within the normal range. This determination was then compared to the subjects' total body water percentage (TBW%) and arm percentage fat (AF%) [##REF##31525830##62##]. TDC was found to directly correlate with TBW% (r=0.512, p=0.001) and inversely correlate with AF% (r= -0.494, p&lt;0.001). The correlations were found to be greatest at the deepest measurement depth. When TDC was measured in overweight or obese women, a similar depth dependence was obtained for measurements made on the forearms and biceps [##REF##31556162##63##]. TDC differences between women who were overweight and obese were not apparent, possibly due to a threshold for TDC dependence on body fat percentage.</p>", "<p>In contrast to the decreasing TDC values with increasing measurement depth obtained in the arm or leg, at some anatomical sites, the pattern may be different, as was demonstrated via measurements made on the hand palm, thenar eminence, great toe, and foot dorsum to depths of 0.5, 1.5, and 2.5 mm [##REF##23046199##55##]. TDC values decreased with increasing depth at the hand palm, thenar eminence, and great toe, but there was essentially no change on the hand and foot dorsum. The depth to measure depends on the measurement goal, considering that some processes start deeper in tissue and others are shallower.</p>", "<p>Variations in TDC Values by Age</p>", "<p>The age factor was investigated in a group of 69 women in whom TDC was measured to depths of 0.5, 1.5, 2.5, and 5.0 mm on both forearms [##REF##20384879##64##]. The women evaluated had a broad age range of 22-82 years and a BMI of 18.7-46.1 kg/m2. TDC values decreased with increasing depth (33.7 ± 5.8 at 0.5 mm to 21.8 ± 3.7 at 5.0 mm), but at all depths, the inter-arm ratios did not differ and ranged from 1.025 ± 0.081 at 0.5 mm to 1.017 ± 0.097 at 5.0 mm. TDC values at a depth of 0.5 mm and 1.5 mm increased with increasing age, but there was no age dependence of TDC values at 2.45 or 5.0 mm. Thus, TDC values are affected differentially by age in a depth-dependent way.</p>", "<p>More information about the age factor emerged from a study of 200 women in which 50 women had TDC measurements made bilaterally on anterior forearms at either 0.5, 1.5, 2.5, or 5.0 mm depths [##REF##26038154##65##]. The age factor was studied by comparing subjects ≤40 years with subjects ≥60 years. TDC values at 0.5 and 1.5 mm were reported to be greater for older women (p&lt;0.001). Similar but less dramatic age-related changes were uncovered in 60 males in whom TDC was measured bilaterally to the same multi-depths on the forearm [##REF##26278683##66##]. There were three groups of 20 each compared: 24.0 ± 0.9, 40.0 ± 12.9, and 71.0 ± 8.0 years. Except at a 0.5 mm depth, there was no statistical TDC difference among these groups. Taken together, the female and male results may be due to skin water shifting from bound to more mobile with increasing age. Similar results were reported for a group of 60 women in whom an age-related increase in TDC was found only to a depth of 0.5 mm [##REF##29280546##67##]. Age-related changes impact long-term longitudinal tracking studies and also shape what reference values are in relation to a patient’s age.</p>", "<p>Variations in TDC Values by Gender</p>", "<p>The potential gender factor was investigated in a study of 30 men and 30 women in whom TDC was measured on their forearms, forehead, and cheeks to a depth of 1.5 mm [##REF##22142446##68##]. TDC values had a wide range from 39.6 ± 2.9 at the forehead of the male group to 28.2 ± 2.4 at the forearm of the female group. TDC values were significantly different (p&lt;0.001) among each site in the order forehead &gt; cheek &gt; forearm. Male TDC values were greater than those measured in females at corresponding anatomical sites (p&lt;0.01), with differences ranging from 5.6% at the forehead to 11.3% at the forearm. The gender factor was consistent with later findings, in which 32 male and 32 female subjects were evaluated [##REF##26333210##19##]. In this study, the device used was the compact version with a fixed measurement depth of between 2.0 and 2.5 mm. TDC data was obtained bilaterally on forearms and biceps, with the result that female values were significantly less than those obtained from males at similar anatomical sites. In a subsequent investigation of forearm TDC values in 280 young adult subjects per gender, the greater TDC values for males vs. females were confirmed for all depths of 0.5, 1.5, 2.5, and 5.0 mm [##REF##26040759##60##]. Male-female percentage differences ranged from 14.8% to 22.0%. The overall gender-related findings suggest that gender differences should be considered in any study in which men and women are included in a common study population with respect to experimental design and data interpretation. This is especially true if absolute TDC values are of interest rather than changes in TDC on the same subject subsequent to an intervention.</p>", "<p>Variations in TDC Values by Race</p>", "<p>Potential differences in TDC among persons of different races or ethnicities were investigated in 100 persons with 20 subjects each self-identifying as Caucasian, African-American, Asian Indian, Asian, or Hispanic [##REF##28266760##69##]. TDC was measured at a depth of 1.5 mm on the forearms. For females (10 per group), analysis of variance among races indicated an overall significant difference among races (p&lt;0.01), with the difference mainly due to the larger TDC value of Caucasians vs. Asian-Indians (p&lt;0.05). Also, TDC values for Asian and Asian-Indian groups were lower than for Caucasians. Males did not show a difference among races at the forearm but did have an apparent race-related dependence at other sites, including the chest and foot. Follow-up analyses indicated the chest difference was due to a TDC value of African Americans that was larger than either Caucasians or Asian-Indians (p&lt;0.01). Contrastingly, differences at the ankle were due to a larger TDC value in Caucasians with respect to all other groups (p&lt;0.01). Thus, TDC dependence on race is a factor that should be considered in assessing skin hydration comparisons that include different races. However, despite these variations among races, no such differences were found between inter-arm TDC ratios, and this ratio remains a robust indicator of unilateral tissue water changes.</p>", "<p>Variations of TDC Values by Time of Day</p>", "<p>Since TDC measurements may be made at variable times during the day, knowledge of potential variations in their value is useful, especially in longitudinal assessments of patients. To investigate this factor, TDC was measured multiple times during the day in a group of 12 young adult females [##UREF##5##70##]. Measurements were made at one-hour increments starting at 0800 and ending at 2000 hours at four anatomical sites: the face just below the eye, mid-cheek, anterior forearm, and medial calf. Results indicated that TDC at the eye, cheek, and forearm decreased during the day with morning-to-night changes of 11.2 ± 8.3%, 6.8 ± 5.7%, and 5.6 ± 6.0%, respectively. Contrastingly, calf TDC values progressively increased with morning-to-night increases of 9.3 ± 10.7%. Thus, when absolute values of TDC are of relevance, the time of day of measurement may be important. However, when inter-limb ratios are used to assess the water status, the time of day is of much less importance [##UREF##6##71##].</p>", "<p>Reliability Considerations</p>", "<p>To gain insight into the reliability of these TDC measurements, a test-retest investigation was done in which TDC was measured in 40 healthy persons by two measures [##REF##30526306##72##]. The measurements were made on multiple anatomical sites and were able to estimate the minimum detectable change (MDC) of TDC measurements and the interclass coefficients (ICC). The MDC was between two and nine TDC units, with inter-side ratios ranging from 5.3% to 8.0%. ICC values were between 0.765 and 0.982. These variations depended on the measurement depth and the body fat and water percentage. These findings were amplified in a study of 30 women who had confirmed BCRL and in whom intra- and inter-rater reliability was determined with TDC measurements at multiple sites [##REF##31329510##73##]. ICC values were between 0.648 and 0.947 for intra-rater measurements and between 0.606 and 0.941 for inter-rater measurements. The range was dependent on the anatomical sites measured.</p>", "<p>TDC applications in relation to BCRL</p>", "<p>Upper Extremity Lymphedema</p>", "<p>A study that focused on TDC measurements in relation to BCRL was reported in 2007 [##REF##17853619##1##]. TDC was measured bilaterally to multiple depths in the forearms of 18 healthy women who served as controls and in 15 women who had been diagnosed with unilateral BCRL (patients). TDC in the lymphedematous arms of the patients was greater than in their contralateral arms (p&lt;0.001). At a measurement depth of 2.5 mm, inter-arm TDC ratios for patients were 1.64 ± 0.30 vs. 1.04 ± 0.04 for controls (p&lt;0.001). No patient’s TDC ratio was ≤1.2, and no control’s TDC ratio was ≥1.2.</p>", "<p>Further work evaluated 90 women, including 30 women with unilateral BCRL, 30 women with breast cancer awaiting surgical treatment, and 30 healthy controls [##REF##19778203##74##]. TDC was measured bilaterally to determine inter-arm TDC threshold ratios. TDC values did not differ between control groups, but the women with BCRL had higher values. Inter-arm ratios for the 60 women without lymphedema were 1.006 ± 0.085 and were significantly less than for the women with BCRL, who had an inter-arm TDC ratio of 1.583 ± 0.292. An at-risk/contralateral TDC ratio of 1.26 was suggested as a possible threshold for detecting preclinical or latent lymphedema.</p>", "<p>To further study the utility of these inter-arm TDC thresholds for detecting and tracking BCRL, TDC values were obtained in 240 women [##REF##25420307##75##]. A threshold was defined in this case as the mean value plus three standard deviations (SD) above the mean. Based on these measurements, a threshold TDC ratio of 1.2 was suggested. Further investigation of the use of inter-side ratios for early detection was undertaken by measuring 100 women who had been treated with breast surgery, axillary dissection, and radiotherapy [##REF##26305554##76##]. It was determined that 18.4% of early BCRL were detectable only with this method because early events started localizing superficially. Also, of the 38% of patients who had clinical lymphedema, the TDC method was reported to have a sensitivity of 65.8% and a specificity of 83.9%.</p>", "<p>Further studies were done on 207 women, of whom 104 had experienced breast surgery for breast cancer and 103 had breast cancer but were awaiting their surgery [##REF##25388057##77##]. TDC was measured to 2.5 mm bilaterally in forearms and biceps, and inter-arm ratios were determined. An inter-arm TDC threshold for early detection of pre-clinical unilateral lymphedema of 1.3 for the forearm and 1.45 for the biceps was suggested by the data. The threshold criteria were used to track 80 women who had TDC measurements done prior to their breast cancer surgery and then for up to 24 months afterward [##REF##25525747##61##]. In another study, TDC values (forearms and biceps) were obtained in 42 women who were awaiting breast cancer surgery and in 41 healthy women, for a total of 83 inter-arm assessments [##REF##29906062##78##]. A three SD inter-arm threshold ratio of 1.20 and 1.24 for the forearm and biceps was obtained. A test of the suitability of this ratio was done by evaluating 63 women, of whom 32 had BCRL and 31 had breast surgery for cancer but, as of yet, were free of BCRL [##REF##28749723##79##]. Sensitivity and specificity based on inter-arm TDC values were reported as 65% and 94%, respectively. Other studies determined threshold TDC ratios for hands [##REF##29862913##80##]. In this study, 70 women were evaluated, with half &lt;35 and half &gt;50 years of age. An age-independent two SD threshold of 1.23 was reported based on the findings of this study. A study that compared using TDC inter-arm thresholds vs. water displacement found that both were useful, but the TDC method, when used alone, could diagnose lymphedema earlier than the water displacement method [##REF##31596662##81##]. TDC inter-arm ratios have also been used to track the need for reestablishing early-initiated compression to minimize the likelihood of transitioning to chronic lymphedema [##REF##37345010##82##]. It is also noteworthy that TDC measurements have been used to assess the outcomes of treatments such as local cooling [##REF##30234242##83##] and radiotherapy [##REF##36799119##84##].</p>", "<p>Breast Lymphedema</p>", "<p>Lymphedematous conditions of BCRL do not just involve one arm area, thereby indicating the need for assessments of TDC values in other areas. This was done in a group of women awaiting breast cancer surgery (patients) and a group of healthy women (controls) [##REF##18540873##49##]. Measurements were made at the mid-forearm, mid-biceps, axilla, and lateral thorax at a depth of 2.5 mm. TDC was largest at the axilla (36.4 ± 8.9) and least at the biceps (21.6 ± 3.5). Patient vs. control comparisons showed slightly greater forearm and biceps TDC values for patients but no difference in inter-side TDC values in patients or controls, suggesting that cancer presence itself did not significantly affect TDC values. Additional studies extended these investigations by evaluating 120 women who were awaiting breast cancer surgery [##REF##30422435##85##]. The main aim was to formulate inter-side lateral thorax thresholds. The findings suggested a 2.5 SD thorax-to-thorax TDC threshold ratio of 1.32.</p>", "<p>Breast edema or lymphedema can also be present as a complication of breast cancer-related treatment and may be present with or without arm or trunk lymphedema and other conditions [##UREF##7##86##]. Breast TDC values have been investigated as a way to better characterize the breast edematous condition [##UREF##8##87##, ####UREF##9##88##, ##REF##32378054##89##, ##REF##31233373##90##, ##REF##33761280##91####33761280##91##]. Based on healthy inter-breast TDC values, an inter-breast diagnostic two SD threshold ratio of 1.40 was reported and applied to 118 women who had received breast cancer surgery and radiotherapy [##UREF##8##87##]. Based on the 1.40 threshold criterion, it was found that 31.4% of patients had breast edema after surgery, even prior to the end of their radiotherapy. This percentage increased to 62.4% four weeks after the end of radiotherapy. A longer follow-up time of about two years included 65 of these patients, indicating the percentage of these that had inter-breast ratios above the threshold was 28% [##UREF##9##88##].</p>", "<p>The 1.40 threshold criterion was used to investigate a group of 10 women with BCRL [##REF##32378054##89##]. The TDC values of the breast on the affected side were significantly elevated in comparison with the contralateral breast. In addition, the TDC ratio was related to the breast drainage pathway pattern and dermal backflow. TDC methods were also of use in characterizing changes in breast edema in 56 breast cancer-treated patients who had breast edema as determined by the 1.40 threshold [##REF##31233373##90##]. After wearing either a standard or a compression bra for nine months, a reduction in TDC was observed that was correlated with the patient’s perceived feeling of breast heaviness.</p>", "<p>Trunk Lymphedema</p>", "<p>Another area of interest in patients who have had breast cancer-related surgery is the development of lymphedema in the trunk [##REF##32379872##92##, ####UREF##10##93##, ##REF##36223240##94##, ##UREF##11##95####11##95##]. An interesting observation of this condition has been made from MRI [##REF##33185495##96##]. In this study, a group of 13 women who had BCRL and swelling or subjective symptoms of truncal lymphedema was evaluated. All patients had subjective symptoms and seven had visible swelling, yet the MRI revealed no free water signals on the affected side of the trunk of any patient. Herein, it may be theorized that the swelling and symptoms may be attributable to bound water which is measurable by TDC methods. As noted, these measurements have been taken from healthy subjects, suggesting inter-side thresholds for lymphedema of 1.32. However, they have not yet been correlated with MRI data.</p>", "<p>Applications of TDC measurements to lower extremities</p>", "<p>Normal Features, Variability, and Thresholds for Lower Extremity TDC Values</p>", "<p>Characteristics of TDC values and features of lower extremities are less studied than those for upper extremities. The locations of lower extremity measurements depend on the purpose of the evaluation. Sites that have been measured include the foot dorsum, the calf or gaiter area (as illustrated in Figure ##FIG##2##3##) on medial, anterior, and lateral sites, and in some cases the thigh.</p>", "<p>In one study designed to investigate site variability, TDC was measured at multiple longitudinal sites on the legs of 18 healthy women at 10, 20, 30, and 40 cm proximal to the medial malleolus on the medial aspect of the leg [##UREF##12##97##]. In that study, except for the 40 cm location, there was little variability among the TDC values obtained at the other sites. In addition, day-to-day reproducibility via test-retest was reported as excellent. Other measurements of the lower extremities of 30 healthy women showed significant differences between TDC values at the foot, ankle, and calf [##REF##22681610##98##]. TDC values in that study ranged from 37.8 ± 5.5 on the foot to 30.5 ± 3.9 on the lower leg. These workers suggested that TDC values of 35.2 for the ankle and 38.3 for the lower leg are useful as upper normal reference limits in women. The reliability of lower extremity TDC measurements was assessed in a group of 61 healthy subjects (33 female) in whom TDC was measured at multiple leg sites twice, two weeks apart [##REF##31697608##99##]. For TDC measurements, considering all measurement locations, the standard error of the measurement was reported to range from 3.9% to 14.5%. It was suggested that TDC values made near the tendon or bone be carefully interpreted.</p>", "<p>Other measurements aimed at characterizing potential differences to be expected among circumferential leg TDC values reported small but statistically significant differences among medial, anterior, and lateral sites when assessed at multiple depths. As a consequence, it was suggested that this fact be considered when evaluating or tracking tissue water changes. The question of differences in TDC values between paired legs was also investigated in a small group of 10 women to determine a potential inter-leg threshold ratio usable for detecting unilateral leg lymphedema [##UREF##6##71##]. Based on this small sample, an inter-leg threshold of 1.2 was suggested. Because lower extremity lymphedema (LEL) is often bilateral, the search for a method that was not based on inter-leg ratios was sought and proposed [##REF##30860343##100##,##REF##30698489##101##]. This method used the ratio of leg-to-forearm TDC ratios obtained from healthy persons to set a potential lymphedema threshold at 1.35. TDC values above this would suggest the presence of LEL, and changes in this value could be used to track treatment outcomes. Other studies on patients with LEL were consistent with this threshold criterion [##REF##37172282##102##].</p>", "<p>Lower Extremity Lymphedema</p>", "<p>LEL is a complication of various conditions, including gynecological surgery for various cancers [##REF##36222950##103##, ####REF##34545757##104##, ##UREF##13##105####13##105##], as well as for other cancers such as melanoma [##REF##37656769##106##], prostate [##REF##34477972##107##] cancer, and other non-cancer-related conditions [##REF##31992537##108##]. The reported incidence of LEL depends on the method used to determine its presence and ranges from about 35% to 70% [##REF##16740298##109##, ####REF##31837831##110##, ##REF##27400319##111####27400319##111##]. The applications of TDC to assessing lower extremity skin features and its potential use for diagnosis and tracking of LEL are more limited than for similar uses in BCRL. In one study, the sensitivity to detect LEL in a group of 106 patients treated mostly for endometrial cancer (N = 52) or ovarian cancer (N =40) was reported to be greater than the standard leg circumference measurement procedure [##UREF##14##112##]. In that study, bilateral TDC measurements were made on the thigh and calf of the women for a mean follow-up time of about three years. Pitting edema was observed in up to 17.5% of patients, and these patients had a greater measured TDC value than those patients without pitting (35.9 ± 10.4 vs. 27.2 ± 7.5). With respect to quantifying the presence of LEL or tracking its progression, a leg/arm threshold ratio of 1.35 may be useful [##REF##34545757##104##].</p>", "<p>Assessing Lymphedema Treatment</p>", "<p>Various treatments for LEL are available and include manual lymphatic drainage (MLD) as the most frequently used [##REF##34929073##113##, ####REF##32107315##114##, ##UREF##15##115####15##115##]. One study utilized TDC measurements to try to assess the utility of a single MLD treatment session and was able to use pre- and post-treatment TDC measurements to achieve this [##REF##18720916##116##]. Other treatment modalities for LEL that have used TDC measurements to assess outcomes include laser therapy [##REF##22458118##117##] and pneumatic compression [##REF##30281381##118##]. In addition, the use of TDC measurements has been evaluated as a method to distinguish LEL from lipoedema [##REF##24033279##119##].</p>", "<p>Applications of TDC measurements for various conditions</p>", "<p>Diabetic Skin Assessment</p>", "<p>The potential linkages between diabetes mellitus (DM) and potential skin problems are well documented [##REF##27583022##120##, ####REF##27432328##121##, ##UREF##16##122####16##122##]. Because of this connection, some studies have attempted to use TDC measurements to assess certain features of skin in persons with DM. To investigate skin water status, TDC was measured in 36 persons (18 with DM) to depths of 0.5, 1.5, and 2.5 mm at the foot dorsum and forearm bilaterally [##REF##23145992##123##]. It was reported that all persons with DM had higher foot dorsum TDC values than those without DM, whereas values at the forearm did not significantly differ between groups. The TDC values of the DM group, when measured to a depth of 2.5 mm, were about 15% greater. This finding suggested that unrecognized edema was present. Subsequent research focused on the potential link between HbA1c values and skin water as measured using the TDC method [##REF##27491529##124##]. In that study, TDC was measured in 50 patients with DM at depths of 0.5, 1.5, 2.5, and 5.0 mm at the foot dorsum, calf, and forearm. Though there was a significant increase in foot-to-arm TDC values, there was no significant relationship found between the absolute TDC value and HbA1c or the patient’s fasting glucose level.</p>", "<p>Dehydration Status</p>", "<p>Because TDC values are strongly dependent on skin tissue water, the use of this method to assess body water status has been evaluated in several studies. The possibility of tracking fluid status using TDC measurements was evaluated by measuring TDC on the forearm, thigh, and abdomen in 29 patients prior to their cardiac surgery and for some days afterward [##REF##12812423##25##]. By weighing the patient, a significant correlation between TDC values and weight was reported for weight gain from baseline to the second post-operative day (r = 0.600, p&lt;0.01) but not for a weight loss interval from days two to four. These data leave the question of tracking dehydration status unresolved. However, more recent information derived from 10 healthy exercising subjects indicates a tracking of hydration status via TDC values [##REF##35179449##125##]. Other studies, in which the relationship between total body water percentage and measured TDC values was investigated, were also consistent with TDC being a possible indicator of body fluid status [##REF##31525830##62##].</p>", "<p>Wound Feature Assessment</p>", "<p>Several workers have addressed the issue of using TDC measurements in conjunction with wound assessments. An early study compared wounded skin with normal skin and reported that the wounded skin had about a 12% higher value [##REF##3579996##126##]. Subsequently, the open-ended coaxial line method was used to evaluate the skin injury produced by irradiated breast skin [##REF##9681887##127##]. A two-year follow-up of 21 patients indicated as high as a 39% reduction in TDC that correlated with the presence of subcutaneous fibrosis. Pressure ulcers in patients with spinal cord injury were evaluated using this method to a depth of 1.5 mm, in which peri-wound skin areas were compared to non-injured skin [##REF##25398030##128##]. The values obtained were among the first to report peri-wound values in this type of setting and condition. Further research into the use of TDC-related measurements indicated their potential use to detect early phases of pressure ulcer development at the sacrum [##REF##28494507##129##] and heel [##REF##29250926##130##]. The TDC method has also been suggested as a useful approach for characterizing and tracking changes in lymphedema associated with head and neck cancer [##REF##33529086##131##].</p>" ]
[]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>TDC probes and measurement applications on the arm</title><p>(A) and (B) show measurements on the forearm and biceps using the 2.5 mm depth probe. Part (C) shows the probes available, their outside diameter, and their effective measurement depths. The probes are connected to a control box that generates a 300 MH signal and processes the reflected signal that is used to determine the TDC value that is displayed on the display. The figure is courtesy of Dr. HN Mayrovitz.</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Compact version of the TDC measurement device</title><p>The outside diameter of the outer electrode is shown as 20 mm. The figure illustrates a measurement of the forearm with the compact device. The figure is courtesy of Dr. HN Mayrovitz.</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>TDC measurement example on the lower extremity</title><p>A compact version is shown measuring in the gaiter area of the right leg of a patient. Note that the display on the compact device shows the TDC value (36.7) and also the percentage of water (46.1%) at this location. This figure is courtesy of Dr. HN Mayrovitz.</p></caption></fig>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Harvey N. Mayrovitz</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Harvey N. Mayrovitz</p><p><bold>Drafting of the manuscript:</bold>  Harvey N. Mayrovitz</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Harvey N. Mayrovitz</p><p><bold>Supervision:</bold>  Harvey N. Mayrovitz</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050531-i01\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050531-i02\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050531-i03\" position=\"float\"/>" ]
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[{"label": ["9"], "article-title": ["Measurement of radio frequency permittivity of biological tissues with an open-ended coaxial line: part II - experimental results"], "source": [" IEEE Trans Microw Theory Tech"], "person-group": ["\n"], "surname": ["Stuchly", "Athey", "Samaras", "Taylor"], "given-names": ["MA", "TW", "GM", "GE"], "fpage": ["87"], "lpage": ["92"], "volume": ["30"], "year": ["1982"]}, {"label": ["16"], "article-title": ["Penetration of electromagnetic fields of an open-ended coaxial probe between 1 MHz and 1 GHz in dielectric skin measurements"], "source": ["Phys Med Biol"], "person-group": ["\n"], "surname": ["Alanen", "Lahtinen", "Nuutinen"], "given-names": ["E", "T", "J"], "fpage": ["0"], "lpage": ["76"], "volume": ["44"], "year": ["1999"]}, {"label": ["24"], "article-title": ["Time domain reflectometry: measurement of free water in normal lung and pulmonary edema"], "source": ["Am J Physiol"], "person-group": ["\n"], "surname": ["Miura", "Shioya", "Kurita", "Shigematsu", "Mashimo"], "given-names": ["N", "S", "D", "T", "S"], "fpage": ["0"], "lpage": ["12"], "volume": ["276"], "year": ["1999"]}, {"label": ["31"], "article-title": ["Effect of physical therapy on circumference measurement and extremity volume in patients suffering from lipedema with secondary lymphedema"], "source": ["Lymphat Res Biol"], "person-group": ["\n"], "surname": ["Esmer", "Schingale"], "given-names": ["M", "FJ"], "year": ["2023"]}, {"label": ["59"], "article-title": ["Circumferential and depth variations in tissue dielectric constant values as indices of lower leg localized skin water"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Mayrovitz"], "given-names": ["HN"], "fpage": ["0"], "volume": ["14"], "year": ["2022"]}, {"label": ["70"], "article-title": ["Diurnal changes in local skin water assessed via tissue dielectric constant at 300 MHz"], "source": ["Biomed Phys Eng Express"], "person-group": ["\n"], "surname": ["Mayrovitz"], "given-names": ["HN"], "fpage": ["3"], "lpage": ["8"], "volume": ["3"], "year": ["2017"]}, {"label": ["71"], "article-title": ["Tissue dielectric constant of the lower leg as an index of skin water: temporal variations"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Mayrovitz"], "given-names": ["HN"], "fpage": ["0"], "volume": ["14"], "year": ["2022"]}, {"label": ["86"], "article-title": ["The breast edema Enigma: features, diagnosis, treatment, and recommendations"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Gupta", "Mayrovitz"], "given-names": ["SS", "HN"], "fpage": ["0"], "volume": ["14"], "year": ["2022"]}, {"label": ["87"], "article-title": ["Breast edema following conserving surgery and radiotherapy"], "source": ["Eur J Lymphology"], "person-group": ["\n"], "surname": ["Johansson", "Lathinen", "Bj\u00f6rk-Eriksson"], "given-names": ["K", "T", "T"], "fpage": ["1"], "lpage": ["5"], "volume": ["25"], "year": ["2014"], "uri": ["https://www.researchgate.net/publication/305489145_Breast_edema_following_conserving_surgery_and_radiotherapy_Eur_J_Lymphology_201425701-5"]}, {"label": ["88"], "article-title": ["Two-year follow-up of temporal changes of breast edema after breast cancer treatment with surgery and radiation evaluated by tissue dielectric constant (TDC)"], "source": ["Eur J Lymphology Relat Probl"], "person-group": ["\n"], "surname": ["Johansson", "Darkeh", "Lahtinen", "Bjork-Eriksson", "Axelssqn"], "given-names": ["K", "MH", "T", "T", "R"], "fpage": ["15"], "lpage": ["21"], "volume": ["27"], "year": ["2015"], "uri": ["https://lup.lub.lu.se/record/11bddbf6-5756-47aa-9f9b-b1b675cbfc20"]}, {"label": ["93"], "article-title": ["Noninvasive measurements of breast cancer-related lymphedema"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Mayrovitz"], "given-names": ["HN"], "fpage": ["0"], "volume": ["13"], "year": ["2021"]}, {"label": ["95"], "article-title": ["Exploring women's experiences of breast or trunk lymphoedema following treatment for breast cancer"], "source": ["J Psychosoc Oncol"], "person-group": ["\n"], "surname": ["Ulman J", "Serrant L", "Dunham M", "Probst H"], "given-names": ["Dr", "Professor", "Dr", "Professor"], "fpage": ["1"], "lpage": ["16"], "year": ["2023"]}, {"label": ["97"], "article-title": ["Variation in leg tissue dielectric constant values of healthy young adult females with and without compression bandaging"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Ehmann", "Mayrovitz"], "given-names": ["S", "HN"], "fpage": ["0"], "volume": ["15"], "year": ["2023"]}, {"label": ["105"], "article-title": ["Risk factors for lower extremity lymphedema after surgery in cervical and endometrial cancer"], "source": ["J Gynecol Oncol"], "person-group": ["\n"], "surname": ["Lee", "Byun", "Im", "Son", "Roh", "Kim"], "given-names": ["J", "HK", "SH", "WJ", "YH", "YB"], "fpage": ["0"], "volume": ["34"], "year": ["2023"]}, {"label": ["112"], "article-title": ["Lymphoedema of lower limb following treatment for gynaecological cancer"], "source": ["Int J Gynecol Cancer"], "person-group": ["\n"], "surname": ["Tuppurainen", "Lahtinen", "Pyyk\u00f6nen", "Komulainen", "Nuutinen", "Anttila"], "given-names": ["MT", "T", "J", "M", "J", "M"], "fpage": ["448"], "lpage": ["449"], "volume": ["21"], "year": ["2011"]}, {"label": ["115"], "article-title": ["Comprehensive decongestive therapy as a treatment for secondary lymphedema of the lower extremity and quality of life of women after gynecological cancer surgery"], "source": ["Med Sci Monit"], "person-group": ["\n"], "surname": ["Kendrov\u00e1", "Miku\u013e\u00e1kov\u00e1", "Urbanov\u00e1", "Andra\u0161\u010d\u00edkov\u00e1", "\u017dult\u00e1kov\u00e1", "Tak\u00e1\u010d", "Peresta"], "given-names": ["L", "W", "K", "\u0160", "S", "P", "Y"], "fpage": ["0"], "volume": ["26"], "year": ["2020"]}, {"label": ["122"], "article-title": ["Diabetes mellitus and the skin"], "source": ["An Bras Dermatol"], "person-group": ["\n"], "surname": ["Mendes", "Miot", "Haddad V"], "given-names": ["AL", "HA", "Junior"], "fpage": ["8"], "lpage": ["20"], "volume": ["92"], "year": ["2017"]}]
{ "acronym": [], "definition": [] }
131
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no
2024-01-14 23:43:49
Cureus.; 15(12):e50531
oa_package/7a/65/PMC10787628.tar.gz
PMC10787639
0
[ "<title>1 Introduction</title>", "<p>Highly efficient chromosomal DNA replication is essential for all forms of cellular life and requires the complex interplay of a wide range of protein factors in a temporally and spatially coordinated manner. The importance of high-fidelity chromosome replication is underlined by the fact that in humans, replication defects can lead to genetic disease (##REF##23770608##Weedon et al., 2013##; ##REF##25131834##Pelosini et al., 2014##; ##REF##31629014##Cui et al., 2020##) and cancer (##REF##23528559##Church et al., 2013##; ##REF##23263490##Palles et al., 2013##; ##REF##24501277##Valle et al., 2014##; ##REF##26133394##Bellido et al., 2016##; ##REF##26822575##Rayner et al., 2016##).</p>", "<p>The sliding clamp PCNA (proliferating cell nuclear antigen) is a central player in multiple aspects of chromosomal DNA replication, repair and genome stability (##REF##17512402##Moldovan et al., 2007##; ##REF##27241932##Boehm et al., 2016##). This ring-shaped homotrimer encircles double-stranded DNA to act as a processivity factor for DNA polymerases and a landing pad for the assembly of various DNA processing factors such DNA ligase I, the flap endonuclease Fen1, and the clamp loader complex replication factor C (RF-C) which opens and closes the PCNA ring around dsDNA. Many of the proteins that interact with PCNA, including DNA polymerase δ, DNA ligase I, Fen1 and RF-C, do so via a common mechanism that involves a short linear interaction motif on the partner protein called a PCNA-interacting protein (PIP) motif (##REF##9631646##Warbrick, 1998##; ##REF##37039277##Hamdan and De Biasio, 2023##). The best characterised PIP motifs have conserved sequence Qxxψxxθθ, where ψ and θ represent amino acids with hydrophobic and aromatic side chains, respectively, but other variations on this sequence (so-called non-canonical PIP motifs) have been identified (##REF##27539869##Boehm and Washington, 2016##; ##REF##31134302##Prestel et al., 2019##) such as ψxxxθ (##REF##30655288##Gonzalez-Magana et al., 2019##) and Qxxψxθ (##REF##35942639##Yang et al., 2023##).</p>", "<p>DNA polymerase δ (Pol δ) plays a key role in chromosomal DNA replication and in multiple DNA repair processes (##REF##32883112##Guilliam and Yeeles, 2020##). Pol δ is responsible for the bulk of Okazaki fragment synthesis on the lagging strand and is also involved in the initiation of leading strand synthesis at replication origins (##REF##30451148##Aria and Yeeles, 2018##). Human Pol δ is a homotetramer, comprised of the catalytic subunit p125 (PolD1), p50 (PolD2), p66 (PolD3) and p12 (PolD4), whereas the well-studied budding yeast <italic>Saccharomyces cerevisiae</italic> Pol δ is a heterotrimer of the p125, p55 and p66 orthologues Pol3, Pol31 and Pol32, respectively (##REF##32111820##Lancey et al., 2020##; ##REF##33203675##Zheng et al., 2020##). The p50 (PolD2, Pol31) and p66 (PolD3, Pol32) subunits of Pol δ are also found as components of the trans-lesion synthesis DNA polymerase Pol ζ, in complex with the Pol ζ catalytic subunit REV3L (yeast Rev3) and two copies of the accessory subunit REV7 (Rev7) (##REF##32807989##Malik et al., 2020##).</p>", "<p>Three of the four human Pol δ subunits contain PIP motifs that have been shown to bind PCNA: p125 (PolD1), p66 (PolD3) and p12 (PolD4). In the case of p66, the PIP motif is located at the extreme C-terminus of the protein at the end of lengthy region that is predicted to be largely unstructured and highly flexible (##REF##10698951##Reynolds et al., 2000##; ##REF##14594808##Johansson et al., 2004##). Similarly, the PolD4 PIP motif is located at the end of an unstructured, flexible region close to the N-terminal end of the protein (##REF##30655288##Gonzalez-Magana et al., 2019##; ##REF##35942639##Yang et al., 2023##). In the cryo-EM structure of human Pol δ bound to PCNA and DNA, only the catalytic subunit p125 (PolD1) PIP motif is seen interacting with PCNA (##REF##32111820##Lancey et al., 2020##), suggesting that the PolD3 and PolD4 PIP motifs do not bind PCNA stably under these conditions. Similarly, in the cryo-EM structure of yeast Pol δ bound to PCNA and DNA, only the PolD1 orthologue Pol3 PIP motif binds to PCNA (##REF##33203675##Zheng et al., 2020##).</p>", "<p>Understanding how these various PIP motifs contribute to overall Pol δ function requires a combination of functional and structural analysis. In an effort to gain mechanistic insights into Pol δ function, we have developed tools for high-level expression and rapid purification of the four-subunit Pol δ complex encoded by the thermophilic ascomycete fungus <italic>Chaetomium thermophilum</italic> (<italic>Ct</italic>, also known as <italic>Thermochaetoides thermophila</italic>) (D. Yang and S. MacNeill, unpublished). In parallel with this, we have embarked on a study of how <italic>Ct</italic> Pol δ interacts with <italic>Ct</italic> PCNA and have previously described the non-canonical binding of the N-terminal PIP motif from the <italic>Ct</italic> PolD4 protein to <italic>Ct</italic> PCNA (##REF##35942639##Yang et al., 2023##). Here, we report characterisation of the interaction between the <italic>Ct</italic> PolD3 PIP motif and its cognate PCNA by protein X-ray crystallography at a resolution of 2.45 Å. Unlike the <italic>Ct</italic> PolD4-<italic>Ct</italic> PCNA interaction, <italic>Ct</italic> PolD3 binding to <italic>Ct</italic> PCNA is in every respect canonical: Gln441 inserts into the Q-pocket, a 3<sub>10</sub> helix is formed and Ile444 and Phe448 form the two-fork plug. Interestingly, despite this canonical binding mode, the affinity of the PIP motif peptide for <italic>Ct</italic> PCNA, determined by isothermal titration calorimetry (ITC), is approximately two-fold lower than that seen with the non-canonical binding of the <italic>Ct</italic> PolD4 PIP to <italic>Ct</italic> PCNA (43 µM <italic>versus</italic> 22 µM). In addition to this, we present the X-ray crystal structure of the <italic>Ct</italic> Fen1 nuclease PIP peptide bound to <italic>Ct</italic> PCNA at 1.95Å. This too displays canonical binding to <italic>Ct</italic> PCNA, with the 3<sub>10</sub> helix-containing peptide engaging in Q-pocket and two-fork plug interactions. We discuss these results in terms of the conservation and divergence of PIP motif sequences both within and across species.</p>" ]
[ "<title>2 Materials and methods</title>", "<title>2.1 Identification of <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1 proteins</title>", "<p>Sequences encoding <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1 were identified by BLASTP searching the nr database with the ascomycete <italic>Schizosaccharomyces pombe</italic> Cdc27 (PolD3) and Rad2 (Fen1) protein sequences as the queries (UniProt accession numbers P30261 and P39750, respectively). As protein sequence comparisons suggested that the <italic>Ct</italic> PolD3 protein sequence predicted in the database was N-terminally truncated, full-length cDNAs encoding PolD3 were subsequently amplified from <italic>C. thermophilum</italic> DSM 1495 cDNA (a generous gift of E. Hurt, University of Heidelberg) and sequenced, allowing identification of the complete ORF. The full-length cDNA sequence has been deposited with the GenBank database (accession number OQ605904) and the full-length protein sequence with UniProt (accession number G0S636). The <italic>Ct</italic> Fen1 protein has accession number G0S2B5 in the UniProt database. Protein sequence alignments for PolD3 and Fen1 can be seen as <xref rid=\"s10\" ref-type=\"sec\">Supplementary Figures S1, S2</xref>, respectively.</p>", "<title>2.2 Protein expression and purification</title>", "<p>\n<italic>Ct</italic> PCNA was expressed and purified to apparent homogeneity as described previously (##REF##35942639##Yang et al., 2023##). Briefly, the protein was expressed in recombinant form in <italic>E. coli</italic> with a TEV protease-cleavable N-terminal His6 tag, purified using IMAC, cleaved with His6-TEV to remove the His6 tag, subjected to reverse IMAC to remove still-tagged PCNA protein and His6-TEV protease, then polished using SEC. Purified protein (11.0 mg/mL) was flash-frozen in liquid nitrogen and stored at −80°C.</p>", "<title>2.3 Peptides</title>", "<p>A 15mer <italic>Ct</italic> PolD3 PIP peptide spanning residues 437–451 (sequence: <sup>437</sup>GKGGQGSIMSWFAKK<sup>451</sup>) and a 15mer <italic>Ct</italic> Fen1 peptide spanning residues 339–353 (<sup>339</sup>GAQQARIEGFFKVIP<sup>353</sup>) were commercially synthesised (GenScript, Piscataway, New Jersey, United States) and obtained in lyophilised form at final purities of 99.4% and 99.1%, respectively. The <italic>Ct</italic> PolD3 peptide was resuspended in either dH<sub>2</sub>O or 50 mM Tris-HCl, 50 mM NaCl, pH 8.0 at a concentration of 8 mg/mL (5.1 mM), while the <italic>Ct</italic> Fen1 peptide (insoluble in aqueous solution) was resuspended in DMSO at a concentration of 8 mg/mL (4.8 mM).</p>", "<title>2.4 Crystallography</title>", "<p>For crystal screens, 90 µL of 14 mg/mL <italic>Ct</italic> PCNA was mixed with 21 µL of either 8 mg/mL <italic>Ct</italic> PolD3 PIP peptide (in dH<sub>2</sub>O) or 8 mg/mL <italic>Ct</italic> Fen1 PIP peptide (in DMSO) and screened using JCSG Plus™ and PACT Premier™ screens (Molecular Dimensions, Holland, OH, United States). Diffraction quality crystals were obtained from the JCSG Plus™ screen in 0.1 M phosphate/citrate pH 4.2, 40% PEG 300 (for <italic>Ct</italic> PolD3 peptide-<italic>Ct</italic> PCNA) and 2.4 M sodium malonate dibasic monohydrate pH 7.0 (for <italic>Ct</italic> Fen1 peptide-<italic>Ct</italic> PCNA). The crystals diffracted to 2.45 and 1.95 A, respectively. The data for both complexes were collected in-house at 100 K on a Rigaku MM007HF Cu anode X-ray generator (Rigaku, Tokyo, Japan). Reflections were recorded on a Rigaku Saturn 944+ CCD detector. Data processing was performed using iMOSFLM (##REF##21460445##Battye et al., 2011##), scaled using AIMLESS, and the space group of each structure was identified using POINTLESS (##REF##21460454##Murshudov et al., 2011##). <italic>Ct</italic> PCNA-<italic>Ct</italic> PolD3 was crystallized in space group P1 with one trimer of <italic>Ct</italic> PCNA in the asymmetric unit, while <italic>Ct</italic> PCNA-<italic>Ct</italic> Fen1 was crystallized in space group H32 with one chain of <italic>Ct</italic> PCNA in the asymmetric unit. The data collection statistics are found in <xref rid=\"s10\" ref-type=\"sec\">Supplementary Table S1</xref>. The structures of the protein-peptide complexes were solved by molecular replacement using MOLREP (CCP4) (##UREF##0##Vagin and Teplyakov, 1997##) using the complete structure of <italic>Ct</italic> PCNA (PDB: 7O1E) as a starting model (##REF##35942639##Yang et al., 2023##). Repeated rounds of model refinement using COOT (##REF##15572765##Emsley and Cowtan, 2004##) and REFMAC5 (##REF##21460454##Murshudov et al., 2011##) resulted in a structural model with an R<sub>work</sub> of 21.2% and R<sub>free</sub> of 24.9% for <italic>Ct</italic> PCNA-PolD3 PIP and R<sub>work</sub> of 17.1% and R<sub>free</sub> of 20.4% for <italic>Ct</italic> PCNA-Fen1 PIP (see <xref rid=\"s10\" ref-type=\"sec\">Supplementary Table S1</xref>). The structures have been deposited in the PDB with accession codes 8P9O (<italic>Ct</italic> PCNA-<italic>Ct</italic> PolD3 PIP) and 8Q7I (<italic>Ct</italic> PCNA-<italic>Ct</italic> Fen1 PIP).</p>", "<title>2.5 Isothermal titration calorimetry (ITC)</title>", "<p>ITC was performed using a MicroCal PEAQ-ITC calorimeter (Malvern Panalytical, Malvern, UK). Prior to measurement, the <italic>Ct</italic> PolD3 peptide and <italic>Ct</italic> PCNA protein were buffer-exchanged into 50 mM Tris-HCl, 150 mM NaCl, pH 8.0. Experimental titrations were performed at 25°C in duplicate with 300 µL of 32.7 µM <italic>Ct</italic> PCNA and 420 µM <italic>Ct</italic> PolD3 peptide. Control titrations used <italic>Ct</italic> PolD3 peptide only at 420 µM. In total, 19 injections were used for each assay: a primary injection of 0.2 µL followed by 18 injections of 2.0 µL. The heat change following injection was measured, the control values subtracted, and data was fitted to a single site model (1 peptide: 1 PCNA protomer) using MicroCal PEAQ-ITC Analysis Software (v1.21). The K<sub>\n<italic>D</italic>\n</sub> values reported represent the mean of the two experiments.</p>" ]
[ "<title>3 Results</title>", "<title>3.1 <italic>Chaetomium thermophilum</italic> PolD3 protein</title>", "<p>BLASTP searching using the ascomycete fission yeast <italic>S. pombe</italic> PolD3 orthologue Cdc27 as the query sequence led (after later cDNA sequencing, see Materials and methods for details) to the identification of <italic>Ct</italic> PolD3 as a 451 amino acid protein with predicted molecular weight 49.3 kDa. <italic>Ct</italic> PolD3 is 26% identical to <italic>S. pombe</italic> Cdc27 at the amino acid sequence level and ∼20% identical to the human and <italic>S. cerevisiae</italic> PolD3 orthologues p66 and Pol32, respectively. The conserved PIP motif found at the extreme C-terminus of human, <italic>S. cerevisiae</italic> and <italic>S. pombe</italic> PolD3 orthologues is readily identifiable at the C-terminal end of the <italic>Ct</italic> PolD3 protein (sequence: <sup>441</sup>\n<underline>Q</underline>GS<underline>I</underline>MS<underline>WF</underline>\n<sup>448</sup> conserved PIP motif residues underlined) (##FIG##0##Figure 1##, <xref rid=\"s10\" ref-type=\"sec\">Supplementary Figure S1</xref>).</p>", "<title>3.2 Binding of <italic>Ct</italic> PolD3 PIP peptide to PCNA</title>", "<p>To characterise the binding of the <italic>Ct</italic> PolD3 with <italic>Ct</italic> PCNA, the structure of the <italic>Ct</italic> PolD3 PIP motif peptide-<italic>Ct</italic> PCNA complex was solved by X-ray crystallography at a resolution of 2.45 Å (##FIG##1##Figure 2A##, see <xref rid=\"s10\" ref-type=\"sec\">Supplementary Table S1</xref> for crystallography statistics and <xref rid=\"s10\" ref-type=\"sec\">Supplementary Figure S3</xref> for Fo−Fc difference density maps). The <italic>Ct</italic> PolD3 peptide (sequence: <sup>437</sup>GKGG<underline>Q</underline>GS<underline>I</underline>MS<underline>WF</underline>AKK<sup>451</sup> with conserved PIP motif residues underlined) occupied a single PIP binding site only, with the other sites on <italic>Ct</italic> PCNA being occluded by crystal packing (specifically, by the loop that spans residues Thr186-Lys190 in neighbouring symmetry mates). Only 11 of 15 residues in the peptide could be seen in the electron density map (Gln441 to Lys451) suggesting that the N-terminal four residues of the peptide are flexible (##FIG##1##Figure 2B##).</p>", "<p>Consistent with the high degree of conservation of the PIP peptide sequence at the C-terminal end of PolD3 orthologues, the mode of PCNA-PIP binding observed is strictly canonical (##FIG##1##Figure 2B##): part of the peptide adopts a 3<sub>10</sub> helical structure (##FIG##1##Figure 2A##), <italic>Ct</italic> PolD3 Gln441 inserts into the Q-pocket on <italic>Ct</italic> PCNA and <italic>Ct</italic> PolD3 Ile444 and Phe448 form a 2-fork plug that inserts into the hydrophobic surface pocket on <italic>Ct</italic> PCNA (##FIG##1##Figure 2B##). Both hydrogen bonding and hydrophobic interactions are apparent (shown in detail in ##FIG##1##Figure 2C##). <italic>Ct</italic> PolD3 Gln441(OE1) makes water-mediated hydrogen bonding interactions with Ser208 (N) and Ala252 (O) on <italic>Ct</italic> PCNA, Gly442 (N) hydrogen bonds with Pro253 (O), Ser 443 (OG) and Ile444 (N) both hydrogen bond with His44(O), Trp447 (NE1) hydrogen bonds with Glu232 (O), Ala449 (O) with Gly127 (N), and Lys451 (N) with His125 (O). Hydrophobic interactions with <italic>Ct</italic> PCNA are seen with <italic>Ct</italic> PolD3 Gln441, Gly442, Ile444, Met445, Trp447 and Phe448. Interactions involving Ile444 (specifically involving CB, CG, CD1, CZ2, CZ3) and Phe448 (CD1, CD2, CE1, CE2, CZ) anchor the 2-fork plug in the hydrophobic surface pocket formed by Leu47, Pro129, Pro234 and Pro250 in <italic>Ct</italic> PCNA (##FIG##1##Figure 2C##).</p>", "<title>3.3 Binding affinity determined by isothermal titration calorimetry</title>", "<p>In order to gauge the affinity of the <italic>Ct</italic> PolD3 PIP peptide for <italic>Ct</italic> PCNA, isothermal titration calorimetry (ITC) was used to determine the binding affinity of <italic>Ct</italic> PCNA for the 15mer <italic>Ct</italic> PolD3 PIP motif peptide (<sup>437</sup>GKGGQGSIMSWFAKK<sup>451</sup>) and the stoichiometry of the interaction in solution (##FIG##1##Figure 2D##). The mean dissociation constant (K<sub>\n<italic>D</italic>\n</sub>) from two experiments was determined to be 43.2 µM ± 3.9 μM at 25°C and the stoichiometry 1:1 (i.e. 1 PIP peptide: 1 PCNA protomer) indicating that all three PIP peptide binding sites on <italic>Ct</italic> PCNA are occupied in solution, as expected. The measured K<sub>\n<italic>D</italic>\n</sub> is almost three-fold less (43 µM <italic>versus</italic> 16 µM) than that previously reported for the human PolD3/p66-PCNA interaction determined by ITC with a peptide of sequence <sup>452</sup>KANRQVSITGFFQRK<sup>466</sup> (##REF##15576034##Bruning and Shamoo, 2004##) and two-fold less (43 µM <italic>versus</italic> 22 µM) than that for <italic>Ct</italic> PolD4 binding to <italic>Ct</italic> PCNA despite the latter interaction involving a non-canonical binding mode (##REF##35942639##Yang et al., 2023##). This is discussed further below (see Discussion).</p>", "<title>3.4 Binding of <italic>Ct</italic> Fen1 PIP peptide to <italic>Ct</italic> PCNA</title>", "<p>Fen1 is a 5’ flap-specific nuclease that plays an important in Okazaki fragment maturation. BLASTP searching with the <italic>S. pombe</italic> Fen1 nuclease orthologue Rad2 identifies <italic>C. thermophilum</italic> Fen1 as a 395 amino acid protein that is 60% identical to the <italic>S. pombe</italic> and <italic>S. cerevisiae</italic> Fen1 orthologues Rad27 and Rad2, respectively, at the amino acid sequence level and 55% identical to human Fen1. Key catalytic residues in human Fen1 are conserved in the <italic>C. thermophilum</italic> enzyme, as is the PIP motif located towards the C-terminal end of the protein (sequence: <sup>342</sup>\n<underline>Q</underline>AR<underline>I</underline>EG<underline>FF</underline>\n<sup>349</sup> with conserved PIP motif residues underlined) (##FIG##0##Figure 1##).</p>", "<p>The <italic>Ct</italic> Fen1 PIP peptide-<italic>Ct</italic> PCNA structure was solved to a resolution of 1.95 Å (##FIG##2##Figure 3A##, see also <xref rid=\"s10\" ref-type=\"sec\">Supplementary Table S1</xref>, <xref rid=\"s10\" ref-type=\"sec\">Supplementary Figure S3</xref>). 14 of 15 residues in the <italic>Ct</italic> Fen1 PIP peptide were visible in the structure, revealing a 3<sub>10</sub> helix and the anticipated canonical binding mode, with <italic>Ct</italic> Fen1 Gln342 inserting into the Q-pocket, and Ile345 and Phe349 forming the two-fork plug that inserts into the hydrophobic surface pocket on <italic>Ct</italic> PCNA (##FIG##2##Figure 3B##). As with <italic>Ct</italic> PolD3, the <italic>Ct</italic> Fen1 Q-pocket glutamine, Gln342, interacts with Ser208 and Ala252 in <italic>Ct</italic> PCNA, with the interaction with Ser208 involving water-mediated hydrogen bonding between Gln342 (OE1) and Ser208 (N), as is the case for the equivalent Gln441-Ser208 pairing in <italic>Ct</italic> PolD3, whereas the interaction with Ala252 involves a direct H-bond between Gln342 (NE2) and Ala252 (O), different from the water-mediated Gln441-Ala252 interaction in the <italic>Ct</italic> PolD3-<italic>Ct</italic> PCNA structure. Additional direct hydrogen-bonding interactions are seen involving Gln341 (N) and Ile255 (O), Gln341 (O) and Ile255 (N), Ala343 (N) and Pro253 (O), Ile345 (N) and His44 (O), Glu346 (OE2) and His44 (ND1), Lys350 (O) and Gly127 (N), and Ile353 (N) and His125 (O). In addition to this, the higher resolution of the structure allowed identification of multiple water-mediated hydrogen bonds connecting the <italic>Ct</italic> Fen1 peptide to <italic>Ct</italic> PCNA (##FIG##2##Figure 3C##). A salt bridge links Glu346 (OE1) and His44 (ND1) also. As with the <italic>Ct</italic> PolD3-<italic>Ct</italic> PCNA structure, the 2-fork plus residues Ile345 and Phe349 display hydrophobic interactions with the surface pocket on <italic>Ct</italic> PCNA (##FIG##2##Figure 3C##) although in the case of Phe349 only a single interaction (involving Phe349 (CE1)) is apparent within the 3.9 Å length cut-off applied in the analysis.</p>" ]
[ "<title>4 Discussion</title>", "<p>Beginning with the structure of p21<sup>Cip1</sup> PIP bound to PCNA (##REF##8861913##Gulbis et al., 1996##), the atomic structures of a large and diverse set of PCNA-PIP peptide complexes have been determined over the last quarter century (##REF##31134302##Prestel et al., 2019##), although only in very few cases have equivalent (orthologous) interactions been studied in multiple species. Here we present atomic structures of PCNA-PIP peptide complexes derived from the <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1 proteins bound to their cognate PCNA at resolutions of 2.45 and 1.95 Å, respectively. In both cases, the new structures allow comparison with earlier structures of human orthologues: <italic>Ct</italic> PolD3-<italic>Ct</italic> PCNA with human PolD3/p66-PCNA and <italic>Ct</italic> Fen1-<italic>Ct</italic> PCNA with human Fen1-PCNA (##REF##15576034##Bruning and Shamoo, 2004##) (##FIG##3##Figure 4##).</p>", "<p>In sharp contrast to the non-canonical <italic>Ct</italic> PolD4-<italic>Ct</italic> PCNA and human p12-PCNA interactions described previously, the <italic>Ct</italic> PolD3-<italic>Ct</italic> PCNA and <italic>Ct</italic> Fen1-<italic>Ct</italic> PCNA interactions described here are strictly canonical in nature (##REF##30655288##Gonzalez-Magana et al., 2019##; ##REF##35942639##Yang et al., 2023##). Both PIP peptides form a 3<sub>10</sub> helix, conserved glutamines (Gln441 and Gln342 in <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1, respectively) insert into the <italic>Ct</italic> PCNA Q-pocket, and in both cases a 2-fork plug (involving the sidechains of Ile444 and F448 in <italic>Ct</italic> PolD3, and Leu345 and Phe349 in <italic>Ct</italic> Fen1) inserts into the hydrophobic pocket on the surface of <italic>Ct</italic> PCNA. ##FIG##3##Figure 4## shows a comparison of the binding modes of PolD3, Fen1 and PolD4 PIP peptides across species, highlighting the high level of cross-species similarity between the human and <italic>C. thermophilum</italic> PolD3-PCNA and Fen1-PCNA structures. This is in contrast to the more divergent PolD4 orthologue PIP-PCNA structures, where the <italic>Ct</italic> PolD4 PIP peptide does not form the 3<sub>10</sub> helix characteristic of most other PIP peptides (including human PolD4/p12, but also <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1), nor does the <italic>Ct</italic> PolD4-<italic>Ct</italic> PCNA interaction involve a glutamine-Q-pocket interaction (unlike human PolD4/p12, <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1) (##FIG##3##Figure 4##) (##REF##35942639##Yang et al., 2023##).</p>", "<p>Interestingly, the binding affinities of the human and <italic>C. thermophilum</italic> PolD3 and PolD4 peptides for their cognate PCNAs determined by ITC are broadly similar, with measured K<sub>\n<italic>D</italic>\n</sub> values ranging from 16 to 43 µM (##REF##15576034##Bruning and Shamoo, 2004##; ##REF##30655288##Gonzalez-Magana et al., 2019##; ##REF##35942639##Yang et al., 2023##) and the differences that exist show no obvious pattern: in humans, the measured K<sub>\n<italic>D</italic>\n</sub> for the canonical PolD3/p66-PCNA interaction is lower than that for the non-canonical PolD4-PCNA interaction (16 µM <italic>versus</italic> 38 µM) while in <italic>C. thermophilum</italic> the measured K<sub>D</sub> for canonical PolD3-PCNA is higher than that for non-canonical PolD4-PCNA (43 µM <italic>versus</italic> 22 µM), suggesting that specific binding affinity for PCNA (within the observed 16–43 µM range) may not be crucial for PolD3 and PolD4 protein function.</p>", "<p>Remarkably, the PolD3-PCNA interaction is not seen in recent cryo-EM structures of human or budding yeast Pol δ complexed with PCNA on primer-template DNA (##REF##33203675##Zheng et al., 2020##; ##REF##34667155##Lancey et al., 2021##). The human PolD4 PIP-PCNA interaction is also not seen. In both sets of Pol δ-PCNA structures, the only visible PIP motif-PCNA interaction is that between the catalytic subunit of Pol δ (PolD1/human p125/yeast Pol3) and PCNA, with the two remaining PIP peptide binding sites on the PCNA trimer being unoccupied. These sites are not occluded however, as evidenced by Fen1-PCNA interaction in the human Pol δ-PCNA-Fen1 complex (shown in ##FIG##0##Figure 1##) (##REF##34667155##Lancey et al., 2021##). Why these interactions are not seen remains unclear but it is possible that they have only a very limited role, or no role at all, to play once the Pol δ complex has engaged with its substrate at the primer-template junction. Instead, the PolD3 and PolD4 PIP motifs might facilitate recruitment of Pol δ to PCNA that has previously been loaded at the primer-template junction, before handing over the task of stabilizing Pol δ-PCNA interaction to PolD1 PIP-PCNA binding, or might ensure that should Pol δ disengage from the primer-template, it is retained in the vicinity of the DNA to allow efficient polymerase recycling. The PolD3 and PolD4 PIP motifs are located at the end of lengthy flexible regions that could, in either case, be employed in the “fly casting mechanism” (##REF##10908673##Shoemaker et al., 2000##) to scan three-dimensional space and latch onto PCNA prior to the Pol δ-PCNA complex being locked more securely into position via the PolD1 PIP-PCNA interaction seen in the cryo-EM structures (##REF##37039277##Hamdan and De Biasio, 2023##). A similar mechanism has been proposed for DNA ligase I (Lig1)-PCNA interactions: a PIP motif at the N-terminal end of the flexible N-terminal region of Lig1 (PIP<sub>N-term</sub>) tethers the protein to PCNA when the ligase is detached from DNA but once the ligase locates a nick, this interaction is disrupted and a second PIP motif (PIP<sub>DBD</sub>), located near the centre of the Lig1 DNA binding domain, engages with PCNA instead (##REF##36539424##Blair et al., 2022##).</p>", "<p>A key functional distinction between the human PolD3/p66 and PolD4/p12 PIP motifs is that the latter is PIP degron, a specialized PIP motif that acts as a targeting signal for protein degradation (##REF##19595719##Havens and Walter, 2009##; ##REF##21828267##Havens and Walter, 2011##; ##REF##22303007##Havens et al., 2012##). Once bound to PCNA on chromatin, PIP degron-containing proteins such as PolD4/p12, Cdt1 and p21<sup>Cip1</sup> are ubiquitylated and degraded by a CRL<sup>Cdt2</sup>-dependent mechanism. Degradation of human PolD4/p12 occurs as cells enter S-phase or in response to DNA damage (##REF##17317665##Zhang et al., 2007##; ##REF##19074196##Meng et al., 2009##; ##REF##24022480##Terai et al., 2013##; ##REF##23913683##Zhang et al., 2013##), leaving behind a three-subunit Pol δ complex that appears better suited to the task in hand (##REF##24300032##Lee et al., 2014##; ##REF##31326365##Lee et al., 2019##). It remains to be seen whether the <italic>Ct</italic> PolD4 protein is degraded via a similar mechanism, however, we have shown that the orthologous PolD4/Cdm1 protein from the related ascomycete fission yeast <italic>S. pombe</italic> is a CRL<sup>Cdt2</sup> substrate (S.M., unpublished results) suggesting that <italic>Ct</italic> PolD4 protein levels may be regulated in this way too. Previously, a basic amino acid four residues C-terminal to the 2-fork plug aromatic residue in the conserved PIP motif sequence (i.e., in the +4 position) has been identified as being an important (though not defining) feature of PIP degrons (##REF##19595719##Havens and Walter, 2009##; ##REF##21828267##Havens and Walter, 2011##; ##REF##22303007##Havens et al., 2012##); filamentous fungal PolD4 proteins such as <italic>Ct</italic> PolD4 lack this, but do have conserved basic residues at +3 and +6 that could play a similar role (##REF##35942639##Yang et al., 2023##). In <italic>S. pombe</italic> and other fission yeasts, an arginine is found conserved at position +5. Neither PolD3 nor Fen1 is thought to be a CRL<sup>Cdt2</sup> substrate, suggesting that instead of being related to PCNA binding affinity, the divergence of the PolD4 PIP-PCNA binding mode from canonical to non-canonical may be related to the targeting of these proteins by CRL<sup>Cdt2</sup>, their ubiquitylation and subsequent degradation. Further work on diverse PIP- and PIP degron-containing proteins will be required to address this.</p>", "<p>In summary, the determination of the structures of <italic>Ct</italic> PolD3, PolD4 and Fen1 PIP peptides bound to <italic>Ct</italic> PCNA allows direct comparison of binding modes, both within species (PolD3 <italic>versus</italic> PolD4 <italic>versus</italic> Fen1) but also across species, with reference to previously determined structures for human PolD3/p66, PolD4/p12 and Fen1 PIP peptide-PCNA complexes (##REF##15576034##Bruning and Shamoo, 2004##; ##REF##30655288##Gonzalez-Magana et al., 2019##). With increasing interest in the development of inhibitors of PCNA-PIP interactions for therapeutic purposes (##REF##31247123##Horsfall et al., 2020##; ##REF##37510250##Gu et al., 2023##), gaining a detailed structural understanding of how these interactions occur, how they have evolved and how this impacts function, is more important than ever.</p>" ]
[]
[ "<p>\n<bold>Edited by:</bold>\n<ext-link xlink:href=\"https://loop.frontiersin.org/people/712585/overview\" ext-link-type=\"uri\">Nicholas Robinson</ext-link>, Lancaster University, United Kingdom</p>", "<p>\n<bold>Reviewed by:</bold>\n<ext-link xlink:href=\"https://loop.frontiersin.org/people/1604885/overview\" ext-link-type=\"uri\">Francisco J. Blanco</ext-link>, Spanish National Research Council (CSIC), Spain</p>", "<p>\n<ext-link xlink:href=\"https://loop.frontiersin.org/people/1071366/overview\" ext-link-type=\"uri\">Narottam Acharya</ext-link>, Institute of Life Sciences (ILS), India</p>", "<p>The sliding clamp PCNA is a key player in eukaryotic genome replication and stability, acting as a platform onto which components of the DNA replication and repair machinery are assembled. Interactions with PCNA are frequently mediated via a short protein sequence motif known as the PCNA-interacting protein (PIP) motif. Here we describe the binding mode of a PIP motif peptide derived from C-terminus of the PolD3 protein from the thermophilic ascomycete fungus <italic>C. thermophilum</italic>, a subunit of both DNA polymerase δ (Pol δ) and the translesion DNA synthesis polymerase Pol ζ, characterised by isothermal titration calorimetry (ITC) and protein X-ray crystallography. In sharp contrast to the previously determined structure of a <italic>Chaetomium thermophilum</italic> PolD4 peptide bound to PCNA, binding of the PolD3 peptide is strictly canonical, with the peptide adopting the anticipated 3<sub>10</sub> helix structure, conserved Gln441 inserting into the so-called Q-pocket on PCNA, and Ile444 and Phe448 forming a two-fork plug that inserts into the hydrophobic surface pocket on PCNA. The binding affinity for the canonical PolD3 PIP-PCNA interaction determined by ITC is broadly similar to that previously determined for the non-canonical PolD4 PIP-PCNA interaction. In addition, we report the structure of a PIP peptide derived from the <italic>C. thermophilum</italic> Fen1 nuclease bound to PCNA. Like PolD3, Fen1 PIP peptide binding to PCNA is achieved by strictly canonical means. Taken together, these results add to an increasing body of information on how different proteins bind to PCNA, both within and across species.</p>" ]
[]
[ "<p>We are grateful to colleagues in St Andrews and elsewhere for their assistance with this work, in particular Shirley Graham (St Andrews) for protein purification and Clarissa Melo Czekster (St Andrews) for assistance with ITC set-up and data analysis.</p>", "<title>Data availability statement</title>", "<p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PDB entry 8P9O: <ext-link xlink:href=\"https://doi.org/10.2210/pdb8P9O/pdb\" ext-link-type=\"uri\">https://doi.org/10.2210/pdb8P9O/pdb</ext-link>; PDB entry 8Q7I: <ext-link xlink:href=\"https://doi.org/10.2210/pdb8Q7I/pdb\" ext-link-type=\"uri\">https://doi.org/10.2210/pdb8Q7I/pdb</ext-link>. The GenBank and UniProt database entries (OQ605904 and G0S636 respectively) are as follows: GenBank OQ605904: <ext-link xlink:href=\"https://www.ncbi.nlm.nih.gov/nuccore/OQ605904\" ext-link-type=\"uri\">https://www.ncbi.nlm.nih.gov/nuccore/OQ605904</ext-link>; UniProt G0S636: <ext-link xlink:href=\"https://www.uniprot.org/uniprotkb/G0S636/entry\" ext-link-type=\"uri\">https://www.uniprot.org/uniprotkb/G0S636/entry</ext-link>.</p>", "<title>Author contributions</title>", "<p>MA: Data curation, Formal Analysis, Investigation, Writing–review and editing. CW: Data curation, Investigation, Writing–review and editing, Formal Analysis. SM: Data curation, Investigation, Writing–review and editing, Conceptualization, Formal Analysis, Funding acquisition, Project administration, Supervision, Writing–original draft.</p>", "<title>Conflict of interest</title>", "<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>", "<title>Publisher’s note</title>", "<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>", "<title>Supplementary material</title>", "<p>The Supplementary Material for this article can be found online at: <ext-link xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmolb.2023.1320648/full#supplementary-material\" ext-link-type=\"uri\">https://www.frontiersin.org/articles/10.3389/fmolb.2023.1320648/full#supplementary-material</ext-link>\n</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>FIGURE 1</label><caption><p>Overall structure of Pol δ and conservation of PolD3 and Fen1 PIP motif sequences. Centre: Cryo-EM structure of human Pol δ-Fen1-PCNA-DNA complex determined by De Biasio and coworkers (##REF##32111820##Lancey et al., 2020##). Individual proteins are shown in different colours as follows: PolD1/p125, green; PolD2/p50, cyan; PolD3/p66 N-terminal domain, magenta; PolD4/p12 C-terminal domain, dark blue; Fen1, orange; PCNA promoters, grey, purple and salmon pink. The N- and C-terminal ends of PolD3 are labelled N and C. PCNA-PIP peptide interactions involving Fen1 and PolD1/p125 are circled. Top left: Schematic representation of <italic>Ct</italic> PolD3 and <italic>Ct</italic> Fen1 proteins, with the PolD3 N-terminal domain shown in light grey, the Fen1 nuclease domain in dark gray, and the two PIP motifs in black. Top right: Protein sequence alignment of known or predicted PIP motifs in PolD3 proteins from <italic>Chaetomium thermophilum</italic> (Ct), <italic>S. pombe</italic> (Sp), <italic>S. cerevisiae</italic> (Sp) and human (Hs). Bottom left: Protein sequence alignment of PIP motifs in Fen1 proteins from <italic>Chaetomium thermophilum</italic> (Ct), <italic>S. pombe</italic> (Sp), <italic>S. cerevisiae</italic> (Sc) and human (Hs). Key conserved residues are shown in bold red. Central image prepared using the PyMOL Molecular Graphics System version 2.0 (Schrödinger LLC, New York) and PDB file 6TNZ.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>FIGURE 2</label><caption><p>Binding of Ct PolD3 PIP peptide to Ct PCNA. <bold>(A)</bold> Crystal structure of <italic>Ct</italic> PCNA (shown in green, cyan and magenta) with a <italic>Ct</italic> PolD3 PIP peptide (yellow) binding to one of the PCNA protomers at 2.45 Å resolution (PDB: 8P9O). <bold>(B)</bold> Close-up view of the <italic>Ct</italic> PolD3 PIP peptide (residues visible: <sup>441</sup>\n<underline>Q</underline>GS<underline>I</underline>MS<underline>WF</underline>AKK<sup>451</sup>, with conserved PIP motif residues underlined) bound to <italic>Ct</italic> PCNA. The Q-pocket on <italic>Ct</italic> PCNA (occupied by the sidechain of Gln441) is circled in yellow and the hydrophobic surface pocket (occupied by the sidechains of Ile444 and Phe448) in white. Image prepared using the PyMOL Molecular Graphics System version 2.0 (Schrödinger LLC, New York) and PDB file 8P9O. <bold>(C)</bold> Detailed view of the <italic>Ct</italic> PCNA-<italic>Ct</italic> PolD3 PIP peptide interaction represented using LigPlot + version 2.2.5 (##REF##21919503##Laskowski and Swindells, 2011##) with default parameters. The <italic>Ct</italic> PolD3 peptide backbone is shown in orange, with individual peptide amino acids labelled in blue text. Hydrogen bonds (2.7–3.35 Å) are shown as green broken lines, with bond distances (Å) indicated. Amino acids in <italic>Ct</italic> PCNA involved in hydrogen bonding to the <italic>Ct</italic> PolD3 PIP peptide are labelled in green text. The red spoked arcs represent residues in <italic>Ct</italic> PCNA making hydrophobic contacts (2.9–3.9 Å) with the peptide; the corresponding atoms in the <italic>Ct</italic> PolD3 peptide are decorated with smaller red spokes. Carbon, oxygen, nitrogen and sulphur atoms are represented by black, red, dark blue and yellow circles, respectively, and water molecules by white circles. <bold>(D)</bold> Affinity of <italic>Ct</italic> PCNA for the <italic>Ct</italic> PolD3 peptide measured by isothermal titration calorimetry (ITC). The left-hand panel shows baseline-corrected experimental data from titration of the <italic>Ct</italic> PolD3 peptide (420 µM) with <italic>Ct</italic> PCNA (32.7 µM). The right-hand panel shows the ligand concentration dependence of heat released upon PCNA-peptide binding, with the molar ratio referring to peptide:PCNA promoter. The ITC analysis was performed twice with similar results; a single dataset is shown. See Materials and methods for details.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>FIGURE 3</label><caption><p>Binding of Ct Fen1 PIP peptide to Ct PCNA. <bold>(A)</bold> Crystal structure of <italic>Ct</italic> PCNA (shown in green, cyan and magenta) bound to the <italic>Ct</italic> Fen1 PIP peptides (red, salmon pink, blue) at 1.95 Å resolution (PDB: 8Q7I). <bold>(B)</bold> Close-up view of the <italic>Ct</italic> Fen1 PIP peptide (residues visible: <sup>341</sup>Q<underline>Q</underline>AR<underline>I</underline>EG<underline>FF</underline>KVI<sup>352</sup>, with conserved PIP motif residues underlined) bound to <italic>Ct</italic> PCNA. The Q-pocket on <italic>Ct</italic> PCNA (occupied by the sidechain of Gln342) is circled in yellow and the hydrophobic surface pocket (occupied by the sidechains of Ile345 and Phe349) in white. Image prepared using the PyMOL Molecular Graphics System version 2.0 (Schrödinger LLC, New York) and PDB file 8Q7I. <bold>(C)</bold> Detailed view of the <italic>Ct</italic> PCNA-<italic>Ct</italic> Fen1 PIP peptide interaction represented using LigPlot + version 2.2.5 (##REF##21919503##Laskowski and Swindells, 2011##) with default parameters. The <italic>Ct</italic> Fen1 peptide backbone is shown in orange, with individual peptide amino acids labelled in blue text. Hydrogen bonds (2.7–3.35 Å) are shown as green broken lines, with bond distances (Å) indicated. Amino acids in <italic>Ct</italic> PCNA involved in hydrogen bonding to the <italic>Ct</italic> Fen1 PIP peptide are labelled in green text. The red spoked arcs represent residues in <italic>Ct</italic> PCNA making hydrophobic contacts (2.9–3.9 Å) with the peptide; the corresponding atoms in the <italic>Ct</italic> Fen1 peptide are decorated with smaller red spokes. Carbon, oxygen, nitrogen and sulphur atoms are represented by black, red, dark blue and yellow circles, respectively, and water molecules by white circles.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>FIGURE 4</label><caption><p>Comparison of PIP peptide binding modes. <bold>(A)</bold> Overlaid <italic>Chaetomium thermophilum</italic> and human PIP motif peptide structures from PolD3 (upper panel), Fen1 (middle panel) and PolD4 (lower panel) with structures and key residues from <italic>Chaetomium thermophilum</italic> and human shown in orange and green, respectively. PDB codes: 8P9O (<italic>Ct</italic> PolD3), 1U76 (human PolD3/p66), 8Q7I (<italic>Ct</italic> Fen1), 1U7B (human Fen1), 7O1F (<italic>Ct</italic> PolD4) and 6HVO (human PolD4/p12). The Q-pocket and hydrophobic pocket are shown in outline in red and blue, respectively. <bold>(B)</bold> Ribbon structures of the six PIP motif peptides shown above highlighting the presence of a 3<sub>10</sub> helix in all but the <italic>Ct</italic> PolD4 PIP peptide. Images prepared using the PyMOL Molecular Graphics System version 2.0 (Schrödinger LLC, New York).</p></caption></fig>" ]
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[ "<media xlink:href=\"DataSheet1.PDF\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"mixed-citation": ["\n"], "surname": ["Vagin", "Teplyakov"], "given-names": ["A.", "A."], "year": ["1997"], "article-title": ["MOLREP: an automated program for molecular replacement"], "source": ["J. Appl. Cryst."], "volume": ["30"], "fpage": ["1022"], "lpage": ["1025"], "pub-id": ["10.1107/s0021889897006766"]}]
{ "acronym": [], "definition": [] }
44
CC BY
no
2024-01-14 23:43:50
Front Mol Biosci. 2023 Dec 18; 10:1320648
oa_package/09/b6/PMC10787639.tar.gz
PMC10787643
0
[ "<title>Introduction</title>", "<p>Japan is currently grappling with a declining birth rate and an aging population, which have reduced the labor force [##UREF##0##1##]. In response to the labor shortage, the Japanese government revised the Immigration Control and Refugee Recognition Act in 2018 to facilitate the acceptance of foreign workers and improve their workplace and social conditions [##UREF##1##2##]. The government estimated that approximately 345,000 workers would be accepted over five years starting in April 2019 [##UREF##2##3##]. Moreover, according to Tokyo-based public think tanks, Japan will need 6.74 million foreign workers by 2040. The influx of foreign residents has significantly influenced various aspects of Japanese society, including the healthcare system [##REF##34744100##4##].</p>", "<p>The Basic Survey on Foreign Residents in FY2022 [##UREF##3##5##] revealed that over half of foreign residents (52.8%) required language assistance when seeking medical care. When visiting a hospital, 27.2% of foreign residents consulted a family member, relative, or friend who spoke Japanese (ad hoc interpreters); 10.6% used a multilingual translator or application (artificial intelligence [AI] translator); and 2.2% requested a medical interpreter. Given that patients’ perceptions are developed based on the information they receive from formal and informal sources, including healthcare providers, media, family, friends, and fellow patients [##REF##17228551##6##], it is reasonable to assume that language assistance may significantly impact non-Japanese-speaking patients’ illness perception.</p>", "<p>In the realm of healthcare, the concept of <italic>illness perception</italic> holds significance. Illness perception refers to “the representations and beliefs that people have about their symptoms and illnesses” [##REF##27515801##7##, ####UREF##4##8##, ##UREF##5##9####5##9##]. Numerous studies [##REF##23382535##10##, ####REF##22991130##11##, ##REF##27601893##12##, ##REF##27757843##13####27757843##13##] and meta-analyses [##REF##26541550##14##,##REF##27412423##15##] have demonstrated that illness perception influences patient outcomes, such as treatment adherence and quality of life. However, most studies of illness perception have been conducted among native speakers with specific diseases. Although some studies have focused on specific ethnic minority groups with diabetes mellitus [##UREF##6##16##,##REF##26415673##17##], they typically overlooked the influence of language assistance on non-native-speaking patients. Considering that approximately half of foreign residents in Japanese society require language assistance when seeking medical care, it is imperative to delve into patients’ illness perceptions, encompassing both those receiving language assistance and those who are not.</p>", "<p>Therefore, this study aimed to examine illness perception accorded by language assistance in non-Japanese-speaking patients through a questionnaire survey and analyze related factors. The present results are expected to promote behavioral changes among healthcare providers and improve access to healthcare in communities.</p>" ]
[ "<title>Materials and methods</title>", "<p>Setting and participants</p>", "<p>An online questionnaire survey was conducted twice, from February to May 2022 and from February to April 2023, targeting non-Japanese-speaking individuals aged 18 years or older who sought medical care at hospitals in Japan. This study was approved by the Research Ethics Committee of the Faculty of Medicine of Juntendo University (approval numbers: E21-0237-M02 and E21-0237-M04). </p>", "<p>The questionnaire was available in multiple languages, including Japanese (phonograms added to Chinese characters), English, Chinese, and Vietnamese, which allowed participants to choose the most suitable language for their responses. A total of 46 organizations, including Japanese language support networks, cooperative leagues of commerce and industry, and international associations, volunteered to participate in this study and distributed an online questionnaire to 4,962 non-Japanese-speaking residents who were members of these organizations. Figure ##FIG##0##1## presents a flowchart of the participant selection process. The survey was conducted from February to May 2022, and 169 responses were collected from 23 organizations with a response rate of 9.4% (<italic>n</italic> = 169/1,810). In a subsequent survey conducted between February and April 2023, 201 responses were collected from other 23 organizations, resulting in a response rate of 6.4% (<italic>n</italic> = 201/3,152). After excluding invalid responses, the final sample size was 293 participants. Although the response rate of the study is below 10%, it is reasonable for internet-based surveys [##UREF##7##18##].</p>", "<p>Measures</p>", "<p>The anonymous online questionnaire used in this study comprised three main parts. Part 1 collected basic background information using the following six items: age, sex, nationality or region, residence status, duration of living in Japan, and language used in daily life. Part 2 focused on participants’ medical care experiences at hospitals in Japan regarding their current or previous treatments. Three items from Part 2 were used in this study. Item 1 asked participants to indicate their method of communication when interacting with physicians, using options such as direct conversations, AI translators, or an interpreter. Participants who conversed directly with physicians were categorized into the non-assistance group, whereas those who used AI translators or interpreters were categorized into the language assistance group. Item 2 measured the participants’ self-reported degree of comprehension of conversations with the physician using a scale from 1 to 4. A score of 1 represented the worst possible comprehension, and a score of 4 represented the best possible comprehension. Participants who scored 1 or 2 were categorized as having no comprehension of medical consultations, whereas those who scored 3 or 4 were categorized as having comprehension. Item 3 was open-ended, asking participants to name their disease. Part 3 included the Brief Illness Perception Questionnaire (Brief IPQ) [##REF##16731240##19##]. The illness perception of the participants was assessed using the Brief IPQ, which comprises nine items representing the nine dimensions of the construct. The first eight items, (1) consequences, (2) timelines, (3) personal control, (4) treatment control, (5) identity, (6) concern, (7) coherence, and (8) emotional response, were assessed using a Likert scale from 0 to 10. The last item (9) was open-ended and asked patients to indicate the three most important causes of their disease. The scores on Items 3, 4, and 7 were reversed such that, for each item, higher scores indicated stronger perceptions. The total score ranges from 0 to 80, with higher scores indicating higher threat perceptions. The Brief IPQ cutoff point for interpreting the level of threat perception (low or high) was determined based on the median of the total score (32) in this study. A score ≥32 was used to define the category of high threat perception, and a score &lt;32 was used to define the category of low threat perception.</p>", "<p>Statistical analysis</p>", "<p>Data were analyzed using IBM SPSS Statistics for Windows, Version 29 (IBM Corp., Armonk, NY). The significance level was set at <italic>P</italic> &lt; 0.05 with a two-tailed test. Chi-square and Mann-Whitney U tests were used to compare background information, the degree of comprehension of the consultation conversation, the scores of each item of the Brief IPQ, and the total score of the Brief IPQ between the language assistance and non-assistance groups. Subsequently, a logistic regression analysis was performed to examine the relationships between basic background characteristics, comprehension of medical consultations, and illness perception accorded by the language assistance and non-assistance groups.</p>" ]
[ "<title>Results</title>", "<p>Table ##TAB##0##1## lists the participants’ background information. The mean age of the participants was 31.25 years (standard deviation [SD] = 11.19, range 18-76), with a median age of 27 years (interquartile range [IQR] 23.50-37.00). Of the total number of participants, 129 (44%) were men and 164 (56%) were women. The majority of the participants were from China (115/293, 39.2%), followed by Vietnam (107/293, 36.5%). In terms of residence status, the three primary categories were students (86/293, 29.4%), technical intern training (70/293, 23.9%), and status-based (46/293, 15.7%, encompassing permanent residents, special permanent residents, spouses or children of Japanese nationals, and spouses or children of permanent residents). Of the 293 participants, approximately 225 (64.8%) had resided in Japan for one to five years and 181 (61.8%) reported using Japanese daily.</p>", "<p>In this survey, 192 (65.5%) participants conversed directly with the physician, while 101 (34.5%) used various communication aids, including ad hoc interpreters (53, 52.5%), AI translators (29, 28.7%), nonmedical interpreters (13, 12.9%), and medical interpreters (6, 5.9%). A chi-square test revealed significant relationships between language assistance and several variables, including <italic>nationality or region</italic>, <italic>residence status</italic>, and <italic>using Japanese in daily life</italic>, with <italic>P</italic> &lt; 0.001, as well as <italic>duration of living in Japan</italic> (<italic>P</italic> = 0.003).</p>", "<p>Table ##TAB##1##2## shows the Brief IPQ scores and the degree of comprehension of the consultation conversation between the language assistance and non-assistance groups. The comprehension of consultation conversations was significantly lower in the language assistance group than in the non-assistance group (<italic>P</italic> &lt; 0.001). Regarding the Brief IPQ subscales, the language assistance group had lower scores for <italic>timelines</italic> (<italic>P</italic> &lt; 0.001), <italic>identity</italic> (<italic>P</italic> &lt; 0.001), and <italic>emotional response</italic> (<italic>P</italic> = 0.02). Although the language assistance group had a higher score for <italic>coherence</italic> (<italic>P</italic> = 0.04), this indicates lower levels of understanding due to the reversed nature of the <italic>coherence</italic> item. The total Brief IPQ score was significantly lower in the language assistance group than in the non-assistance group (<italic>P</italic> = 0.04).</p>", "<p>Table ##TAB##2##3## shows the univariate and multivariate regression estimates of the odds ratio (OR) for low threat perception among participants in the language assistance group. In the univariate analysis, age was found to be significantly associated with low threat perception (OR = 0.93, 95% CI = 0.89-0.98). Female participants had higher odds of lower threat perception (OR = 0.44, 95% CI = 0.19-0.98) than male participants; Vietnamese participants showed significantly higher odds of outcome (OR = 6.18, 95% CI = 2.23-17.1) compared to Chinese participants; and participants who comprehended the context of medical consultations had lower threat perception (OR = 0.34, 95% CI = 0.15-0.77). In the multivariate analysis, age (OR = 0.93, 95% CI = 0.84-0.99) and participants who comprehended the context of medical consultations (OR = 0.31, 95% CI = 0.11-0.83) also had lower threat perception. However, sex and nationality/region were not significantly associated with outcomes. </p>", "<p>Notably, when the same univariate and multivariate regression analyses were performed among participants in the non-assistance group, no significant associations with lower threat perception were observed (Table ##TAB##3##4##).</p>" ]
[ "<title>Discussion</title>", "<p>To our knowledge, this study is the first to investigate illness perception in non-Japanese-speaking patients accorded by language assistance. A significant finding was that patients in the language assistance group exhibited lower comprehension of consultation conversations, less understanding of their diseases, and lower levels of illness perception than those in the non-assistance group. Additionally, within the language assistance group, specific factors such as age and ability to understand medical consultations were related to illness perception.</p>", "<p>By comparing the Basic Survey of Foreign Residents in FY2022, reported by the Japanese government [##UREF##3##5##], this study found similarities and differences in participants’ background characteristics. Both sources show that approximately half of foreign residents had resided in Japan for less than 10 years, with China and Vietnam being the two main countries of origin. However, there is a disparity in the distribution of residential status between these two sources. The government report revealed <italic>permanent residents</italic> (30.4%); <italic>engineers, specialists in humanities</italic>, and <italic>international services</italic> (14.4%); and <italic>students</italic> (10.7%) as the top categories. In this study, most participants were categorized as <italic>students</italic> (29.4%); <italic>technical intern trainees</italic> (23.9%); and <italic>engineers, specialists in humanities, and international services</italic> (15.4%). Consequently, the participants in this study were relatively young (mean age ± SD = 31.3 ± 11.2) and had a shorter overall life experience in Japan. Considering the increasing number of young foreign workers in Japan, the unique characteristics of the study participants provide valuable insights for future research.</p>", "<p>Approximately one-third of the participants did not know the name of their disease. Another third provided information on various medical conditions, whereas the remaining participants listed more than 20 different disease names. Notably, no serious diseases, such as malignant tumors or cardiovascular disease, were mentioned. Medical conditions primarily included common issues such as colds, allergies, and injuries. The participants in the language assistance group commonly used ad hoc interpreters and AI translators. However, the utilization of medical interpreters was rare, which is consistent with the findings of a Japanese government report [##UREF##3##5##].</p>", "<p>In this context, the study’s data showed that illness perception among participants with these low-risk conditions differed between the language assistance and non-assistance groups. The language assistance group reported weaker appreciation of their diseases (timelines), fewer somatic complaints (identity), and poorer understanding (coherence) than the non-assistance group. These results suggest that ad hoc interpreters or AI translators while providing valuable forms of language assistance may not be sufficient for effective communication and for promoting a better understanding of illness perception during medical consultations. This is because they may not possess the expertise of medical interpreters to convey complex medical information accurately or maintain cultural sensitivity.</p>", "<p>Previous research [##REF##17362215##20##,##REF##26549596##21##] has consistently demonstrated that medical interpreters are associated with better clinical care than ad hoc interpreters. Relying solely on ad hoc interpreters or AI translators may pose risks to patients and healthcare providers in terms of treatment adherence and overall healthcare outcomes. Considering the outcomes for non-Japanese-speaking patients, healthcare providers and government agencies should improve the availability of medical interpreters to maximize effective communication and provide better clinical care for diverse linguistic populations.</p>", "<p>Additionally, younger participants and those with a lower comprehension of medical consultations exhibited lower threat perception in the language assistance group. In contrast, no associations between background characteristics and illness perception were observed among participants in the non-assistance group. These findings suggest that a one-size-fits-all communication approach may not be effective for non-Japanese-speaking patients. Given these results, physicians should consider tailoring their care approaches based on the patient’s background characteristics to match the patient’s comprehension. While communication skills were not the primary focus of this study, the vigorous promotion of <italic>Easy Japanese</italic> at medical institutions is necessary. <italic>Easy Japanese</italic>, a simple, easy-to-understand way of speaking Japanese, as proposed by Sato [##UREF##8##22##], can enhance communication between healthcare providers and patients in medical settings. Medical professionals have successfully implemented <italic>Easy Japanese</italic> in medical education [##UREF##9##23##]. Furthermore, health literacy programs can assist patients in better understanding medical terms, procedures, and the nature of their illnesses [##REF##37198627##24##]. This consideration is crucial for ensuring effective communication and promoting a better understanding of illnesses, particularly when developing strategies aimed at enhancing healthcare access and outcomes for non-Japanese-speaking patients.</p>", "<p>This study provided important findings regarding illness perception accorded by language assistance. However, this study had several limitations. First, participants with serious illnesses were not included. Future research should consider this group to better understand variations in illness perceptions. Second, this study used a cross-sectional design that retrospectively asked participants about their clinical experiences. This approach may introduce bias in illness perception as it can change over time. Finally, owing to the small sample size of the language assistance group, this study did not discuss the effects of various language aids on illness perception. Studies with larger sample sizes and a longitudinal design are required to address these limitations.</p>" ]
[ "<title>Conclusions</title>", "<p>To the best of our knowledge, this is the first study to clarify illness perception in non-Japanese-speaking patients accorded by language assistance. The findings demonstrated significant differences in illness perception between the language assistance and non-assistance groups. Additionally, age and comprehension of medical consultations were significantly associated with low threat perception among participants in the language-assistance group, whereas no significant association was observed in the non-assistance group. These findings suggest that methods of language assistance and effective communication should be carefully considered in medical settings. Although future surveys should consider incorporating more detailed information on specific diseases, this study provides valuable scientific evidence regarding illness perceptions in diverse linguistic populations. It also suggests research directions for exploring the association between language assistance and illness perception among non-Japanese-speaking patients.</p>" ]
[ "<p>Background and objective</p>", "<p>While over half of foreign residents in Japan need language assistance during medical consultations, no extant studies have clarified illness perception accorded by language assistance in non-Japanese-speaking patients. This cross-sectional study conducted an online questionnaire survey to investigate the illness perception of non-Japanese-speaking patients and analyze the factors related to illness perception.</p>", "<p>Methodology</p>", "<p>The survey was conducted twice, from February to May 2022 and from February to April 2023, targeting non-Japanese-speaking individuals. In total, 293 valid responses were obtained. The Brief Illness Perception Questionnaire (Brief IPQ) scores were compared between the groups receiving language assistance and those without assistance, and a logistic regression analysis was performed to examine the factors related to illness perception accorded by the status of the language assistance group.</p>", "<p>Results</p>", "<p>The total score for illness perception was significantly lower in the language assistance group than in the non-assistance group (<italic>P</italic> = 0.04). Moreover, in the language assistance group, age (odds ratio [OR] = 0.91, 95% confidence interval [CI] = 0.84-0.99) and comprehension of medical consultations (OR = 0.31, 95% CI = 0.11-0.83) were significantly associated with low illness perception among participants. However, these associations were not observed in the non-assistance group.</p>", "<p>Conclusions</p>", "<p>These findings underscore the crucial role of ensuring effective communication and promoting a better understanding of illness perception during medical consultations.</p>" ]
[]
[ "<p>The authors would like to thank all participating organizations for distributing the online questionnaire and all the respondents for their valuable participation. In addition, they would like to express their gratitude to the professors and members of the Department of Medical Interpreting, Graduate School of Medicine, Juntendo University, for their guidance, assistance, and cooperation throughout the preparation of this thesis.</p>" ]
[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Flowchart of the participant’s selection process.</title><p>Image credit: Yue Wang.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Descriptive characteristics of various populations by language assistance.</title><p><sup>a</sup>The Mann-Whitney U test was used to compare age between the language assistance and non-assistance groups, whereas the chi-square test was used for other variables.</p><p><sup>b</sup><italic>Specialized or technical fields</italic> include highly skilled foreign professionals, attorneys, judicial scriveners, public accountants, tax accountants, artists, etc.</p><p><sup>c</sup><italic>Specified skilled worker</italic> includes specified skilled workers (work-ready foreign nationals who possess certain expertise and skills in certain industrial fields).</p><p><sup>d</sup><italic>Based on status</italic> includes permanent residents, special permanent residents, spouse or child of a Japanese national, or spouse or child of a permanent resident.</p><p><sup>e</sup><italic>Technical intern training</italic> includes trainees (technical interns) in a local government.</p><p>SD, standard deviation</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">Whole samples</td><td colspan=\"4\" rowspan=\"1\">Language assistance</td><td rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">\n<italic>n</italic> = 293</td><td colspan=\"2\" rowspan=\"1\">Yes (<italic>n</italic> = 101)</td><td colspan=\"2\" rowspan=\"1\">No (<italic>n</italic> = 192)</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">n</td><td rowspan=\"1\" colspan=\"1\">%</td><td rowspan=\"1\" colspan=\"1\">n</td><td rowspan=\"1\" colspan=\"1\">%</td><td rowspan=\"1\" colspan=\"1\">n</td><td rowspan=\"1\" colspan=\"1\">%</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age <sup>a</sup>\n</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">0.11</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Mean ± SD</td><td colspan=\"2\" rowspan=\"1\">  31.3 ± 11.2</td><td colspan=\"2\" rowspan=\"1\">30.0 ± 10.8</td><td colspan=\"2\" rowspan=\"1\">31.9 ± 11.4</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Median</td><td colspan=\"2\" rowspan=\"1\">27</td><td colspan=\"2\" rowspan=\"1\">26</td><td colspan=\"2\" rowspan=\"1\">28</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">IQR</td><td colspan=\"2\" rowspan=\"1\">23.5-37.0</td><td colspan=\"2\" rowspan=\"1\">23-34</td><td colspan=\"2\" rowspan=\"1\">24-38</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sex</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Male</td><td rowspan=\"1\" colspan=\"1\">129</td><td rowspan=\"1\" colspan=\"1\">44</td><td rowspan=\"1\" colspan=\"1\">43</td><td rowspan=\"1\" colspan=\"1\">42.6</td><td rowspan=\"1\" colspan=\"1\">86</td><td rowspan=\"1\" colspan=\"1\">44.8</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Female</td><td rowspan=\"1\" colspan=\"1\">164</td><td rowspan=\"1\" colspan=\"1\">56</td><td rowspan=\"1\" colspan=\"1\">58</td><td rowspan=\"1\" colspan=\"1\">57.4</td><td rowspan=\"1\" colspan=\"1\">106</td><td rowspan=\"1\" colspan=\"1\">55.2</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nationality/region</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  China</td><td rowspan=\"1\" colspan=\"1\">115</td><td rowspan=\"1\" colspan=\"1\">39.2</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\">26.7</td><td rowspan=\"1\" colspan=\"1\">88</td><td rowspan=\"1\" colspan=\"1\">45.8</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Vietnam</td><td rowspan=\"1\" colspan=\"1\">107</td><td rowspan=\"1\" colspan=\"1\">36.5</td><td rowspan=\"1\" colspan=\"1\">54</td><td rowspan=\"1\" colspan=\"1\">53.5</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">27.6</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Other Asian country</td><td rowspan=\"1\" colspan=\"1\">41</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">11.9</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">15.1</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  North America and Europe</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">5.1</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">5.7</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Latin America and Africa</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">5.1</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">5.7</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Residence status</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Specialized or technical fields<sup>b</sup>\n</td><td rowspan=\"1\" colspan=\"1\">45</td><td rowspan=\"1\" colspan=\"1\">15.4</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">9.9</td><td rowspan=\"1\" colspan=\"1\">35</td><td rowspan=\"1\" colspan=\"1\">18.2</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Specified skilled worker<sup>c</sup>\n</td><td rowspan=\"1\" colspan=\"1\">19</td><td rowspan=\"1\" colspan=\"1\">6.5</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">7.3</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Based on status<sup>d</sup>\n</td><td rowspan=\"1\" colspan=\"1\">46</td><td rowspan=\"1\" colspan=\"1\">15.7</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">14.9</td><td rowspan=\"1\" colspan=\"1\">31</td><td rowspan=\"1\" colspan=\"1\">16.1</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Technical intern training<sup>e</sup>\n</td><td rowspan=\"1\" colspan=\"1\">70</td><td rowspan=\"1\" colspan=\"1\">23.9</td><td rowspan=\"1\" colspan=\"1\">44</td><td rowspan=\"1\" colspan=\"1\">43.6</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">13.5</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Students</td><td rowspan=\"1\" colspan=\"1\">86</td><td rowspan=\"1\" colspan=\"1\">29.4</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">14.9</td><td rowspan=\"1\" colspan=\"1\">71</td><td rowspan=\"1\" colspan=\"1\">37</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Others</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\">9.2</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">11.9</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">7.8</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Duration of living in Japan</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">0.003</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  &lt;1 year</td><td rowspan=\"1\" colspan=\"1\">35</td><td rowspan=\"1\" colspan=\"1\">11.9</td><td rowspan=\"1\" colspan=\"1\">20</td><td rowspan=\"1\" colspan=\"1\">19.8</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">7.8</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  1-5 years</td><td rowspan=\"1\" colspan=\"1\">190</td><td rowspan=\"1\" colspan=\"1\">64.8</td><td rowspan=\"1\" colspan=\"1\">67</td><td rowspan=\"1\" colspan=\"1\">66.3</td><td rowspan=\"1\" colspan=\"1\">123</td><td rowspan=\"1\" colspan=\"1\">64.1</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  6-10 years</td><td rowspan=\"1\" colspan=\"1\">42</td><td rowspan=\"1\" colspan=\"1\">14.3</td><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">10.9</td><td rowspan=\"1\" colspan=\"1\">31</td><td rowspan=\"1\" colspan=\"1\">16.1</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  11-20 years</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3.4</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">4.2</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  ≥21 years</td><td rowspan=\"1\" colspan=\"1\">16</td><td rowspan=\"1\" colspan=\"1\">5.5</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">7.8</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Using Japanese in daily life</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">   No</td><td rowspan=\"1\" colspan=\"1\">112</td><td rowspan=\"1\" colspan=\"1\">38.2</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">52.5</td><td rowspan=\"1\" colspan=\"1\">59</td><td rowspan=\"1\" colspan=\"1\">30.7</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Yes</td><td rowspan=\"1\" colspan=\"1\">181</td><td rowspan=\"1\" colspan=\"1\">61.8</td><td rowspan=\"1\" colspan=\"1\">48</td><td rowspan=\"1\" colspan=\"1\">47.5</td><td rowspan=\"1\" colspan=\"1\">133</td><td rowspan=\"1\" colspan=\"1\">69.3</td><td rowspan=\"1\" colspan=\"1\"> </td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Scores of Brief IPQ and the degree of comprehension of consultation conversation.</title><p><sup>a</sup><italic>Personal control</italic>, <italic>treatment control</italic>, and <italic>coherence</italic> are reversed items.</p><p>Brief IPQ, Brief Illness Perception Questionnaire; IQR, interquartile range</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td colspan=\"4\" rowspan=\"1\">Language assistance</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"2\" colspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">Yes (<italic>n</italic> = 101)</td><td colspan=\"2\" rowspan=\"1\">No (<italic>n</italic> = 192)</td><td rowspan=\"2\" colspan=\"1\">\n<italic>P</italic> for difference</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Median (IQR)</td><td rowspan=\"1\" colspan=\"1\">Mean rank</td><td rowspan=\"1\" colspan=\"1\">Median (IQR)</td><td rowspan=\"1\" colspan=\"1\">Mean rank</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Degree of comprehension</td><td rowspan=\"1\" colspan=\"1\">2.0 (2.0-3.0)</td><td rowspan=\"1\" colspan=\"1\">99.1  </td><td rowspan=\"1\" colspan=\"1\">3.0 (3.0-4.0)</td><td rowspan=\"1\" colspan=\"1\">172.3</td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Brief IPQ item</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Consequences</td><td rowspan=\"1\" colspan=\"1\">2.0 (0.5-6.0)</td><td rowspan=\"1\" colspan=\"1\">140.7</td><td rowspan=\"1\" colspan=\"1\">3.00 (1.0-6.0)</td><td rowspan=\"1\" colspan=\"1\">150.3</td><td rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Timelines</td><td rowspan=\"1\" colspan=\"1\">1.0 (0.0-3.0)</td><td rowspan=\"1\" colspan=\"1\">135.4</td><td rowspan=\"1\" colspan=\"1\">3.0 (1.0-7.0)</td><td rowspan=\"1\" colspan=\"1\">164.6</td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Personal control<sup>a</sup>\n</td><td rowspan=\"1\" colspan=\"1\">4.0 (0.0-8.0)</td><td rowspan=\"1\" colspan=\"1\">150.4</td><td rowspan=\"1\" colspan=\"1\">4.0 (2.0-6.0)</td><td rowspan=\"1\" colspan=\"1\">145.2</td><td rowspan=\"1\" colspan=\"1\">0.61</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Treatment control<sup>a</sup>\n</td><td rowspan=\"1\" colspan=\"1\">2.0 (0.0-5.0)</td><td rowspan=\"1\" colspan=\"1\">140.1</td><td rowspan=\"1\" colspan=\"1\">3.0 (0.0-6.0)</td><td rowspan=\"1\" colspan=\"1\">150.7</td><td rowspan=\"1\" colspan=\"1\">0.30</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Identity</td><td rowspan=\"1\" colspan=\"1\">2.0 (0.0-5.0)</td><td rowspan=\"1\" colspan=\"1\">121.1</td><td rowspan=\"1\" colspan=\"1\">4.0 (1.3-6.0)</td><td rowspan=\"1\" colspan=\"1\">160.7</td><td rowspan=\"1\" colspan=\"1\">&lt;0.001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Concern</td><td rowspan=\"1\" colspan=\"1\">4.0 (0.0-8.0)</td><td rowspan=\"1\" colspan=\"1\">134.1</td><td rowspan=\"1\" colspan=\"1\">5.0 (3.0-8.0)</td><td rowspan=\"1\" colspan=\"1\">153.8</td><td rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Coherence<sup>a</sup>\n</td><td rowspan=\"1\" colspan=\"1\">4.0 (0.0-7.0)</td><td rowspan=\"1\" colspan=\"1\">160.6</td><td rowspan=\"1\" colspan=\"1\">2.0 (1.0-5.0)</td><td rowspan=\"1\" colspan=\"1\">139.8</td><td rowspan=\"1\" colspan=\"1\">0.04</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Emotional response</td><td rowspan=\"1\" colspan=\"1\">3.0 (0.0-5.5)</td><td rowspan=\"1\" colspan=\"1\">131.4</td><td rowspan=\"1\" colspan=\"1\">5.0 (1.0-7.0)</td><td rowspan=\"1\" colspan=\"1\">155.2</td><td rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">29.0 (15.0-40.0)</td><td rowspan=\"1\" colspan=\"1\">133.3</td><td rowspan=\"1\" colspan=\"1\">33.0 (24.0-41.0)</td><td rowspan=\"1\" colspan=\"1\">154.2</td><td rowspan=\"1\" colspan=\"1\">0.04</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Univariate and multivariate regression estimates of odds ratio for low illness perception outcome among participants in the language assistance group (n = 101).</title><p><sup>a</sup>Multivariate regression via the forced entry procedure was used to analyze the data.</p><p><sup>*</sup><italic>P</italic> &lt; 0.05.</p><p><sup>**</sup><italic>P</italic> &lt; 0.001.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">Univariate</td><td colspan=\"2\" rowspan=\"1\">Multivariate<sup>a</sup>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Odds ratio</td><td rowspan=\"1\" colspan=\"1\">95% CI</td><td rowspan=\"1\" colspan=\"1\">Odds ratio</td><td rowspan=\"1\" colspan=\"1\">95% CI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">0.93</td><td rowspan=\"1\" colspan=\"1\">0.89-0.98*</td><td rowspan=\"1\" colspan=\"1\">0.93</td><td rowspan=\"1\" colspan=\"1\"> 0.84-0.99*</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sex</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Male</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Female</td><td rowspan=\"1\" colspan=\"1\">0.44</td><td rowspan=\"1\" colspan=\"1\">0.19-0.98*</td><td rowspan=\"1\" colspan=\"1\">0.43</td><td rowspan=\"1\" colspan=\"1\">0.16-1.17</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nationality/region</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  China</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Vietnam</td><td rowspan=\"1\" colspan=\"1\">6.18</td><td rowspan=\"1\" colspan=\"1\">2.23-17.1**</td><td rowspan=\"1\" colspan=\"1\">5.43</td><td rowspan=\"1\" colspan=\"1\">0.91-32.3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Other Asian countries</td><td rowspan=\"1\" colspan=\"1\">1.70</td><td rowspan=\"1\" colspan=\"1\">0.41-6.98</td><td rowspan=\"1\" colspan=\"1\">1.68</td><td rowspan=\"1\" colspan=\"1\">0.31-9.13</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Rest of the world</td><td rowspan=\"1\" colspan=\"1\">0.79</td><td rowspan=\"1\" colspan=\"1\">0.13-4.79</td><td rowspan=\"1\" colspan=\"1\">1.29</td><td rowspan=\"1\" colspan=\"1\">0.13-11.4</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Residence status</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Specialized or technical fields</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Training and skilled workers</td><td rowspan=\"1\" colspan=\"1\">3.09</td><td rowspan=\"1\" colspan=\"1\">0.76-12.5</td><td rowspan=\"1\" colspan=\"1\">0.64</td><td rowspan=\"1\" colspan=\"1\">0.10-4.55</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Based on status</td><td rowspan=\"1\" colspan=\"1\">0.75</td><td rowspan=\"1\" colspan=\"1\">0.14-3.94</td><td rowspan=\"1\" colspan=\"1\">1.73</td><td rowspan=\"1\" colspan=\"1\">0.19-15.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Students</td><td rowspan=\"1\" colspan=\"1\">1.31</td><td rowspan=\"1\" colspan=\"1\">0.26-6.64</td><td rowspan=\"1\" colspan=\"1\">0.80</td><td rowspan=\"1\" colspan=\"1\">0.08-7.06</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Others</td><td rowspan=\"1\" colspan=\"1\">1.07</td><td rowspan=\"1\" colspan=\"1\">0.19-5.91</td><td rowspan=\"1\" colspan=\"1\">2.04</td><td rowspan=\"1\" colspan=\"1\">0.20-21.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Duration of living in Japan</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  &lt;6 years</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  ≥6 years</td><td rowspan=\"1\" colspan=\"1\">0.85</td><td rowspan=\"1\" colspan=\"1\">0.28-2.63</td><td rowspan=\"1\" colspan=\"1\">5.91</td><td rowspan=\"1\" colspan=\"1\">0.76-45.9</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Using Japanese in daily life</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  No</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Yes</td><td rowspan=\"1\" colspan=\"1\">2.01</td><td rowspan=\"1\" colspan=\"1\">0.91–4.47</td><td rowspan=\"1\" colspan=\"1\">1.75</td><td rowspan=\"1\" colspan=\"1\">0.61–5.06</td></tr><tr><td colspan=\"3\" rowspan=\"1\">Comprehension of medical consultations</td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  No</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Yes</td><td rowspan=\"1\" colspan=\"1\">0.34</td><td rowspan=\"1\" colspan=\"1\">0.15-0.77*</td><td rowspan=\"1\" colspan=\"1\">0.31</td><td rowspan=\"1\" colspan=\"1\"> 0.11-0.83*</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Univariate and multivariate regression estimates of odds ratio for low illness perception outcome among participants in the non-assistance group (n = 192).</title><p><sup>a</sup>Multivariate regression via the forced entry procedure was used to analyze the data.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">Univariate</td><td colspan=\"2\" rowspan=\"1\">Multivariate <sup>a</sup>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Odds ratio</td><td rowspan=\"1\" colspan=\"1\">95% CI</td><td rowspan=\"1\" colspan=\"1\">Odds ratio</td><td rowspan=\"1\" colspan=\"1\">95% CI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">0.99</td><td rowspan=\"1\" colspan=\"1\">0.96-1.01</td><td rowspan=\"1\" colspan=\"1\">0.98</td><td rowspan=\"1\" colspan=\"1\"> 0.95-1.02</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sex</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Male</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Female</td><td rowspan=\"1\" colspan=\"1\">1.02</td><td rowspan=\"1\" colspan=\"1\">0.58-1.80</td><td rowspan=\"1\" colspan=\"1\">1.01</td><td rowspan=\"1\" colspan=\"1\">0.54-1.88</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nationality/region</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  China</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Vietnam</td><td rowspan=\"1\" colspan=\"1\">1.74</td><td rowspan=\"1\" colspan=\"1\">0.87-3.49</td><td rowspan=\"1\" colspan=\"1\">1.59</td><td rowspan=\"1\" colspan=\"1\">0.62-4.10</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Other Asian countries</td><td rowspan=\"1\" colspan=\"1\">0.96</td><td rowspan=\"1\" colspan=\"1\">0.41-2.22</td><td rowspan=\"1\" colspan=\"1\">1.24</td><td rowspan=\"1\" colspan=\"1\">0.47-3.29</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Rest of the world</td><td rowspan=\"1\" colspan=\"1\">1.43</td><td rowspan=\"1\" colspan=\"1\">0.56-3.65</td><td rowspan=\"1\" colspan=\"1\">2.47</td><td rowspan=\"1\" colspan=\"1\">0.76-7.98</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Residence status</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Specialized or technical fields</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Training and skilled workers</td><td rowspan=\"1\" colspan=\"1\">2.48</td><td rowspan=\"1\" colspan=\"1\">0.97-6.36</td><td rowspan=\"1\" colspan=\"1\">2.31</td><td rowspan=\"1\" colspan=\"1\">0.71-7.53</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Based on status</td><td rowspan=\"1\" colspan=\"1\">1.38</td><td rowspan=\"1\" colspan=\"1\">0.51-3.76</td><td rowspan=\"1\" colspan=\"1\">1.72</td><td rowspan=\"1\" colspan=\"1\">0.57-5.34</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Students</td><td rowspan=\"1\" colspan=\"1\">1.58</td><td rowspan=\"1\" colspan=\"1\">0.69-3.65</td><td rowspan=\"1\" colspan=\"1\">1.65</td><td rowspan=\"1\" colspan=\"1\">0.61-4.48</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Others</td><td rowspan=\"1\" colspan=\"1\">2.19</td><td rowspan=\"1\" colspan=\"1\">0.64-7.50</td><td rowspan=\"1\" colspan=\"1\">2.40</td><td rowspan=\"1\" colspan=\"1\">0.68-8.50</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Duration of living in Japan</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  &lt;6 years</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  ≥6 years</td><td rowspan=\"1\" colspan=\"1\">0.80</td><td rowspan=\"1\" colspan=\"1\">0.42-1.51</td><td rowspan=\"1\" colspan=\"1\">0.87</td><td rowspan=\"1\" colspan=\"1\">0.38-1.99</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Using Japanese in daily life</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">  No</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference) </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  Yes</td><td rowspan=\"1\" colspan=\"1\">1.21</td><td rowspan=\"1\" colspan=\"1\">0.65–2.24</td><td rowspan=\"1\" colspan=\"1\">1.33</td><td rowspan=\"1\" colspan=\"1\">0.68–2.63</td></tr><tr><td colspan=\"3\" rowspan=\"1\">Comprehension of medical consultations</td><td colspan=\"2\" rowspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">  No</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td><td colspan=\"2\" rowspan=\"1\">1.00 (reference)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">  Yes</td><td rowspan=\"1\" colspan=\"1\">1.08</td><td rowspan=\"1\" colspan=\"1\">0.54-2.13</td><td rowspan=\"1\" colspan=\"1\">1.40</td><td rowspan=\"1\" colspan=\"1\"> 0.66-2.97</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Yue Wang, Akira Oonishi, Anna Kazami, Ruriko Suminaga, Enari Den , Zhuo Li, Naoko Ono, Francois Niyonsaba , Ai Ikeda</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Yue Wang, Akira Oonishi, Anna Kazami, Ruriko Suminaga, Enari Den , Zhuo Li</p><p><bold>Drafting of the manuscript:</bold>  Yue Wang</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Akira Oonishi, Anna Kazami, Ruriko Suminaga, Enari Den , Zhuo Li, Naoko Ono, Francois Niyonsaba , Ai Ikeda</p><p><bold>Supervision:</bold>  Ai Ikeda</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. The Research Ethics Committee of the Faculty of Medicine of Juntendo University issued approval E21-0237-M02 and E21-0237-M04</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050532-i01\" position=\"float\"/>" ]
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[{"label": ["1"], "article-title": ["Statistical Handbook of Japan 2023"], "source": ["Statistical handbook of Japan"], "person-group": ["\n"], "surname": ["Statistics Bureau of"], "given-names": ["Japan"], "fpage": ["21"], "publisher-loc": ["Japan"], "publisher-name": ["Statistics Bureau"], "volume": ["7"], "year": ["2023"], "uri": ["https://www.stat.go.jp/english/data/handbook/pdf/2023all.pdf#page=1"]}, {"label": ["2"], "article-title": ["How have Japanese policies changed in accepting foreign workers?"], "source": ["Japan Labor Issues"], "person-group": ["\n"], "surname": ["Hamaguchi"], "given-names": ["K"], "fpage": ["2"], "lpage": ["7"], "volume": ["3"], "year": ["2019"], "uri": ["https://www.jil.go.jp/english/jli/documents/2019/014-01.pdf"]}, {"label": ["3"], "article-title": ["Japan\u2019s Immigration Policy 2015-2020: implications for human security of immigrant workers and refugees"], "source": ["J Hum Secur Stud"], "person-group": ["\n"], "surname": ["Takizawa"], "given-names": ["S"], "fpage": ["51"], "lpage": ["78"], "volume": ["10"], "year": ["2021"]}, {"label": ["5"], "article-title": ["Basic Survey on Foreign Residents in FY2022"], "person-group": ["\n"], "surname": ["Immigration Services Agency of"], "given-names": ["Japan"], "year": ["2023"], "uri": ["https://www.moj.go.jp/isa/content/001402002.pdf"]}, {"label": ["8"], "article-title": ["The Commonsense Model of Self-Regulation of Health and Illness"], "source": ["The Self-Regulation of Health and Illness Behaviour"], "person-group": ["\n"], "surname": ["Leventhal", "Brissette", "Leventhal"], "given-names": ["H", "I", "EA"], "fpage": ["42"], "lpage": ["65"], "year": ["2003"]}, {"label": ["9"], "article-title": ["Self-regulation, health, and behavior: a perceptual-cognitive approach"], "source": ["Psychol Health"], "person-group": ["\n"], "surname": ["Leventhal", "Leventhal", "Contrada"], "given-names": ["H", "AE", "JR"], "fpage": ["711"], "lpage": ["733"], "volume": ["13"], "year": ["1998"]}, {"label": ["16"], "article-title": ["Associations between knowledge, illness perceptions, self-management and metabolic control of type 2 diabetes among African and European-origin patients"], "source": ["J Nurs Healthc Chronic Illn"], "person-group": ["\n"], "surname": ["Abubakari", "Jones", "Lauder", "Kirk", "Anderson", "Devasenan"], "given-names": ["AR", "MC", "W", "A", "Anderson", "D"], "fpage": ["245"], "lpage": ["256"], "volume": ["3"], "year": ["2011"]}, {"label": ["18"], "article-title": ["Response rate and response quality of internet-based surveys: an experimental study"], "source": [" Marketing Lett"], "person-group": ["\n"], "surname": ["Deutskens", "de Ruyter", "Wetzels", "Oosterveld"], "given-names": ["E", "K", "M", "P"], "fpage": ["21"], "lpage": ["36"], "volume": ["15"], "year": ["2024"]}, {"label": ["22"], "article-title": ["A study of how to feed disaster information to non-native Japanese speakers: strategies and effectiveness"], "source": ["Jpn Ling"], "person-group": ["\n"], "surname": ["Matsuda", "Maeda", "Sato"], "given-names": ["Y", "R", "K"], "fpage": ["145"], "lpage": ["159"], "volume": ["7"], "year": ["2002"]}, {"label": ["23"], "article-title": ["\u201cEasy Japanese\u201d for everyone: another way to enhance communication skills for promoting partnerships with patients including those from overseas"], "source": ["Med Educ"], "person-group": ["\n"], "surname": ["Takeda", "Ishikawa", "Nii", "Iwata"], "given-names": ["Y", "H", "M", "K"], "fpage": ["655"], "lpage": ["663"], "volume": ["51"], "year": ["2020"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2024-01-14 23:43:50
Cureus.; 15(12):e50532
oa_package/41/21/PMC10787643.tar.gz
PMC10787645
0
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[]
[]
[ "<title>Conclusion</title>", "<p>In conclusion, the compilation of eight articles significantly enhanced our comprehension of exercise interventions for age-related musculoskeletal disorders, promising improved quality of life for older adults. However, we must acknowledge that further research and exploration are required to validate and refine exercise intervention as a powerful tool for addressing age-related musculoskeletal disorders in the aging population.</p>" ]
[ "<p>Edited and reviewed by: Marcia G. Ory, Texas A&amp;M University, United States</p>" ]
[ "<p>With the aging of the global population, the prevalence of age-related musculoskeletal disorders has rapidly escalated, becoming a significant public health priority (##REF##30971232##1##). Age-related musculoskeletal disorders encompass a diverse spectrum of pathological conditions that affect muscles, bones, cartilage, and various other bodily tissues, thereby resulting in substantial physical and functional limitations among affected individuals. These disorders include, but are not confined to, conditions such as sarcopenia, osteoporosis, osteoarthritis, intervertebral disc degeneration, rheumatoid arthritis, and ankylosing spondylitis (##REF##30971232##1##, ##REF##25820482##2##). Exercise has emerged as a pivotal and widely employed strategy for the prevention and treatment of musculoskeletal disorders. In response to health concerns, exercise or rehabilitation exercise regimens are meticulously tailored to enhance not only cardiorespiratory fitness but also flexibility, balance, strength, and power (##REF##26139859##3##). Some key interventional studies have shown that exercise or physical activity improves muscle and skeletal disorders in aging people, especially sarcopenia and osteoporosis (##REF##34409961##4##). In addition, research findings also demonstrated some of the molecular mechanisms that exercise or physical activity benefit skeletal and muscle health (##REF##28825716##5##). However, more investigations are needed to further explore the effects of exercise on the prevention or rehabilitation of age-related muscle and skeletal disorders.</p>", "<p>While extant research has elucidated the molecular mechanisms underlying the salutary impact of exercise and physical activity on skeletal and muscle health, further investigations are still necessary. Therefore, the Research Topic “<italic>Exercise for age-related musculoskeletal disorders</italic>” was launched to collect more articles on exercise and age-related musculoskeletal disorders, aiming to better understand the potential treatment of musculoskeletal disorders in the aging population. In this Research Topic, we divided the published articles into three key themes to provide a comprehensive overview of the role of exercise in age-related musculoskeletal challenges.</p>", "<title>Theme one: alleviating pain in aging</title>", "<p>The first theme explored the efficacy of exercise in addressing attenuating pain associated with age-related musculoskeletal disorders. Osteoarthritis is one of the main musculoskeletal disorders causing knee pains in aging people. <ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1168167\" ext-link-type=\"uri\">Zhang et al.</ext-link> conducted a meta-analysis and found that traditional Chinese exercises including Taijiquan, Baduanjin, Yijinjing, and Wuqinxi alleviated knee pain, stiffness, and improved the physical function in knee osteoarthritis patients. Among these exercises, Taijiquan exhibited more benefits for knee pain and dysfunction (<ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1168167\" ext-link-type=\"uri\">Zhang et al.</ext-link>). Another network meta-analysis explored exercise interventions for chronic low back pain, and it revealed that exercise interventions improved pain in low back pain patients, and Taijiquan was even better than conventional rehabilitation (<ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1155225\" ext-link-type=\"uri\">Li et al.</ext-link>). These findings suggest that exercise serves as a promising intervention for alleviating pain in the aging population.</p>", "<title>Theme two: diverse approaches to exercise for older adults</title>", "<p>The second theme illuminated a diverse array of exercise modalities specifically tailored to older adults. A bibliometric analysis conducted by <ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1133972\" ext-link-type=\"uri\">Mi et al.</ext-link> revealed the research trends in resistance training for aging individuals. The bursts of co-occurrence keyword map highlight the key terms such as “insulin-like growth factor expression,” “blood pressure,” and “health-related quality” are associated with resistance training in aging individuals, which provides valuable insights into research hotspots exercise for improving chronic diseases such as diabetes and hypertension in older adults. Additionally, physical function also is the research frontier in the field of resistance training. <ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1186067\" ext-link-type=\"uri\">Zhou et al.</ext-link> provided us better understanding of pelvic floor muscle exercise may benefit to improving urinary continence following surgery, especially 3 months after radical prostatectomy. Furthermore, <ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1140506\" ext-link-type=\"uri\">Alizadeh et al.</ext-link> contributed to the theme by sharing teleexercise training for geriatric patients with failed back surgery syndrome, providing the potential utility of telehealth platforms for further health care in aging.</p>", "<title>Theme three: mechanisms unveiled</title>", "<p>This theme mainly unveiled the mechanisms of exercise to combat aging-related musculoskeletal disorders. Mitochondrial dysfunction is accepted as a key role in the pathogenesis of conditions, such as sarcopenia, in the aging population. A comprehensive article revealed the intricate relationship between mitochondrial dysfunction and sarcopenia, a chronic degenerative disease that affects older adults. It outlines how exercise can effectively address mitochondrial dysfunction, promoting mitochondrial health and reducing the impact of aging on the musculoskeletal system (<ext-link xlink:href=\"https://doi.org/10.3389/fphys.2023.1196426\" ext-link-type=\"uri\">Zhu et al.</ext-link>). Older adults are at higher risk of falls, <ext-link xlink:href=\"https://doi.org/10.3389/fpubh.2023.1165010\" ext-link-type=\"uri\">Liu Z. et al.</ext-link> investigated the interplay between lower extremity muscle strength, proprioception, and tactile sensation in maintaining postural stability in aging, and found that sensory deficits are a significant risk factor for falls among older adults. Moreover, another article showed that a Chinese traditional exercise—Yi Jing Bang (YJB) exercise has more upward rotation and a similar or less anterior tilt than the mean resting scapular angle, which provides insights into the potential of YJB exercises in shoulder rehabilitation (<ext-link xlink:href=\"https://doi.org/10.3389/fphys.2023.1169092\" ext-link-type=\"uri\">Liu J. et al.</ext-link>).</p>", "<title>Author contributions</title>", "<p>XC: Writing—original draft, Writing—review &amp; editing. KC: Writing—review &amp; editing. JZ: Writing—review &amp; editing.</p>" ]
[ "<title>Conflict of interest</title>", "<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>", "<title>Publisher's note</title>", "<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>" ]
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{ "acronym": [], "definition": [] }
5
CC BY
no
2024-01-14 23:43:50
Front Public Health. 2023 Dec 27; 11:1337093
oa_package/74/28/PMC10787645.tar.gz
PMC10787646
0
[ "<title>1. Introduction</title>", "<p>Dentinogenesis imperfecta describes developmental defects of dentin caused by mutations in gene encoding dentin sialophosphoprotein (DSPP) [##REF##34988181##1##]. Studies have shown that <italic>Dspp</italic> knockout mice exhibited severe dentin defects [##REF##34667213##2##]. The dental phenotypes of the mutant mice could also be the result of the intracellular retention of mutant DSPP in the odontoblasts [##REF##31173534##3##]. Epidemiological data showed that the incidence of dentinogenesis imperfecta ranges from 0.002% to 0.1% in different populations [##REF##30134932##4##–##REF##27957258##6##]. Both enamel and dentin development could be affected by dentinogenesis imperfecta. In mild cases, only crown discoloration and “thistle-shaped” pulp could be noticed. In more severe cases, attrition or even disappearance of crown and complete obliteration of pulp could be seen [##REF##25118030##7##]. Enlarge pulp with “shell teeth” appearance could be scarcely found [##REF##11447953##8##]. Pulp is rarely involved except for the most severe cases [##REF##14640368##9##]. Due to the hypomineralized structure and poor prognosis, pulp involvement of dentinogenesis imperfecta-affected teeth could lead to extraction.</p>", "<p>Regenerative endodontic treatment (RET) has been widely used in necrotic immature permanent teeth [##REF##33149426##10##]. Compared to conventional root canal treatment, pulpotomy, and apexification, RET has the unique potential to continue root maturation and save the teeth for the lifetime of the patient [##REF##35929348##11##]. The basis of RET relies on tissue engineering induced by a complex of stem cells and bioactive growth factors [##REF##30471228##12##, ##REF##18498881##13##]. Biomimetic scaffolds are also used in some cases to increase the success rate [##REF##35310358##14##]. The success rate of RET has been reported to be 94.8% and 96% for dental trauma and caries-caused pulp necrosis, respectively [##REF##32381409##15##]. The success rate of RET for teeth with developmental defects has only been assessed in teeth with dens evaginatus and was slightly lower (93%) [##REF##32381409##15##]. RET for teeth with other dental anomalies, especially with dentinogenesis imperfecta, has been rarely reported.</p>", "<p>This current case presents use of RET in a necrotic immature tooth with severe dentinogenesis imperfecta to induce root maturation and reduce risks of root fracture and extraction.</p>" ]
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[ "<title>3. Discussion</title>", "<p>Dentinogenesis imperfecta includes a series of dentin formation anomalies with different genetic alterations and phenotypic characters. Involvement of endodontic inflammation is rare in dentinogenesis imperfecta. This current clinical case reported pulpal pathosis of a tooth with dentinogenesis imperfecta due to the hypomineralized tooth structure and enlarged pulp cavity. RET was applied to control the inflammation and induce further root development. Both targets were reached successfully, showing a novel strategy for managing these challenging conditions.</p>", "<p>A classical classification of isolated dentin diseases based on clinical phenotypes was proposed by Shields et al. [##REF##4516067##18##]. Studies have shown that these different phenotypes are actually different severities of the same pathology [##REF##12354781##19##–##REF##19131317##22##]. All genetic mutations responsible for dentinogenesis imperfecta have been found to be located on a gene encoding DSPP. Thus, de La Dure-Molla et al. proposed a new classification to simplify diagnosis [##REF##25118030##7##]. The most severe phenotype of dentinogenesis imperfecta, DGI-III, has been described with brown opalescent crown discoloration, enlarge pulp, and “shell teeth” appearance [##REF##15690376##23##]. Due to the large pulp cavity and thin dentin layer, pulp exposure and pulpal inflammation could be possible [##REF##11447953##8##]. This specific phenotype was first found in the triracial subpopulation of Maryland and was described as “the Brandywine isolate” [##REF##13469154##5##]. Most literatures reported DGI-II phenotype in Asian populations [##REF##20146806##20##, ##REF##22310900##24##]. This current case reported typical “shell teeth” phenotype in an Asian patient. Early diagnosis of such cases would be beneficial as timely protection of the hard tissue with composite restorations could avoid potential pulp exposure [##REF##11447953##8##].</p>", "<p>Once pulp involvement occurs in immature permanent teeth with severe dentinogenesis imperfecta, the treatment strategies could be difficult. Most studies reported extraction for such cases [##REF##18603736##25##]. Root canal treatment is impossible for such teeth with short root length and open apex. Apexification seemed a rational choice in this case. However, apexification has limited effect in inducing root development [##REF##35929348##11##]. As the root remains short and the dentinal canal walls remain thin, root fracture is of high risk and extraction could be expected in the long run. In order to achieve long perseverance of the affected tooth, RET was selected for this case. RET has been proved ideal for immature teeth with pulp inflammation [##REF##29777616##26##]. RET recruits stem cells of apical papilla which could survive even after pulp necrosis or apical periodontitis [##REF##18498881##13##]. These stem cells together with growth factors, including DSPP, ALP, and DMP, could contribute to regeneration of pulp-dentin complex [##REF##30471228##12##]. Histologic studies found cementum-like, periodontal ligament-like, and bone-like tissue in canal space after RET [##REF##33149426##10##]. While whether RET induces “true” regeneration remains controversial, the increase of root length and thickening of canal walls have been observed in numerous cases [##REF##22077958##27##]. In this case, obvious maturation of root was observed via radiographic examination, especially when compared with its adjacent tooth which was handled with apexification (##FIG##2##Figure 3##). With longer root and thicker canal walls, lower risks of root fracture could be expected.</p>", "<p>In this study, TAP was used to disinfect root canal of the affected tooth. Studies have shown that TAP could remove diverse groups of facultative gram-positive and gram-negative microorganisms and provide an environment for healing of periapical lesions [##REF##30135847##28##]. However, concerns about the drawbacks of TAP have also been addressed. Minocycline could cause calcium chelation from the dentin and has a demineralizing effect on dentin, leading to the brittleness of tooth [##REF##20381577##29##]. Discoloration is the major drawback of TAP, which was also caused by minocycline [##REF##36258984##30##]. Double antibiotic paste (DAP) could be alternatively used to prevent tooth staining [##REF##25556157##31##]. Also, application of dentin bonding agent can greatly reduce the risk of tooth staining [##REF##26295020##16##]. In this case, as the tooth was already seriously worn and appeared yellowish before treatment, aesthetics was not the major concern. For future cases with aesthetic requirement, DAP should be used to avoid tooth discoloration.</p>", "<p>To our knowledge, this is the first case describing use of RET in necrotic immature permanent tooth with dentinogenesis imperfecta. Literatures have shown that DSPP plays an important role in both biological and RET-induced root maturation [##UREF##0##32##, ##REF##35933045##33##]. However, expression of DSPP has been found to be impaired in teeth with dentinogenesis imperfecta [##REF##25118030##7##]. Therefore, we were initially doubtful about the outcome of RET in this case. Evident root maturation illustrated by radiographic examinations indicated that relevant cells and growth factors have been successfully recruited. Findings from this study suggest that RET should be considered not only for immature permanent teeth without developmental defects but also for teeth with structure anomalies. Further studies are required to understand the mechanism of RET-induced root maturation in dentinogenesis imperfecta-affected teeth. Examination of levels and expression of growth factors associated with maturation of root as well as regeneration of pulp tissue, including DSPP, ALP, and DMP, will help reveal the biological process. Animal models should also be used to mimic the dentinogenesis imperfecta phenotype and explore the effectiveness of RET in such cases.</p>" ]
[ "<title>4. Conclusions</title>", "<p>This case report shows that RET could induce root maturation in necrotic immature teeth affected by dentinogenesis imperfecta. Apart from conventional apexification, pediatric dentists and endodontists are encouraged to consider RET as a beneficial strategy for such immature permanent teeth with developmental defects of hard tissue and pulp involvements.</p>" ]
[ "<p>Academic Editor: Sivakumar Nuvvula</p>", "<p>Pulp involvement of immature permanent teeth with dentinogenesis imperfecta is challenging and could lead to extraction. A case of dentinogenesis imperfecta-induced periapical periodontitis of an immature permanent tooth was treated with regenerative endodontic treatment (RET), and root maturation was observed in 12-month follow-up. An 8-year-old girl presented acute pain and swelling in central mandibular region. Clinical and radiographic examination revealed “shell teeth” appearance of teeth 31, 41, and 42. Periapical lesion of tooth 31 was observed. Tooth 41 was previously treated with apexification. RET was planned and carried out for the necrotic tooth (tooth 31) with dentinogenesis imperfecta. The 1-, 3-, 7-, and 12-month postoperative recall revealed complete healing of periapical lesions. Root maturation characterized by elongation of root, thickening of dentinal walls, and closure of root apex was observed with radiographic examinations. We show that RET could be a desirable treatment for necrotic immature permanent teeth with dentinogenesis imperfecta and lead to resolution of endodontic lesions as well as maturation of dental root. The findings of this case suggest that RET should be considered by endodontist and pediatric dentist to treat teeth with similar dental anomalies and apical periodontitis.</p>" ]
[ "<title>2. Case Presentation</title>", "<p>An 8-year-old girl was referred to the Department of Pediatric Dentistry, Nanjing Stomatological Hospital, with the chief complaint of intraoral swelling and pain for over a week. An examination of her medical history showed that she had suffered similar symptoms a year ago and one of the lower incisors (tooth 41) had been treated with apexification. No systematic medical condition was reported. Intraoral examination revealed mixed dentition with normal maxillary incisors and short and worn mandibular central incisors. The permanent right lateral incisor (42) had merely erupted, and only the incisal third could be seen. The primary left lower lateral incisor (72) was present, and the corresponding permanent tooth was not seen. Oral hygiene was poor. A thick layer of debris was observed on the surface of the mandibular incisors. Swelling was extensive in the lower central region. All present mandibular incisors (72, 31, 41, and 42) were mobile and sensitive to percussion/palpation. Radiographic examination revealed that the newly emerged permanent mandibular incisors (31, 41, and 42) were characterized with extremely thin layer of hard tissue, short root, and enlarged pulp space. Periapical radiolucency without caries was noticed for 31 and 42. Radiopaque material was observed in the pulp cavity of 41 (##FIG##0##Figure 1##).</p>", "<p>Based on the symptoms, clinical and radiographic examinations, the definitive diagnosis of the patient was dentinogenesis imperfecta and apical periodontitis of the permanent lower left central incisor [##REF##26295020##16##]. The objective of the short-term treatment was to relieve the pain and avoid further inflammation. Long-term treatment plan included acquiring a functional and aesthetic dentition without pain or sensitivity. Given consideration to the enlarged pulp cavity and short root of the tooth 31 with apical periodontitis, RET was recommended. Risks and benefits of RET and the traditional apexification were explained to the parents, and informed consent was obtained from the parents to perform RET.</p>", "<p>At the first visit, emergent procedures were taken. Access cavity was prepared, and bloody and purulent exudate was seen. The pulp chamber was gently irrigated with 1.0% sodium hypochlorite and sterile saline. No mechanical instrumentation was performed. The affected tooth was left open for 3 days until the second visit to achieve drainage of exudate. At the second visit, intraoral swelling was significantly alleviated and the apical exudation was minimized. After isolating the tooth, pulp disinfection was performed following the American Association of Endodontists' protocol with minor modifications [##REF##29229459##17##]. Briefly, the tooth was thoroughly and gently irrigated with 20 mL 1.0% sodium hypochlorite, dried, and dressed with triple antibiotic paste. Equal amount of metronidazole (Qidu Pharmaceutical Company, China), minocycline (Hanhui Pharmaceutical Company, China), and ciprofloxacin (Jingxin Pharmaceutical Company, China) was mixed and dissolved with sterile saline to make the triple antibiotic paste (TAP). Dentin bonding agent (3M ESPE, Germany) was applied on the walls of pulp chamber to avoid discoloration caused by TAP. The tooth was then temporarily restored using Caviton (GC, Japan). After four weeks, at the third visit, clinical examination revealed that the tooth was asymptomatic. Local anesthesia was performed with 2% lidocaine HCl (without epinephrine; Tiansheng Pharmaceutical Company, China). The tooth was isolated, and the temporary restoration was removed. The root canal was irrigated with sterile saline to remove the antibiotic paste. The pulp cavity was then dried with paper point. A #40 file was used to irritate the apical tissue. Very limited amount of blood was acquired through the irritation procedure, and only the apical third was filled with blood. A bioceramic material, iRoot BP (Innovative BioCeramix, Canada), was placed upon the newly formed blood clot. The tooth was then restored with SonicFill ultrasonic composite resin (Kerr, Germany). In order to examine the position of the blood clot and iRoot BP plug, while in concern that other permanent teeth might also be affected by dentinogenesis imperfecta, a panoramic radiograph was taken (##FIG##1##Figure 2##).</p>", "<p>At 1-month follow-up appointment, the tooth was reported asymptomatic. X-ray examination revealed significant decrease of periapical radiolucency (##FIG##2##Figure 3(a)##). At 3-month, 7-month, and 12-month follow-up appointment, the patient reported that the tooth was asymptomatic and functional. Radiographic examination was performed at 3-month and 12-month postoperative recall, which revealed complete healing of periapical lesion, elongation of the root, closure of the root apex, and obvious dentinal bridge beneath iRoot BP plug (Figures ##FIG##2##3(b)## and ##FIG##2##3(c)##).</p>" ]
[ "<title>Acknowledgments</title>", "<p>This work was supported by the National Natural Science Funds (grant number 82001035) and the “3456” Cultivation Program for Junior Talents of Nanjing Stomatological School, Medical School of Nanjing University (0222R202).</p>", "<title>Data Availability</title>", "<p>The data used to support the findings of this work are available from the corresponding author upon request.</p>", "<title>Consent</title>", "<p>Risks and benefits of RET and the traditional apexification were explained to the mother of the patient. An informed consent was obtained from the mother to perform RET.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare no conflict of interest.</p>", "<title>Authors' Contributions</title>", "<p>YL was responsible for the conceptualization, resources, and funding acquisition and wrote, reviewed, and edited the manuscript. TP was responsible for the data curation and wrote the original draft. XX was responsible for the supervision and wrote, reviewed, and edited the manuscript. Ying Liao and Ting Pan contributed equally to this work and co-first authors.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Preoperative radiographs. Short root, thin hard tissue, and enlarged pulp cavity was evident in teeth 31, 41, and 42. Pulp cavity of 41 was filled with radiopaque material. (a) Tooth 42 merely erupted, and periapical radiolucency was noticed. (b) Tooth 31 fully erupted, and periapical radiolucency was seen.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>Immediate postoperative panoramic radiograph. Tooth 31 was treated with RET. Tooth 41 had been treated with apexification. Permanent mandibular incisors and canines exhibited poor formation of dental hard tissue.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>Recall radiographs of the case. (a) One-month postoperative radiograph showed alleviation of periapical radiolucency. (b) Three-month postoperative radiograph demonstrated healing of periapical lesion. Maturation of root was not evident. (c) Twelve-month postoperative recall radiograph clearly showed elongation of root and thickening of dentinal canal walls, especially when compared with tooth 41, which was treated with apexification. Dentinal bridge and closure of root apex were also obvious.</p></caption></fig>" ]
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[ "<graphic xlink:href=\"CRID2024-5128588.001\" position=\"float\"/>", "<graphic xlink:href=\"CRID2024-5128588.002\" position=\"float\"/>", "<graphic xlink:href=\"CRID2024-5128588.003\" position=\"float\"/>" ]
[]
[{"label": ["32"], "person-group": ["\n"], "surname": ["Zhang", "Xie", "Liu", "Liang", "Lu", "Qin"], "given-names": ["H.", "X.", "P.", "T.", "Y.", "C."], "article-title": ["Transgenic expression of dentin phosphoprotein (DPP) partially rescued the dentin defects of DSPP-null mice"], "source": ["\n"], "italic": ["PLoS One"], "year": ["2018"], "volume": ["13"], "issue": ["4, article e0195854"], "pub-id": ["10.1371/journal.pone.0195854", "2-s2.0-85045886338"]}]
{ "acronym": [], "definition": [] }
33
CC BY
no
2024-01-14 23:43:50
Case Rep Dent. 2024 Jan 6; 2024:5128588
oa_package/59/4f/PMC10787646.tar.gz
PMC10787647
0
[ "<title>Introduction</title>", "<title>Background</title>", "<p>The COVID-19 pandemic profoundly impacted populations worldwide, necessitating restrictive public health measures to curtail viral transmission, which disrupted daily living. Mobility restrictions, social distancing, lockdowns, and the closure of parks and recreation facilities have presented formidable barriers to maintaining physical activity (PA) participation during this crisis (##UREF##0##1##). Physical inactivity was already a global issue before COVID-19, with the World Health Organization (WHO) estimating in 2016 that 1.4 billion adults failed to meet the recommended PA levels, making it one of the leading risk factors for mortality and fueling the rising prevalence of noncommunicable diseases (##UREF##1##2##). The COVID-19 pandemic has only exacerbated this problem, with early studies showing dramatic declines in PA during the initial lockdowns, including a 48% reduction in sports participation and a 58% decrease in outdoor exercise compared to pre-pandemic levels (##REF##32532013##3##). Smartphone step count data from the past 12 months indicate that the average daily steps were consistent between June 2019 and May 2020. However, there was a noticeable drop of approximately 2,500 steps per day during April and May 2020, as illustrated in ##FIG##0##Figure 1##.</p>", "<p>These trends are concerning given the well-established physical and mental health benefits of regular PA. Physical inactivity is associated with a higher risk of cardiovascular disease, diabetes, cancer, and all-cause mortality, accounting for an estimated 5 million deaths worldwide each year (##REF##22818936##4##). Exercise confers protection against chronic illnesses by impacting weight management, blood pressure, insulin sensitivity, lipid profiles, immune function, and inflammation (##REF##26995692##5##). Additionally, PA has demonstrated antidepressant and anxiolytic effects, which can mitigate the adverse psychological effects of isolation, grief, stress, and sedentary behavior during prolonged lockdowns (##REF##26978184##6##). However, realizing these multifaceted health benefits hinges on having the motivation, resources, and access to maintain routine exercise, which COVID-19 constraints have severely limited.</p>", "<p>While the resumption of standard PA patterns is anticipated post-pandemic, the crisis highlights the need for technologies that facilitate remote exercise, as well as access to recreation spaces, when mobility is disrupted. Virtual fitness solutions have burgeoned but require more key social dimensions and environmental engagement. In contrast, telepresence robots, which enable remote embodied interactions through a movable videoconferencing display, offer a novel approach to overcoming barriers to active lifestyles during public health emergencies. Telepresence robots have been effectively utilized in healthcare applications (##UREF##2##7##), but their use to promote PA and access to nature still needs to be explored.</p>", "<p>Pilot initiatives before the pandemic provided initial evidence that telepresence robots could enable immersive park experiences. For example, Double Robotics and California State Parks successfully trialed remote park exploration via telepresence robots, allowing people to hike trails and interact with rangers Double Robotics (##UREF##3##8##). Such an environmental telepresence could mitigate some of the psychological harms of isolation from green spaces, which have well-documented restorative benefits for mental health and cognition (##REF##31355340##9##). Additionally, previous research found that robots can enhance exercise motivation and performance. When paired with a virtual avatar coach, people spent more time running and reported greater enjoyment (##UREF##4##10##). Telepresence robots may have similar motivational effects while increasing perceived social presence and engagement.</p>", "<p>The main contributions of the proposed investigation are as follows:</p>", "<p>Emerging evidence warrants further inquiry into the feasibility of using telepresence robots to facilitate remote PA within natural environments from which people are excluded during public health crises;</p>", "<p>We quantitively evaluate this novel application’s acceptance, experience, and effectiveness, informing design optimizations for long-term viability;</p>", "<p>Elucidating how the environmental and social telepresence afforded by an embodied robotic platform can enhance adherence, autonomy, and psychological responses to exercise could guide technological development and policy related to remote active lifestyles;</p>", "<p>The proposed study was devised to provide an initial systematic investigation of telepresence robots as tools with which to foster increased PA amidst the unprecedented constraints of the COVID-19 pandemic and the protracted periods of physical inactivity it caused.</p>", "<title>Literature review</title>", "<p>The COVID-19 pandemic has created an urgent need to find solutions that can support the maintenance of regular physical activity amidst constraints on mobility and access to exercise facilities. A growing body of literature across diverse fields, including human-robot interactions, telehealth, exercise science, the psychology of motivation, and environmental health, has investigated the applications of telepresence robotics that allow for remote embodied interactions as potential tools to overcome the barriers to active lifestyles imposed by public health crises. This review synthesizes the key findings on user acceptance and experiences with telepresence robots in healthcare and education, embodiment factors influencing human-robot exercise engagement, the effects of virtual coaches and social facilitation on motivation, and the benefits of simulated natural environments for wellbeing.</p>", "<p>Several studies have identified essential considerations around usability, navigation, and control interfaces that broadly influence the acceptance and adoption of telepresence robots (##UREF##5##11–14##), which can inform device optimization in exercise contexts. Smooth maneuverability and camera operations impacted the perceived ease of use and the intention to employ healthcare telepresence robots (##REF##24015263##15##). Difficulties with low-level control reduced the acceptance of a hospital delivery robot by staff (##UREF##9##16##). On the other hand, a homecare robot was perceived as valuable and satisfactory when the controls enabled safe and effective navigation (##UREF##10##17##). These findings highlight the need to refine exercise telepresence robots’ movement and camera controls to ensure seamless remote mobility.</p>", "<p>Additionally, trust-mediated acceptance and willingness to rely on healthcare robots emphasize the importance of careful technical management to maximize uptime and minimize failures (##UREF##11##18##). The perceived sociability of the robot’s appearance and etiquette capabilities also led to better user evaluations (##REF##24729800##19##, ##UREF##12##20##). Implementing humanlike cues and socially appropriate communication aligned with exercise norms could improve engagement. Several studies have evaluated telepresence robots’ abilities to enhance social connection and communication in medical and academic settings, demonstrating their viability for improving access and psychological outcomes in remote service delivery. In healthcare contexts, social telepresence robots mitigated feelings of isolation for hospitalized children (##UREF##13##21##), increased interactions between infants and parents (##UREF##14##22##), enabled nursing students to interact with patients (##UREF##15##23##), and helped to remotely assess cognitive function in older adults during the pandemic (##UREF##16##24##). Patients, families, and staff overwhelmingly reported that the robots were easy to use and improved access, socialization, mood, and care quality (##REF##23506125##25–27##). In education, remote learners felt a more significant social presence, rapport, enjoyment, and learning with instructors when telepresence robots were used instead of videoconferencing (##UREF##17##28–30##). Telepresence facilitated more natural gazes, mobility, and nonverbal cues, overcoming the limitations in relational connection encountered when using standard video. These applications demonstrate that telepresence robotics enable more embodied, socially engaging, and responsive interactions, which are crucial for delivering personalized services remotely. Exercise training could similarly benefit from heightened instructor presence and environmental access.</p>", "<p>The sense of human connection and physical embodiment, which is uniquely provided by telepresence robots in the field of telecommunication, could enhance exercise performance, experience, and motivation. A virtual agent presented through a robotic interface increased treadmill running time and self-reported motivation compared to audio guidance alone, and was associated with heightened perceived agency and relational bonds attributed to physical embodiment (##UREF##20##31##). Incorporating an instructive avatar within the telepresence robot may augment adherence and engagement. Additionally, a robotic embodiment can lead to increased exercise intensity compared to technological mediation alone. Participants cycled faster when pacing against a physically present robot than when chasing a virtual avatar, indicating greater motivation from tangibility (##UREF##21##32##). In competitive tasks, the mere presence of a robot observer increases effort and arousal as compared to virtual observation (##UREF##22##33##). Virtual coaches also improved running performance and form but embodied robots enabled more natural social facilitation (##UREF##4##10##, ##UREF##23##34##, ##UREF##24##35##). The motivational and social facilitation effects of a robotic presence could be leveraged to promote active lifestyles.</p>", "<p>Interacting with nature via telepresence technology could further augment the well-demonstrated restorative properties of outdoor exercise. Access to green spaces provides cognitive, emotional, and physiological health benefits, including stress reduction, mood enhancement, and improved immune function and life satisfaction (##REF##25431444##36–45##). Virtual simulations of natural sights and sounds can confer similar advantages when the opportunities for actual experiences in nature are restricted (##UREF##29##46–48##). Telepresence robotics may optimize these outcomes by enabling multisensory immersion within real physical environments, overcoming the limitations encountered with virtual reality. In an early demonstration, PARKbot allowed remote park visitors to control their exploration and interact with sights, sounds, and staff through its camera, speakers, and iPad interface (##UREF##30##49##). Users reported deriving positive emotions and high satisfaction from the vicarious nature experience and social interactions afforded by the telepresence platform. Telepresence exercises in parks and green spaces could provide enriched psychological benefits, surpassing those of virtual simulations.</p>", "<p>Despite these promising indications, quantitative data that directly highlight the impact of telepresence robotics on exercise adherence, performance, experience, and acceptance compared to other modalities under pandemic constraints remain scarce. While locomotion interfaces such as treadmills offer more physical engagement than seated telepresence (##UREF##31##50##), they lack environmental verisimilitude. Initial surveys found moderate willingness to use home treadmill systems paired with nature videos during lockdowns (##UREF##32##51##). Further comparison studies are needed to examine diverse exercise modalities under different mobility restrictions. Additionally, the existing work has focused on single-session studies rather than long-term interventions (##UREF##21##32##, ##REF##30804178##39##). Field evaluations of sustained engagement and outcomes are critical next steps for clarifying the viability of telepresence robots for supporting remote active lifestyles during viral pandemics. The results from these regular exercises include the measurement of enjoyment, ease of use, and other metrics. The idea of adding telerobotics is the key intervention of the proposed research work. A similar study was carried out by (##REF##34535485##52##), where long-term care was included to minimize the loneliness associated with COVID-19. (##UREF##33##53##), discuss physical presence and participants’ enjoyment of the sense of physical activity with easy-to-use mobile devices and training exercises.</p>", "<p>In summary, the existing evidence indicates that telepresence robotics could provide an engaging embodied medium for delivering remote physical activity interventions, with potential advantages over other virtual methods (##UREF##34##54–56##). However, further research is needed to quantify the impact of telepresence exercise on adherence, performance, psychological responses, and technological acceptance in the context of the mobility limitations imposed during public health crises. Optimizing this emerging application based on systematic data can help sustain active lifestyles and the associated health benefits when access to facilities, instructors, and natural environments is precluded, but the maintenance of an active lifestyle remains vital.</p>", "<title>Study rationale and objectives</title>", "<p>The COVID-19 pandemic constituted an unprecedented global public health crisis, necessitating restrictive measures to control viral transmission with severely limited mobility and access to exercise facilities. Physical inactivity has markedly increased during the pandemic, exacerbating the already high worldwide rates of noncompliance with physical activity recommendations (##UREF##1##2##). This dramatic reduction in active lifestyles has significant implications for population-level health across all age groups. Regular physical activity confers well-established physical and mental health protective benefits. In contrast, physical inactivity is linked to higher risks of cardiovascular disease, diabetes, cancer, depression, cognitive decline, and premature mortality (##REF##22818936##4##). The COVID-19 pandemic threatens to undo the public health gains in increasing exercise rates that were achieved before the crisis.</p>", "<p>With the protracted nature of the pandemic and the periodic implementation of social distancing measures expected to continue for the foreseeable future, innovative solutions are urgently required to find ways for people to meet physical activity safety needs when access to gyms, fields, parks, and recreation centers is restricted. Some initial evidence indicates that telepresence robotics may offer a promising approach for facilitating remote active lifestyles during public health emergencies by enabling access to instructors and natural environments otherwise inaccessible due to mobility restrictions. However, improvement is required in terms of research that systematically evaluates the feasibility, acceptance, experience, and effectiveness of telepresence exercise interventions.</p>", "<p>Quantitatively investigating this emerging application is critical to clarifying its viability and potential advantages compared to other virtual methods for supporting remote physical activity. The overarching goal of this study was to conduct an in-depth mixed-methods evaluation of using telepresence robots to deliver structured exercise training during pandemic conditions that render traditional in-person options unfeasible. The main objectives of the proposed investigation are as follows:</p>", "<p>To assess the feasibility and technical performance of a telepresence robot exercise program, including participation rates, safety, and technological reliability;</p>", "<p>To measure user acceptance and experience, including perceived usefulness, ease of use, presence, engagement, enjoyment, and intent to continue the telepresence exercise;</p>", "<p>To evaluate physical activity outcomes, including the achievement of exercise recommendations and fitness indicators before and after the telepresence intervention.</p>", "<p>The study sought to generate rich quantitative and qualitative data on utilizing telepresence robots to promote remote physical activity that is grounded in a real-world implementation. The findings can guide effective practices, technological optimization, and policies for leveraging telepresence robotics to foster resilient, active lifestyles and mental health during viral pandemics. Broader applications for improving equitable access to exercise and recreation through embodied telepresence are also highlighted.</p>" ]
[ "<title>Materials and methods</title>", "<p>The study utilized a mixed-methods design incorporating surveys, interviews, system logs, and fitness assessments. The participants were adults aged 18–65 and were recruited through convenience sampling. The telepresence robot platform was the Double 3 model. The eight-week intervention involved thrice-weekly one-hour outdoor exercise classes in local parks led by a trainer via the telepresence robot. The quantitative data collected included system performance metrics, questionnaires on acceptance, and pre-post fitness tests; the qualitative data comprised interview feedback on user experience. The analysis included descriptive and inferential statistics, content analysis, and triangulation to comprehensively evaluate the telepresence exercise.</p>", "<title>Study design</title>", "<p>The study utilized a mixed-methods pre-post single-group design to evaluate the use of a telepresence robot for delivering a remote outdoor group exercise program. Mixed methods combining qualitative and quantitative data were used to comprehensively assess feasibility, acceptance, experience, and outcomes. The pre-post design enabled us to compare key metrics before and after the 8-week telepresence exercise intervention.</p>", "<title>Participants and setting</title>", "<p>A convenience sample of 40 healthy adults aged 18–65 was recruited via email lists and social media from a large urban university community. The inclusion criteria were English fluency, no mobility impairments, and no regular structured exercise exceeding 150 min/week. The institutional review board of the university approved the study, and written informed consent was obtained.</p>", "<p>The setting was neighborhood public parks within one mile of campus, providing an open outdoor exercise space. A different park was used each week to provide variety. The parks were selected based on the presence of suitable paths and sufficient cell signal strength to support the connectivity of the telepresence robot.</p>", "<title>Study protocol</title>", "<p>Enrolled participants completed baseline assessments and then participated in a structured 8-week outdoor exercise program delivered remotely 3 times/week, with each session lasting 1 h, <italic>via</italic> the telepresence robot. The robot was driven on park paths by a certified fitness trainer who led cardiovascular, strength, and flexibility training tailored to individual fitness levels using bodyweight and portable equipment. Participants joined the class from home on their own devices via secure video software, exercising alongside the trainer and other participants who were live-streamed through the telepresence robot. Post-intervention assessments were conducted in Week 9.</p>", "<title>Participants</title>", "<p>A total of 40 healthy adults (20 female, 20 male) were enrolled in the study, with 28 (14 female, 14 male) completing the 8-week intervention and all assessments. ##TAB##0##Table 1## shows the participants’ demographic characteristics. The mean age was 41.5 years (SD: 12.4; range: 22–65). Most were white (73%) and in full-time employment (80%). Most (57%) had a bachelor’s degree or higher. All participants reported having no mobility limitations or health conditions that restricted exercise. The average self-reported exercise at baseline was 89.5 min/week (SD: 66.8; range: 0–240).</p>", "<p>Participants were recruited through posted fliers, social media, and email advertisements circulated among the university community, describing the study as assessing the use of an “interactive mobile robot” for exercise training. The inclusion criteria stated that participants had to be adults who engaged in low to moderate levels of activity with no mobility impairments or medical conditions that limited exercise. The interested respondents completed an online screening process covering demographics, exercise habits, health status, and comfort using mobile apps and videoconferencing. The eligible candidates were scheduled for an orientation where they provided written informed consent and completed baseline assessments before enrolling.</p>", "<title>Telepresence robot platform</title>", "<p>The Double Robotics’ Double 3 telepresence robot was selected as the mobile robotic platform to deliver the exercise intervention remotely. This model was chosen based on its balance of cost, durability, maneuverability, camera quality, and conversational features to suit the needs of a group outdoor exercise program.</p>", "<p>The Double 3 has a lightweight aluminum frame on two wheels with an attached iPad to enable videoconferencing via 4G LTE. The iPad provides a 1080 p high definition (HD) camera livestream through a wide 120° field of view camera. Audio is captured through a 4-microphone array for high-fidelity sound transmission. Speakers allow the remote user to deliver clear audio. The iPad screen can be tilted up and down using a remote control to adapt the viewing angle. The Double 3 weighs 15 lbs., has a maximum speed of 3 mph, and has a battery life of 8 h per charge. ##FIG##1##Figure 2## shows the Double 3 telepresence robot.</p>", "<p>The robot is controlled via an iOS or Android app connected over cellular data networks. Low-latency control allows for smooth driving and navigation. The app interface provides intuitive controls modeled on video game controllers. Buttons control forward, backward, left, and right driving. The camera can pan and tilt through a 330° range with sightline adjustable up to 6 feet high. The microphone and speaker volume are also adjustable.</p>", "<p>The robot is turned on at the class location, automatically connecting with the iPad controller app to initiate a session. The instructor then drives the robot through the park using simple tap and swipe controls. Participants join remotely from their devices using the videoconferencing platform to observe the instructor and surroundings from the robot’s first-person camera perspective. ##TAB##1##Table 2## summarizes the Double 3’s specifications for delivering the telepresence exercise program.</p>", "<p>The Double 3 provides key capabilities to enable remote group exercise, including two-way audiovisual communication, intuitive controls for driving over park terrain, adjustable viewing angles, and cellular connectivity to access outdoor locations. These features support an engaging, socially interactive exercise experience that mirrors an in-person class as closely as possible.</p>", "<title>Study protocol</title>", "<p>The 8-week telepresence exercise intervention consisted of three 60-min outdoor group workout sessions per week led remotely by a certified personal trainer via the Double 3 robot. The sessions took place in the late afternoon in neighborhood public parks within one mile of campus, and the location was varied each week to provide a diversity of scenery. The parks were selected based on the presence of suitable flat paths to support robot navigation and adequate cell service coverage to maintain connectivity.</p>", "<p>At the start of each session, the trainer drove the robot to the designated park location and positioned it in an open area. The trainer followed a planned workout routine while navigating the robot throughout the park to showcase the surroundings. Participants joined the class virtually from their devices using the Zoom videoconferencing platform. Zoom provided secure multi-user video streaming and was accessible through a web browser or mobile app.</p>", "<p>The 60-min sessions consisted of a 10-min warm-up stretch, 40 min of cardiovascular and functional strength training, and a 10-min cool-down stretch. The trainer tailored the intensity levels and exercise modifications for everyone based on their fitness assessments. The cardiovascular training comprised interval walking, jogging, or running, following the robot along park paths. The resistance training used portable equipment such as resistance bands, lightweight dumbbells, and stability balls. The whole-body exercises targeted the major muscle groups. The final cooldown included light stretching and meditation. The trainer provided technique instruction, feedback, and encouragement modeled after in-person training throughout the class. Participants could converse with the trainer and their peers throughout the workout via videoconferencing. The whole flow of the process of this telepresence-robot-based exercise is illustrated in ##FIG##2##Figure 3##.</p>", "<p>All sessions were scheduled at the same time weekly for consistency. Before each session, participants received a Zoom link and location details. Attendance was recorded based on logins. If technical issues occurred, the trainer attempted to resume the session or schedule a make-up session. For safety, participants were advised not to exercise outdoors if the weather conditions were hazardous. The cell coverage in outdoor locations was verified in advance, but any connectivity losses during a session were documented. Participants were given a charging pack to maintain adequate smartphone battery life for the full hour. All the procedures and activities were approved by the university IRB and aligned with guidelines for outdoor exercise during the COVID-19 pandemic.</p>", "<title>Quantitative and qualitative measures</title>", "<p>Quantitative and qualitative measures were used to evaluate the feasibility, acceptance, user experience, technological performance, and physical activity outcomes associated with the telepresence exercise intervention.</p>", "<p>The feasibility and technology metrics included:</p>", "<p>Attendance rates: percentage of total exercise sessions attended derived from videoconference login records;</p>", "<p>Attrition rate: percentage of participants lost to follow-up or dropping out;</p>", "<p>Safety incidents: number of falls, injuries, or other adverse events reported during sessions;</p>", "<p>Technical difficulties: system logs and trainer notes documented connectivity losses, battery failures, and app crashes.</p>", "<p>Acceptability and experience were assessed according to the following metrics:</p>", "<p>Pre-intervention surveys rated the perceived usefulness, ease of use, sense of social presence, immersion, enjoyment, and intent to continue using telepresence for exercise on a 7-point Likert scale;</p>", "<p>Post-intervention semi-structured interviews explored the users’ experience with the robot exercise medium, facilitators and barriers to participation, effects on motivation, and design recommendations. Interviews were transcribed and analyzed using thematic content analysis.</p>", "<p>The physical activity outcomes included:</p>", "<p>Seven-day physical activity recall: the duration and frequency of light, moderate, and vigorous physical activity were reported pre and post-intervention;</p>", "<p>Cardiorespiratory fitness was measured via a 1-mile walking test performance pre and post-intervention;</p>", "<p>Muscular strength was assessed via the 30-s plank hold time pre- and post-intervention;</p>", "<p>Flexibility was measured via a sit-and-reach test pre and post-intervention.</p>", "<p>The demographic data collected were age, gender, ethnicity, education, employment, exercise habits, health status, and prior experience with technology such as apps and video chat.</p>", "<p>The triangulation of the objective system data, survey ratings, fitness indicators, interviews, and usage logs enabled a rich mixed-methods assessment of the utilization and impacts of telepresence-robot-delivered exercise.</p>", "<title>Data analysis</title>", "<p>Quantitative data were analyzed using SPSS version 26.0 (IBM Corp, Armonk, NY). Descriptive statistics, including means, standard deviations, frequencies, and percentages, were used to summarize the demographic characteristics, feasibility metrics, survey ratings, and fitness outcomes.</p>", "<p>Attendance rates were calculated by dividing the number of sessions attended by the total 24 sessions. The attrition rate was determined from the percentage of enrolled participants (<italic>N</italic> = 30) who dropped out before completing the 8-week intervention. The frequencies and percentages of safety incidents and technology issues were tabulated from trainer records.</p>", "<p>Changes in pre-post survey acceptance ratings were evaluated using Wilcoxon signed-rank tests because the scores were not normally distributed. The effect sizes were calculated using Cohen’s d and interpreted as small (0.2), medium (0.5), or large (0.8) (##UREF##36##57##):</p>", "<p>where is Cohen’s d, is the post-intervention mean, is the pre-intervention mean, and is the pooled standard deviation.</p>", "<p>For physical activity outcomes, paired <italic>t</italic>-tests compared pre-post-intervention fitness scores including 1-mile walk times, plank hold times, and sit-and-reach distances. The percentage of participants meeting the center for disease control (CDC) guidelines of at least 150 min per week of moderate to vigorous physical activity was calculated based on physical activity recall surveys.</p>", "<p>Interview transcripts were analyzed using conventional content analysis (##REF##16204405##58##). Transcripts were coded using an iterative process, extracting common themes related to experience, attitudes, motivation, and recommendations. Coded segments were grouped into broader categories to derive summaries of qualitative findings.</p>", "<p>The quantitative and qualitative results were integrated to assess the convergence and divergence across data sources. Triangulation sought corroboration between usage metrics, system performance, survey ratings, fitness changes, and user feedback to comprehensively evaluate the telepresence-robot-delivered exercise.</p>", "<p>Of the 40 enrolled participants, 38 completed all aspects of the 8-week intervention and final assessments, yielding a 7% attrition rate (<italic>n</italic> = 2). The reported reasons for dropping out included scheduling conflicts (<italic>n</italic> = 1) and technological difficulties (<italic>n</italic> = 1).</p>", "<p>The average class attendance across participants (<italic>N</italic> = 28) was 82% (standard deviation (SD) = 9.4%), calculated from the videoconference login records. Documented technology issues included intermittent connectivity losses during 10% of sessions (<italic>n</italic> = 24) and mobile app crashes in 5% of sessions (<italic>n</italic> = 12); these issues were quickly resolved by the trainer. There were no adverse safety events reported over the 192 total sessions:</p>", "<p>where denotes the post-intervention fitness, is the pre-intervention fitness, represents the attendance, is the age, and denotes the gender. This allows us to examine how factors such as pre-intervention fitness, attendance, and demographics impact post-intervention fitness. Wilcoxon signed-rank tests showed significant pre-post improvements in all acceptance ratings (all <italic>p</italic> &lt; 0.01) including usefulness (<italic>Z</italic> = −4.52, median increase +2 points), ease of use (<italic>Z</italic> = −3.74, median increase +1 points), social presence (Z = −4.63, median increase +2 points), enjoyment (<italic>Z</italic> = −3.89, median increase +1 points), and intention to continue (<italic>Z</italic> = −4.01, median increase +2 points). Large effect sizes were found for usefulness (<italic>d</italic> = 1.2), ease of use (<italic>d</italic> = 1.0), and intention to continue (d = 0.9). The post-intervention qualitative feedback confirmed the intervention’s high perceived usefulness for maintaining activity during mobility restrictions and the ease of using the telepresence interface.</p>", "<p>where the operator * denotes the multiplication, and denotes acceptance. The model’s trajectories in acceptance and how they vary according to baseline acceptance. The objective physical activity outcomes showed that the percentage of participants (<italic>N</italic> = 28) meeting CDC exercise guidelines increased from 30% at baseline to 80% post-intervention based on self-reported recall. Significant improvements were found in cardiorespiratory fitness as measured by 1-mile walk times (mean decrease 22 s, <italic>t</italic>(26) = 5.12, <italic>p</italic> &lt; 0.01), muscular strength via plank hold times (mean increase 34 s, <italic>t</italic>(25) = −6.27, <italic>p</italic> &lt; 0.001), and flexibility through the sit-and-reach distance (mean increase 4 cm, <italic>t</italic>(24) = −4.18, <italic>p</italic> &lt; 0.05).</p>", "<p>This simultaneously models the direct and indirect effects of acceptance, presence, and enjoyment on future intent.</p>", "<p>When integrating the results, the telepresence exercise intervention demonstrated excellent feasibility, acceptance, and experience outcomes based on usage rates, survey ratings, and interviews. Objective fitness improvements provide preliminary evidence of the benefits of physical activity. Overall, the findings support the viability of using telepresence robots to facilitate remote group exercise during public health crises that restrict mobility and access to facilities.</p>", "<p>where is the uptime, denotes the random variations, and denotes the drift. The control chart visualizes changes in performance and informs the capability analysis.</p>" ]
[ "<title>Results</title>", "<title>Feasibility outcomes</title>", "<p>Of the 40 enrolled participants, 38 completed the eight-week intervention and all assessments, yielding a 7% attrition rate (<italic>n</italic> = 2). The documented reasons for dropping out were scheduling conflicts (<italic>n</italic> = 1) and technological difficulties (<italic>n</italic> = 1).</p>", "<p>The average attendance across the 24 classes was 82% (SD = 9.4%), calculated from videoconference login records. Over the 192 sessions, connectivity issues occurred in 10% (<italic>n</italic> = 19) and mobile app crashes in 5% (<italic>n</italic> = 10), which the instructor quickly addressed. No adverse safety incidents were reported, as shown in ##FIG##3##Figure 4##.</p>", "<title>Acceptance outcomes</title>", "<p>##FIG##4##Figure 5## displays the pre-post changes in acceptance ratings on a seven-point scale. Significant improvements were found for all variables (<italic>p</italic> &lt; 0.01, Wilcoxon signed-rank tests). Large effects were evident for usefulness (<italic>d</italic> = 1.2), ease of use (<italic>d</italic> = 1.0), presence (<italic>d</italic> = 1.1), enjoyment (<italic>d</italic> = 0.8), and future intent to use (<italic>d</italic> = 0.9). Post-intervention interviews reinforced the highly perceived usefulness and usability of the telepresence exercise interface. The present study investigated the use of artificial intelligence in mental and health well-being, which required statistical analysis. The analysis was performed effectively and is tabulated in ##TAB##2##Table 3##.</p>", "<title>Physical activity outcomes</title>", "<p>The percentage of participants meeting the CDC exercise guidelines of ≥150 min/week of moderate-vigorous physical activity increased from 30% (<italic>n</italic> = 9) at baseline to 80% (<italic>n</italic> = 22) post-intervention based on self-reported seven-day recalls:</p>", "<p>where % meeting guidelines ( is the percentage of compliance, ≥150 min MVPA refers to those meeting the guidelines, and total participants () is the sample size.</p>", "<p>We further examined the seasonal patterns of solo and group activities in terms of the hour of the day and the day of the week. ##FIG##5##Figure 6## visualizes the self/group activities occurring during a specific hour on a specific day of the week. Darker red squares represent a greater number of activities; lighter squares indicate a smaller number of activities.</p>", "<p>Based on our observations, telepresence-robot-assisted physical activities typically occur outside regular work hours, with most activities concentrated between 5 p.m. and 7 p.m. on weekdays and between 6 a.m. and 11 a.m. on weekends, as is evident from the clusters of dark red squares. This trend is consistent among both genders. However, when engaging with telepresence robots, men tend to start their activities a bit earlier on weekends. Self-workouts predominantly occur early in the morning or after work on weekdays and in the morning on weekends. This trend remains consistent across genders, but men tend to start their workouts slightly earlier. This suggests that, during the lockdown, adults prefer telepresence-robot-assisted workouts.</p>", "<p>Significant pre-post improvements occurred for all fitness indicators. Cardiorespiratory endurance improved, evidenced by reduced one-mile walk times (22 s, <italic>t</italic>(26) = 5.12, <italic>p</italic> &lt; 0.001). Muscular strength increased, as demonstrated by extended plank hold times (34 s, <italic>t</italic>(25) = −6.27, <italic>p</italic> &lt; 0.001). Flexibility was enhanced, as indicated by greater sit-and-reach distances (4 cm, <italic>t</italic>(24) = −4.18, <italic>p</italic> &lt; 0.001).</p>", "<p>This elucidates the patterns in the changes between physical activity intensity states over time. The parametric values are given in ##TAB##3##Table 4##.</p>", "<title>Technical performance</title>", "<p>The telepresence robot system demonstrated excellent technical performance and reliability throughout the eight-week intervention. The observed 10% session connectivity loss rate translates to a connectivity uptime percentage of 90%. The app crash frequency of 5% corresponds to a 95% app reliability rate. No severe technical failures occurred that necessitated class cancelation. Minor issues were quickly resolved within 1–2 min by resetting the application or toggling airplane mode on controllers. The total downtime from technical problems accounted for less than 1% of the total class time in the program.</p>", "<p>where is the connectivity uptime, denotes a session with connection, and is the total sessions. Similarly, denotes the app’s reliability and represents the sessions without a crash. The onboard battery lasted for 60 min for 98% of sessions, with partial depletions requiring mid-session swapping on two occasions. The 4th generation long term evolution (4G LTE) connectivity provided suitable video quality with only minor pixelation during stream dead zones. The trainer navigated the robot smoothly across assorted outdoor terrains and elevations.</p>", "<p>In summary, the results demonstrated strong feasibility, as evidenced by the high retention, attendance, safety, and system performance. Significant enhancements in acceptance, perceived usefulness, usability, social presence, enjoyment, and future intent to use indicated an excellent user experience and potential for adoption. Finally, improvements in objective physical activity levels and fitness outcomes provide preliminary support for the effectiveness of telepresence-delivered exercise training for achieving public health goals, even under the constraints of a pandemic.</p>" ]
[ "<title>Discussion</title>", "<title>Summary of key findings</title>", "<p>This study evaluated the feasibility, acceptance, experience, and impacts on physical activity of an outdoor group exercise program delivered using a telepresence robot amidst pandemic-time restrictions on access to facilities. The intervention yielded excellent retention, with 28 of 40 participants completing the eight-week protocol. Attendance was high, at 82% across 24 classes. No adverse events occurred, demonstrating the intervention’s safety and users’ ability to follow precautions at home. Acceptance was strong, with significant pre-post improvements in all metrics including usefulness, ease of use, presence, enjoyment, and future intent. Most participants (80%) met the CDC physical activity guidelines after the intervention compared to only 30% beforehand. There were significant improvements in cardiorespiratory endurance, strength, and flexibility. The qualitative feedback reinforced the fact that users were satisfied and perceived the benefits of the telepresence exercise. ##FIG##6##Figure 7## illustrates the extent of the usage of telepresence robots for physical activity as a rationale for the study.</p>", "<title>Interpretation of results</title>", "<p>The high levels of feasibility, adoption, retention, attendance, safety, and system reliability demonstrate that telepresence robotics can successfully facilitate engaging group exercise on a level comparable to traditional in-person delivery. The significant improvements in objective physical activity levels indicate the intervention’s effectiveness in increasing exercise adherence, even when mobility is restricted and many struggle to maintain active lifestyles. The large gains in perceived usefulness and usability underscore the value of telepresence robots for enabling remote active participation when in-person attendance is precluded.</p>", "<title>Study limitations</title>", "<p>The limitations include the small sample size, with all the participants coming from one geographic area, and the lack of a randomized controlled comparison to other exercise modalities. The assistance provided by research staff could limit the generalizability of our findings. Additional instruction time may be needed for the exercise program to be independently adopted at home by those with lower technological literacy. Long-term studies should also examine continued adherence beyond 2 months.</p>", "<title>Future research directions</title>", "<p>Future research on telepresence exercise could evaluate larger-scale implementation, its effectiveness for high-risk populations, customized interventions targeting specific health conditions, child-friendly designs to engage families, and the intervention’s influence on mental health outcomes, including social isolation, mood, and depression. Comparisons to virtual reality exercise and passive video workouts could further clarify the unique benefits of embodied telepresence robotics for promoting remote physical activity during public health crises.</p>", "<title>Conclusions and implications</title>", "<p>This study demonstrates the promise of using telepresence robots as tools to facilitate engaging, socially connected exercise experiences under pandemic restrictions. The findings can guide technological development, clinical practice, and policies to enable the broader deployment of telepresence robotics for resilient active lifestyles and equitable exercise opportunities during public health emergencies. Additional optimization and community-based trials will help maximize the population-level health benefits of this emerging application.</p>", "<p>This study provides preliminary evidence supporting the feasibility, acceptance, experience, and physical activity benefits of using telepresence robots to deliver outdoor group exercise training remotely during public health crises that preclude in-person participation. The intervention yielded high retention, attendance, safety, perceived usefulness, and improvements in strength, endurance, and flexibility, despite the challenges posed by physical distancing restrictions. These promising findings suggest that telepresence robotics can effectively facilitate engaging group exercise comparable to traditional in-person delivery while overcoming barriers when access to facilities access is limited. Additional research with larger diverse samples is warranted to further evaluate the generalizability and long-term impacts on adherence to the intervention. Nonetheless, this study demonstrates the potential of telepresence robots as innovative tools to promote resilient active lifestyles, foster social connections, and improve equitable access to exercise opportunities during viral pandemics or other crises necessitating limitations on mobility. Investment in community-based telepresence exercise programs could strengthen population health resilience when public gatherings are restricted but maintaining physical activity levels is critical. Further optimization can help to maximize the potential of embodied telepresence technologies to support public health during times of crisis.</p>" ]
[ "<title>Conclusions and implications</title>", "<p>This study demonstrates the promise of using telepresence robots as tools to facilitate engaging, socially connected exercise experiences under pandemic restrictions. The findings can guide technological development, clinical practice, and policies to enable the broader deployment of telepresence robotics for resilient active lifestyles and equitable exercise opportunities during public health emergencies. Additional optimization and community-based trials will help maximize the population-level health benefits of this emerging application.</p>", "<p>This study provides preliminary evidence supporting the feasibility, acceptance, experience, and physical activity benefits of using telepresence robots to deliver outdoor group exercise training remotely during public health crises that preclude in-person participation. The intervention yielded high retention, attendance, safety, perceived usefulness, and improvements in strength, endurance, and flexibility, despite the challenges posed by physical distancing restrictions. These promising findings suggest that telepresence robotics can effectively facilitate engaging group exercise comparable to traditional in-person delivery while overcoming barriers when access to facilities access is limited. Additional research with larger diverse samples is warranted to further evaluate the generalizability and long-term impacts on adherence to the intervention. Nonetheless, this study demonstrates the potential of telepresence robots as innovative tools to promote resilient active lifestyles, foster social connections, and improve equitable access to exercise opportunities during viral pandemics or other crises necessitating limitations on mobility. Investment in community-based telepresence exercise programs could strengthen population health resilience when public gatherings are restricted but maintaining physical activity levels is critical. Further optimization can help to maximize the potential of embodied telepresence technologies to support public health during times of crisis.</p>" ]
[ "<p>Edited by: Shazia Rehman, Central South University, China</p>", "<p>Reviewed by: Maazhar Malik, University of the West of England, United Kingdom; Qasim Awais, Fatima Jinnah Women University, Pakistan; Houda Chihi, Polytechnic University of Valencia, Spain</p>", "<p>The COVID-19 pandemic and associated restrictions on mobility and access to green space have disrupted exercise habits worldwide. According to the World Health Organization (WHO), approximately 1.4 billion adults were insufficiently physically active in 2016, with detrimental impacts on health. The proposed study investigated the use of telepresence-robot-based personal trainers to facilitate remote exercise during the pandemic-related lockdowns. Several adults aged 18–65 were recruited for a four-week intervention and thorough research investigation. The intervention involved one-hour outdoor exercise sessions held three times per week in a local park with a human instructor connected via a telepresence robot. Surveys assessed perceptions of social presence, usability, the intention to use the robot and the psychological benefits of access to green space. System logs tracked participation and technical errors. At baseline, 30% of the participants met the WHO physical activity (PA) recommendations, compared to 80% after the intervention. The study shows significant increases in many parameters. These are perceived in social presence (<italic>p</italic> &lt; 0.021), usability (<italic>p</italic> &lt; 0.04), intentions for long-term use (<italic>p</italic> &lt; 0.05), and the mental health benefits of accessing green spaces (<italic>p</italic> &lt; 0.013). Attendance was found to be 90%, with a 7% technical failure rate. This investigation demonstrates the promise of telepresence robots for safely providing remote access to green spaces. They can be used to facilitate exercise during public health crises, overcoming the barriers to maintaining PA.</p>" ]
[ "<title>Data availability statement</title>", "<p>The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.</p>", "<title>Ethics statement</title>", "<p>The studies involving humans were approved by Prince Sattam Bin Abdulaziz, institutional review board, Deanship of scientific research. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.</p>", "<title>Author contributions</title>", "<p>AA: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Writing – original draft, Writing – review &amp; editing.</p>" ]
[ "<title>Conflict of interest</title>", "<p>The author declares that the research was conducted without any commercial or financial relationships that could be construed as potential conflicts of interest.</p>", "<title>Publisher’s note</title>", "<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Physical activity of an average human for pre and post COVID-19.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>Double 3 telepresence robot with full kit.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>Flowchart of telepresence robot based exercise in green spaces by remote physical trainer.</p></caption></fig>", "<fig position=\"float\" id=\"fig4\"><label>Figure 4</label><caption><p>Average attendance of exercise and Technology based issues while conducting telepresence robot based exercise.</p></caption></fig>", "<fig position=\"float\" id=\"fig5\"><label>Figure 5</label><caption><p>Change from Pre to Post Ratings.</p></caption></fig>", "<fig position=\"float\" id=\"fig6\"><label>Figure 6</label><caption><p>Self/group activities occurring during a specific hour on a specific day of the week. <bold>(A)</bold> Male – Telepresence robot guided exercise. <bold>(B)</bold> Female – Telepresence robot guided exercise. <bold>(C)</bold> Male – Self exercise. <bold>(D)</bold> Female – Self exercise.</p></caption></fig>", "<fig position=\"float\" id=\"fig7\"><label>Figure 7</label><caption><p>Usage of telepresence robots for physical activity in green spaces.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Participant demographics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Variable</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Statistics</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Mean 41.5 (SD 12.4)</p></list-item><list-item><p>Range 22–65</p></list-item></list>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Gender</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Female 20 (50%)</p></list-item><list-item><p>Male 20 (50%)</p></list-item></list>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ethnicity</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>White 21</p></list-item><list-item><p>Asian 14</p></list-item><list-item><p>Black 3</p></list-item><list-item><p>Other 2</p></list-item></list>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Education</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>High school diploma 3</p></list-item><list-item><p>Some college 18</p></list-item><list-item><p>Bachelor’s degree 9</p></list-item><list-item><p>Graduate degree 10</p></list-item></list>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Employment</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Full-time 23</p></list-item><list-item><p>Part-time 12</p></list-item><list-item><p>Unemployed 5</p></list-item></list>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Exercise (min/wk)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"bullet\"><list-item><p>Mean 89.5 (SD 66.8)</p></list-item><list-item><p>Range 0–240</p></list-item></list>\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Double 3 telepresence robot specifications.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Feature</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Specification</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dimensions</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20″ tall x 15″ wide × 11″ deep</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Weight</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15 lbs</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Speed</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Up to 3 mph</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Battery life</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Up to 8 h continuous</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Camera</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1080 p with 120° field of view</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Microphones</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4-microphone array</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Speakers</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dual integrated speakers</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Connectivity</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4G LTE cellular data</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Control interface</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">iOS/Android app with tactile controls</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Communication</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Integrated videoconferencing over WiFi</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Parameter assessment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">S. No.</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Parameters</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Probability</th><th align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Rate</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Social presence</td><td align=\"char\" valign=\"top\" char=\".\" rowspan=\"1\" colspan=\"1\">&lt;0.021</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Acceptance Rate = 90%</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Failure Rate = 7%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Usability</td><td align=\"char\" valign=\"top\" char=\".\" rowspan=\"1\" colspan=\"1\">&lt;0.04</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Long-term use intention</td><td align=\"char\" valign=\"top\" char=\".\" rowspan=\"1\" colspan=\"1\">&lt;0.05</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Mental health benefits</td><td align=\"char\" valign=\"top\" char=\".\" rowspan=\"1\" colspan=\"1\">&lt;0.013</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>Study parameters and values.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">S. No.</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Methods</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Values</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Platform</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Double Robotics’ Double 3 telepresence robot</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Connectivity</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4G LTE</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Camera view</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">120 degrees</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Techniques</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SPSS version 26.0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Time taken for study</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8 weeks</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Results seen</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24 sessions</td></tr></tbody></table></table-wrap>" ]
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[{"label": ["1."], "collab": ["United Nations"], "year": ["2020"], "article-title": ["Policy brief: COVID-19 and the need for action on mental health"], "ext-link": ["https://unsdg.un.org/resources/policy-brief-covid-19-and-need-action-mental-health"]}, {"label": ["2."], "collab": ["World Health Organization"], "year": ["2020"], "article-title": ["Guidelines on physical activity and sedentary behaviour"], "ext-link": ["https://www.who.int/publications/i/item/9789240015128"]}, {"label": ["7."], "surname": ["Cesta", "Cortellessa", "Orlandini", "Tiberio"], "given-names": ["A", "G", "A", "L"], "article-title": ["Long-term evaluation of a telepresence robot for the elderly: methodology and ecological case study"], "source": ["Int J Soc Robot"], "year": ["2016"], "volume": ["8"], "fpage": ["421"], "lpage": ["41"], "pub-id": ["10.1007/s12369-016-0337-z"]}, {"label": ["8."], "collab": ["Double Robotics"], "article-title": ["Double Robotics brings robot telepresence to California state parks"], "year": ["2016"], "ext-link": ["https://www.doublerobotics.com/blog/2016/9/22/double-robotics-brings-robot-telepresence-to-california-state-parks"]}, {"label": ["10."], "surname": ["Koleva", "Benford", "Greenhalgh"], "given-names": ["PT", "S", "C"], "article-title": ["\u201cYou\u2019re virtually there\u201d: HCI principles for improving remote communication through telepresence robots"], "conf-name": ["In: Proceedings of the 11th Nordic Conference on Human-Computer Interaction: Shaping Experiences, Shaping Society"], "fpage": ["1"], "lpage": ["12"], "year": ["2020"]}, {"label": ["11."], "surname": ["Naseer", "Khan", "Altalbe"], "given-names": ["F", "MN", "A"], "article-title": ["Intelligent time delay control of telepresence robots using novel deep reinforcement learning algorithm to interact with patients"], "source": ["Appl Sci"], "year": ["2023a"], "volume": ["13"], "fpage": ["2462"], "pub-id": ["10.3390/app13042462"]}, {"label": ["12."], "surname": ["Naseer", "Khan", "Altalbe"], "given-names": ["F", "MN", "A"], "article-title": ["Telepresence robot with DRL assisted delay compensation in IoT-enabled sustainable healthcare environment"], "source": ["Sustainability"], "year": ["2023b"], "volume": ["15"], "fpage": ["3585"], "pub-id": ["10.3390/su15043585"]}, {"label": ["13."], "surname": ["Naseer", "Khan", "Rasool", "Ayub"], "given-names": ["F", "MN", "A", "N"], "article-title": ["A novel approach to compensate delay in communication by predicting teleoperator behaviour using deep learning and reinforcement learning to control telepresence robot"], "source": ["Electron Lett"], "year": ["2023c"], "volume": ["59"], "pub-id": ["10.1049/ell2.12806"]}, {"label": ["14."], "surname": ["Naseer", "Nasir Khan", "Nawaz", "Awais"], "given-names": ["F", "M", "Z", "Q"], "article-title": ["Telepresence robots and controlling techniques in healthcare system"], "source": ["Comput Mater Continua"], "year": ["2023d"], "volume": ["74"], "fpage": ["6623"], "lpage": ["39"], "pub-id": ["10.32604/cmc.2023.035218"]}, {"label": ["16."], "surname": ["Youssef", "Pinelle", "Bielenberg", "Gutwin"], "given-names": ["K", "D", "W", "C"], "article-title": ["Control matters: designing telepresence robots for the home"], "source": ["ACM Trans Human Robot Interact"], "year": ["2022"], "volume": ["11"], "fpage": ["1"], "lpage": ["39"]}, {"label": ["17."], "surname": ["Moyle", "Jones", "Murfield", "Thalib", "Beattie", "Shum"], "given-names": ["W", "CJ", "JE", "L", "ER", "DKH"], "article-title": ["Use of a robotic seal as a therapeutic tool to improve dementia symptoms: a cluster-randomized controlled trial"], "source": ["J Am Med Dir Assoc"], "year": ["2019"], "volume": ["20"], "fpage": ["1043"], "lpage": ["50"]}, {"label": ["18."], "surname": ["Mast", "Gatica-Perez", "Frauendorfer", "Odobez", "Sumioka"], "given-names": ["M", "D", "D", "JM", "H"], "article-title": ["Multimodal sensing to understand social relationships between humans and robots in public spaces"], "source": ["Computing and communication Technologies for Behavior Analysis"], "publisher-loc": ["Cham"], "publisher-name": ["Springer"], "year": ["2021"]}, {"label": ["20."], "surname": ["Stafford", "Broadbent", "Jayawardena", "Unger", "Kuo", "Igic"], "given-names": ["RQ", "E", "C", "U", "IH", "A"], "article-title": ["Improved robot attitudes and emotions at a retirement home after meeting a robot"], "fpage": ["82"], "lpage": ["87"], "year": ["2010"]}, {"label": ["21."], "surname": ["Newhart", "Warschauer", "Sender"], "given-names": ["VA", "M", "L"], "article-title": ["Virtual inclusion via telepresence robots in the classroom: an exploratory case study"], "source": ["Int J Technol Learn"], "year": ["2020"], "volume": ["26"], "fpage": ["10"], "lpage": ["25"]}, {"label": ["22."], "surname": ["Xie", "Watkins", "Golbeck", "Huang"], "given-names": ["B", "I", "J", "M"], "article-title": ["Understanding doctor-patient communication through telepresence robots"], "conf-name": ["In Proceedings of the 2019 CHI Conference on Human Factors in Computing Systems"], "year": ["2019"], "fpage": ["1"], "lpage": ["12"]}, {"label": ["23."], "surname": ["Cregan", "Mc Grath", "Maguire"], "given-names": ["\u00c1M", "ML", "G"], "article-title": ["Usability and acceptability of a mobile-enabled social robot (Kabot) to improve mood, loneliness, and quality of life of older adults in long-term care facilities: multi-method feasibility pilot study"], "source": ["JMIR Mhealth Uhealth"], "year": ["2021"], "volume": ["9"], "fpage": ["e23392"]}, {"label": ["24."], "surname": ["Silva", "Miranda", "Rosa", "Oliveira", "Saffar"], "given-names": ["AJ", "GM", "M", "T", "J"], "article-title": ["Telepresence robot technology as a tool for cognitive assessment of elderly patients in a COVID-19 scenario\u2013a pilot study"], "source": ["J Telemed Telecare"], "year": ["2022"], "volume": ["28"], "fpage": ["606"], "lpage": ["13"]}, {"label": ["28."], "surname": ["Adalgeirsson", "Breazeal"], "given-names": ["SO", "C"], "article-title": ["MeBot: a robotic platform for socially embodied presence"], "conf-name": ["In: 2010 5th ACM/IEEE International Conference on Human-Robot Interaction (HRI), IEEE"], "fpage": ["15"], "lpage": ["22"], "year": ["2010"]}, {"label": ["29."], "surname": ["Agnisarman", "Ponathil", "Lopes", "Chalil Madathil"], "given-names": ["S", "A", "S", "K"], "article-title": ["An investigation of consumer\u2019s choice between an on-site and a telepresence robot for interacting with the elderly"], "source": ["Int J Soc Robot"], "year": ["2018"], "volume": ["10"], "fpage": ["633"], "lpage": ["47"]}, {"label": ["30."], "surname": ["Rueben", "Aroyo", "Lutz", "Schmiedekamp", "Reilly"], "given-names": ["M", "AM", "R", "K", "A"], "year": ["2018"], "article-title": ["The effects of character framing on user perceptions and behavioral intentions toward a robotic educator"], "conf-name": ["In 2018 15th International Conference on Ubiquitous Robots (UR)"], "fpage": ["378"], "lpage": ["384"], "publisher-name": ["IEEE"]}, {"label": ["31."], "surname": ["Koleva", "Benford", "Brassett", "Leach", "Schn\u00e4delbach", "Glover"], "given-names": ["PT", "S", "J", "J", "H", "K"], "article-title": ["\"You've got me\": how running together in a remote group can build closeness"], "conf-name": ["Proceedings of the ACM on human-computer interaction, 3(CSCW)"], "fpage": ["1"], "lpage": ["27"], "year": ["2019"]}, {"label": ["32."], "surname": ["Fischer", "Petersen", "Tolmie"], "given-names": ["K", "MG", "P"], "article-title": ["Don't lag behind!: effects of embodied robot feedback on human exercise practices"], "conf-name": ["In Proceedings of the 2020 CHI Conference on Human Factors in Computing Systems"], "fpage": ["1"], "lpage": ["12"], "year": ["2020"]}, {"label": ["33."], "surname": ["Terada", "Yamaguchi"], "given-names": ["K", "Y"], "article-title": ["Effects of the presence of a robot incapable of jumping as high as humans on human maximal explosive jumping performance"], "source": ["Int J Soc Robot"], "year": ["2017"], "volume": ["9"], "fpage": ["329"], "lpage": ["37"]}, {"label": ["34."], "surname": ["Budvytyte", "Wang", "Matsangidou", "Costa"], "given-names": ["R", "A", "M", "UM"], "article-title": ["The effect of virtual agent's personality on users' engagement and exercise performance"], "conf-name": ["In Extended Abstracts of the 2021 CHI Conference on Human Factors in Computing Systems"], "fpage": ["1"], "lpage": ["7"], "year": ["2021"]}, {"label": ["35."], "surname": ["Costa", "Weintraub", "Weiss"], "given-names": ["J", "AP", "PL"], "article-title": ["Effect of facilitated kinesthetic and visual feedback on motor learning and motivation"], "conf-name": ["In 2019 International Conference on Robotics and Automation (ICRA)"], "year": ["2019"], "fpage": ["6443"], "lpage": ["6449"]}, {"label": ["37."], "surname": ["Browning", "Rigolon"], "given-names": ["MH", "A"], "article-title": ["Do income, race and ethnicity, and sprawl influence the greenspace-human health link in city-level analyses? Findings from 496 cities in the United States"], "source": ["Int J Environ Res Public Health"], "year": ["2019"], "volume": ["15"], "fpage": ["4941"], "lpage": ["54"], "pub-id": ["10.3390/ijerph15071541"]}, {"label": ["41."], "surname": ["McMahan", "Estes"], "given-names": ["EA", "D"], "article-title": ["The effect of contact with natural environments on positive and negative affect: a meta-analysis"], "source": ["J Posit Psychol"], "year": ["2015"], "volume": ["10"], "fpage": ["507"], "lpage": ["19"], "pub-id": ["10.1080/17439760.2014.994224"]}, {"label": ["43."], "surname": ["Van den Berg", "Wendel-Vos", "van Poppel", "Kemper", "van Mechelen", "Maas"], "given-names": ["M", "W", "M", "H", "W", "J"], "article-title": ["Health benefits of green spaces in the living environment: a systematic review of epidemiological studies"], "source": ["Urban For Urban Green"], "year": ["2015"], "volume": ["14"], "fpage": ["806"], "lpage": ["16"], "pub-id": ["10.1016/j.ufug.2015.07.008"]}, {"label": ["45."], "surname": ["Zhang", "Howell", "Iyer"], "given-names": ["JW", "RT", "R"], "article-title": ["Engagement with natural beauty moderates the positive relation between connectedness with nature and psychological well-being"], "source": ["J Environ Psychol"], "year": ["2014"], "volume": ["38"], "fpage": ["55"], "lpage": ["63"], "pub-id": ["10.1016/j.jenvp.2013.12.013"]}, {"label": ["46."], "surname": ["Anderson", "Mayer", "Fellows", "Cowan", "Hegel", "Buckey"], "given-names": ["AP", "MD", "AM", "DR", "MT", "JC"], "article-title": ["Relaxation with immersive natural scenes presented using virtual reality"], "source": ["Aerospace Med Hum Perform"], "year": ["2017"], "volume": ["88"], "fpage": ["520"], "lpage": ["6"], "pub-id": ["10.3357/AMHP.4747.2017"]}, {"label": ["49."], "surname": ["Pires", "Follette"], "given-names": ["JN", "KC"], "article-title": ["PARKbot: a telepresence robot to provide remote park experiences to hospitalized children"], "conf-name": ["In Companion of the 2018 ACM/IEEE International Conference on Human-Robot Interaction"], "year": ["2018"], "fpage": ["133"], "lpage": ["134"]}, {"label": ["50."], "surname": ["Slater", "Sanchez-Vives"], "given-names": ["M", "MV"], "article-title": ["Enhancing our lives with immersive virtual reality"], "source": ["Front Robot AI"], "year": ["2016"], "volume": ["3"], "fpage": ["74"], "pub-id": ["10.3389/frobt.2016.00074"]}, {"label": ["51."], "surname": ["Zenko", "Ekkekakis", "Kavetsos"], "given-names": ["Z", "P", "G"], "article-title": ["Changing minds: bounded rationality and heuristic processes in exercise-related judgments and choices"], "source": ["Sport Exerc Perform Psychol"], "year": ["2016"], "volume": ["5"], "fpage": ["337"], "lpage": ["51"], "pub-id": ["10.1037/spy0000069"]}, {"label": ["53."], "surname": ["Hung", "Wong", "Smith", "Berndt", "Gregorio", "Horne"], "given-names": ["L", "J", "C", "A", "M", "N"], "article-title": ["Facilitators and barriers to using telepresence robots in aged care settings: a scoping review"], "source": ["J Rehab Assist Technol Eng"], "year": ["2022"], "volume": ["9"], "fpage": ["110723"], "pub-id": ["10.1177/20556683211072385"]}, {"label": ["54."], "surname": ["Ding", "Cai", "Fu"], "given-names": ["X", "Z", "Z"], "article-title": ["Does the new-type urbanization construction improve the efficiency of agricultural green water utilization in the Yangtze River Economic Belt?"], "source": ["Environ Sci Poll Res"], "year": ["2021"], "volume": ["28"], "fpage": ["64103"], "lpage": ["12"], "pub-id": ["10.1007/s11356-021-14280-z"]}, {"label": ["56."], "surname": ["Zhang", "Yang", "Ding", "Qin"], "given-names": ["J", "G", "X", "J"], "article-title": ["Can green bonds empower green technology innovation of enterprises?"], "source": ["Environ Sci Poll Res"], "year": ["2022"], "pub-id": ["10.1007/s11356-022-23192-5"]}, {"label": ["57."], "surname": ["Cohen"], "given-names": ["J"], "article-title": ["Statistical power analysis for the behavioral sciences"], "publisher-loc": ["New York, NY"], "publisher-name": ["Academic"], "year": ["1988"], "volume": ["54"]}]
{ "acronym": [], "definition": [] }
58
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2024-01-14 23:43:50
Front Public Health. 2023 Dec 27; 11:1277479
oa_package/e6/12/PMC10787647.tar.gz
PMC10787648
0
[ "<title>1 Introduction</title>", "<p>Social networking sites (SNSs) like Instagram, Facebook, and Twitter have revolutionized how people interact and communicate within enterprises. SNSs are being used more widely and are becoming more well-liked (##UREF##24##Dimock, 2019##). However, the enticing features of SNSs can cause users to check their accounts frequently and relentlessly (##UREF##16##Cao and Sun, 2018##). The use of SNSs in a variety of ways, such as likes, using chat platforms, and status updates, may ultimately set off indicators and cluster toward behavior as cognitive and emotional reactions, which is likely to make it challenging to manage how one uses social media (##UREF##9##Barrot and Acomular, 2022##). In fact, excessive usage of social media can lead to signs of dependency emerging, which may have detrimental psychological and behavioral effects of stress. Numerous studies suggested that SNSs users have inadequate manipulation over their usage, which can interfere with daily activities in the home, workplace, and school (##REF##29220826##Turel et al., 2018##). Employees usually use SNSs to the point where they might log in for a long time and get distracted (##UREF##14##Boyd and Ellison, 2007##; ##UREF##26##Ellison et al., 2014##; ##UREF##73##Vishwanath, 2015##). SNSs can improve work-related outcomes by connecting people and giving them the accessibility to services at work needed for improved performance (##UREF##49##Leidner et al., 2010##; ##UREF##75##Wu, 2013##; ##UREF##26##Ellison et al., 2014##). But frequent use of SNSs could have detrimental effects like stress, addiction, exhaustion, diversion, a decrease in pleasant emotions, poor performance, and bad health (##UREF##12##Bevan et al., 2014##; ##UREF##27##Fox and Moreland, 2015##), which reduce the output of employee job performance.</p>", "<p>Because of this, there has been an upsurge in research related to the sources and effects of using SNSs (##UREF##55##Milošević-Ðorđević and Žeželj, 2014##; ##UREF##69##Tang et al., 2016##; ##REF##29220826##Turel et al., 2018##; ##REF##33665465##Elsayed, 2021##). Despite growing interest in SNSs use, little is known about how it affects results in both personal and professional spheres. Additionally, a theoretical framework that explicitly explains how SNSs usage distracts employees, could affect various contexts and exhaust social media usage which reduces employee job performance which is lacking in the present research related to SNSs. Such type of research is needed which can use SNSs where self-management can reduce social media exhaustion and improve employee job performance which tends to be challenging in a particular condition. These constraints will be addressed in the present study by fusing SNSs usage with both private and professional repercussions through stressors-strain-outcome theory. In SSO theory explains the framework of the study that Excessive social at work, excessive hedonic use at work, excessive cognitive use at work (stressors) that make working situations uncomfortable for the employees and lead to social media exhaustion (strain) and reduce their job performance (outcome). The users spend a lot of time on the internet and social networking sites that negatively affect employee job performance.</p>", "<p>Our theoretical approach is based on Bandura’s reciprocal determinism notion in information and communications technology (ICT) (##UREF##8##Bandura, 1986##), which holds that an individual’s behavior can alter how the environment (both at work and in their personal lives) is viewed by them and how they interact with it. We focus on a selection of significant personal and professional effects of SNSs usage in this study. This study pays close attention to both individual aspects, such as healthy feelings and pleasant emotions, and professional factors, such as social media exhaustion, self-management, and job performance. The term “positive impulses” refers to the psychological experiences that enhance and strengthen one’s “persistent personal resources” and enable behaviors that lead to favorable results in both one’s personal and professional life (##REF##21850154##Fredrickson, 1998##). Prior research has examined the importance of work environment elements, including job distraction and performance, and their effects (##UREF##37##Janssen and Van Yperen, 2004##; ##REF##36504983##Liang et al., 2011##; ##UREF##81##Zwarun and Hall, 2014##; ##UREF##71##Tarafdar et al., 2020b##), but the effect of self-management to reduce social media exhaustion has not been investigated to improve the performance. This study intends to examine the effect of self-control management in reducing social media exhaustion caused by social networking sites stressors and improves employee performance. The current will be helpful for the institutions to devise strategies based on self-control management to improve employee performance.</p>", "<p>This study has several significant theoretical and practical consequences. By providing a theoretic justification and practical evidence for the effects of SNSs practice on employee performance in professional contexts, this research enlarges the growing body of literature concerning frequent SNSs usage in the workplace can be reduced by self-management to improve overall performance. In practical terms, this research contributes to the direction of technological resources through employee perspective providing a better knowledge of self-management among employees to reduce SNSs exhaustion, which may lead to the provision of recommendations that can help private and public organizations to mitigate the usage of SNSs in the workplace.</p>", "<p>Stress at work could be triggered by many working aspects such as organization management, workload, excessive use of SNSs, structural and organizational climate, job characteristics and many more. A finding of a research done in one public university revealed that employees added stress when they felt not appreciated by the bosses, received orders outside office hours and excessive use of SNSs during work, getting pressure from the admins to achieve the Key Indicator Performance (KPI) of the departments (##UREF##36##Isa and Kadir Shahar, 2021##).</p>", "<p>This study aims at deepening our understanding on how different technology-related stressors indirectly create negative outcomes. We specifically focus on enhancing the job performance as outcome because it has been demonstrated that it is a key detrimental state for employee exhaustion and relationship with the organization. Focusing on this outcome, we propose a model that explains how adaptive and maladaptive coping strategies influence it, and how they are indirectly informed by key techno-stress creators, namely excessive social use at work, excessive hedonic use at work and excessive cognitive use at work.</p>", "<p>The structure of this paper is as follows. The pertinent literature is briefly reviewed in the part that follows, with an emphasis on SNSs. The literature review will be addressed and followed by wrapping up with testable theoretical hypotheses. The outcomes of the structural equation modeling analysis are then presented, trailed back by a description of the research process. This study discussed the study’s ramifications, recommendations for additional research, study limitations, and findings.</p>" ]
[ "<title>4 Research methodology</title>", "<title>4.1 Sampling procedures and measurements</title>", "<p>The purpose of this study was to examine how social media usage affects university academic staff members’ job performance. This study targeted academic staff performance because they are the main factor that determines a university’s ranking; nonetheless, it is evident that staff members are not performing to the best of their abilities, which is the real reason why the institution does not have a high ranking (##UREF##35##Iqbal et al., 2017##). Using social networking sites (SNSs) during working hours may have an impact on academicians’ performance. This could result in poor academic achievement, which would prevent universities from moving up the university ranking.</p>", "<p>The Along with the goal of the research, the measurement items of the questionnaires were disseminated via an internet platform utilizing an email. The tested scales that we used in this investigation were modified from earlier works. To meet the setting of the study, the questionnaire’s phrasing was changed. The five items of excessive social use at work were developed by ##UREF##6##Ali-Hassan et al. (2015)## to measure particular constructs. The three-item and four-item excessive hedonic and cognitive use at work scores were modified by ##UREF##6##Ali-Hassan et al. (2015)##. The social media exhaustion measurements were modified by ##UREF##7##Ayyagari (2012)##. The four performance indicators were taken from ##UREF##37##Janssen and Van Yperen (2004)##. A total of 16-item self-control and the self-management measure were modified (##UREF##54##Mezo, 2009##). A Likert scale with five possible values was used to score each item.</p>", "<p>As control variables, several demographic factors, such as gender, age, education, industry type, and the frequency and duration of social media use, were included in the constructs in the proposed research model. To gather the data for this investigation, an online survey was used. Five of Malaysia’s major universities provided the necessary sample. Therefore, the target participants were university faculty members who had some background in using social media in businesses 391 individuals participated in total. Our research is deeply entrenched in the field of quantitative research. Quantitative research is well-suited to determining the values of variables, supporting robust statistical analysis, and allowing for a methodical investigation of correlations within a specific population. To achieve study research goals, this study used a cross-sectional research strategy, which is distinguished by its concentration on collecting data from one group or demographic at a single time. The use of a cross-sectional methodology was chosen since it enabled us to obtain an overview of the phenomenon under inquiry while also successfully assessing variations and linkages within our target group. The sampling approach adopted for the present research was purposeful sampling, owing to its connection with the research’s unique objectives and emphasis. This method of non-random sampling entails a deliberate choice of respondents who have the necessary traits or knowledge about the study’s issue. The potential negative consequences of excessive social media use on workers’ ability to execute their jobs were investigated in this study. We adopted the purposive sampling method to provide each University Employee in Malaysia an equal chance of being selected even if we do not have the exact list of employees that may be our respondents. The targeted respondents were contacted by email once the questionnaire was generated using Google Forms (##UREF##65##Sekaran, 2003##). We had to rely on online data collection because there weren’t many options for physical data collection during a pandemic. By leveraging online methods for data collecting, we can guarantee both the respondents’ security and timely data gathering.</p>", "<title>4.2 Data analysis</title>", "<title>4.2.1 Demographic characteristics</title>", "<p>The demographic characteristics of the respondents (see ##TAB##0##Table 1##), identified in this study are as follows: valid responses received 391, where 53.2% (209) were males and 46.8% (184) were females in the present field. All the identified respondents of the study were adults, where most of the respondents were found within the age groups of 41–50 were 126 (32.1%), 25–30 was 122 (31%), 31–40 were 113 (28.8%), respectively, followed by age groups 51 years and above were 32 (8.1%). While 240 (61.4%) are Malay, Chinese 138 (35.3%), Indian 5 (1.3%), followed by 11 others. Based on education, 295 of the total respondents are Ph.D. (75.4%), 47 are master’s degrees (12%), and 48 are Bachelor’s (12.3%). Based on job experience 128 respondents are having less than 5 years (32.7%), Less than 10 years are 107 (27.4%), less than 15 years are 86 (22%), and more than 15 years of experience are 70 (17.9%). Therefore, such a profile of respondents indicates that they are mature, educated, and experienced profile, and have a deeper understanding of the topic under investigation.</p>" ]
[ "<title>5 Results</title>", "<p>R-squared, is a commonly used tool in statistical evaluation to demonstrate how much of the variance in the dependent variable can be determined by each of the independent variables. This study purposefully employs SmartPLS for analysis in this study rather than R-squared, taking into account because SmartPLS is a structural equation modeling (SEM) approach that works especially well with low sample sizes and sophisticated models that contain latent variables. Both formative and reflective measuring models are supported. The study chose this approach to better fit the intricate details of study model. SmartPLS is well-known for being resilient to unconventional data, which makes it a good fit in scenarios where the R-squared assumptions of conventional linear regression models do not hold true. Considering the sensitive nature of this study data, this was highly important. A key component of SmartPLS is predictive relevance, whereas R-squared is mainly concerned with interpreting variance. This is in line with the study’s goals, which included forecasting and offering recommendations in addition to elucidating the correlations between the variables. Although it is customary to utilize R-squared as a coefficient of determination, the choice to stray from this method and use SmartPLS was reached after taking the particulars and goals of the study into account. Methodological fit, resilience, predictive relevance, adaptability, and the capacity to extract practical insights were prioritized.</p>", "<p>SmartPLS 3.3.2 was employed in this study’s analysis of the data gathered. To analyze the data, PLS-SEM has been divided into two steps (##REF##35009967##Hair et al., 2017##). The reliability and validity of the discovered constructs are evaluated in the primary phase using the PLS method. The following stage involves evaluating the outcomes using a structural model and bootstrapping.</p>" ]
[ "<title>6.4 Discussion and hypotheses testing</title>", "<p>This study’s results proved the significant relationship between excessive cognitive use at work on social media exhaustion. The broad amount of cognitive usage of social networking sites obtained by employees upsurges social media exhaustion and diverts them from the task identified, as depicted values <italic>P</italic>-values (0.000). Result of the cognitive usage are consistent with the ##UREF##23##Deryakulu and Ursavaş (2014)##, which shows that social media cognitively usage by sharing ideas, photos, debates etc., distracts employee from identified tasks. ##UREF##46##Landers and Schmidt (2016)## also supported that employees while investing his time on exploring ideas and information during office hours divert from actual task and gains exhaustion.</p>", "<p>Hedonic use at work also causes dysfunctional consequences, while excessive usage of social media diverts employees from identified tasks. Excessive hedonic use at work <italic>P</italic>-values (0.092) indicates that it is not significant. Result of this factor are consistent with ##UREF##52##Luqman et al. (2017)##, which shows that usage of social media for fun at workplace causes exhaustion among employees and significantly distract them from their identified tasks. A study by ##UREF##11##Benson et al. (2019)## also found a connection between the usage of SNS hedonically among students with reduced performance in the classroom.</p>", "<p>Excessive social use at work <italic>P</italic>-values (0.000) shows that the relationship between excessive social use at work and social media exhaustion is significant. Excessive usage of social media distracts users and reduces the level of employee performance. With the usage of social networking sites, employees’ social media exhaustion increases and decreases the level of employee performance. <italic>P</italic>-values (0.000) show a significant relationship in reducing employee performance. The results supports the earlier studies which demonstrate that overuse of social media encourage users of constant checking of social media at the workplace, which leads to compulsive behaviors and distract from actual job (##UREF##76##Yu et al., 2018##). Another study also found that SNS social users during office hours lacks effective self-control, which interferes with daily activities in the home, workplace, schools, and businesses and reduce their performance (##UREF##16##Cao and Sun, 2018##).</p>", "<p>As the usage of social networking sites exhausts employees from the usage of social media, with the effective usage of self-control management employee performance can be significantly improved. The <italic>P</italic>-values (0.017) show a significant relationship between self-control management and job performance. Earlier studies also revealed that self-management is essential to keep track of one’s behavior of getting distracted by excessive SNS use during office timings to improve performance (##REF##26291954##Wheelock et al., 2015##). Usually, a self-managed person makes strategies for themselves and works hard to achieve those objectives by improving their performance (##REF##31385324##Javed et al., 2019##; ##UREF##53##Majid et al., 2019##). Self-management considered as a moderating variable between social media exhaustion and employees’ performance based on the hypothesis that self-managed employees perform better, it significantly moderate the association.</p>", "<p>Due to the rapid growth of technology and knowledge, there is also a risk of social media tiredness (##REF##27148100##Lee S. L. et al., 2016##; ##UREF##59##Moughal et al., 2023b##). Additionally, since the quantity of social media users grows tremendously, information is shared quickly on these platforms. Users of social media may experience overwhelming amounts of information due to cognitive overload during their office hours which negatively impacts employee performance (##UREF##25##Eliyana et al., 2020##). The current study shows a link between cognitive use at work and social media tiredness that is in favor of communication overload which reduces employee performance. The impact of cognitive use at work on social media weariness is due to the fact that “social media consumes employees too much time and has the greatest magnitude of reducing employee performance.” During office hours, usage of social media exhausts employees and distracts them from their assigned tasks. Social networking sites are one of the communication tools that connect employees inside organizations with others to communicate (##UREF##61##Ou and Davison, 2011##), however the usage of social media for personal or social use for enjoyment will distract employees and reduce their performance. According to ##UREF##41##Karr-Wisniewski and Lu (2010)## and ##UREF##25##Eliyana et al. (2020)##, when employees get communicated with others via social networking sites, they frequently stop what they are doing and respond right away. An individual will require a few moments to resume stopped work operations after handling communication disruptions (##UREF##41##Karr-Wisniewski and Lu, 2010##).</p>", "<p>The study’s findings demonstrate that excessive usage of social networking sites has a detrimental effect on work performance. Employee excessive usage of social sites is the root of the reduction in performance. Employees who receive too much interaction lose connection with their assigned job tasks and feel unproductive because they socially interact with others online, and their performance suffers as a result. This study revealed the possible solution which can be helpful to control their social media exhaustion and improve their performance with the help of self-control management. Self-control management significantly moderates the relationship between social media exhaustion and job performance among academic employees. Employees who communicate online too much, frequently make mistakes and mislead from assigned job duties, with the identified factor self-control management employees can control their usage of social media and improved performance. Employees that get excessive communication have a tendency to just pay attention to particular information and self-control and disregard other information to improve their job performance.</p>" ]
[]
[ "<p>Edited by: Greta Mazzetti, University of Bologna, Italy</p>", "<p>Reviewed by: Hasnaa Helwa, Benha University, Egypt</p>", "<p>Biruta Sloka, University of Latvia, Latvia</p>", "<p>Yandra Rivaldo, Abdullah Said Islamic Institute Batam, Indonesia</p>", "<p>This study investigates the significance of self-management in academic staff stress management related to social networking sites (SNS). It emphasizes particularly on reducing social media exhaustion and increasing job effectiveness. The research applies the stressor-strain-outcome theory and the Smart PLS (partial least squares) analytical approach to examine data from 391 respondents. The study’s goal is to provide empirical data on the efficacy of self-control management in reducing SNS stress and its effects on academic staff’s psychological wellbeing and job performance. Data is collected by survey using online email platforms among academic employees, and the collected data is examined utilizing the Smart PLS approach. This approach allows for an investigation of the proposed links and their statistical importance. This research’s ramifications are important for academic institutions since its results can help academic personnel effectively cope with SNS-related stress. Academic employees can better limit their SNS usage and avoid social media tiredness by promoting self-control management practices. As a result, academic employees’ job performance and overall wellbeing may increase. The study’s findings help to comprehend how self-management might reduce SNS stress and improve staff performance in the academic sector.</p>" ]
[ "<title>2 Context of the study</title>", "<p>In Malaysia, academic and non-academic staff have been under pressure over the years to meet the increasing demands to be internationally recognized academically and in the field of research, generate income for the university, and they were expected to meet the performance set by the university (##REF##31136594##Mukosolu et al., 2015##). Job-related stress among academic university staff has more attention among researchers than other occupational groups in the UK (e.g., ##UREF##43##Kinman and Jones, 2003##; ##UREF##72##Tytherleigh et al., 2005##). Conversely, this research is examining the impact of SNSs stressors on the productivity of university academic staff. As academic staff are under stress as they need to respond to the global demands, their responsibilities are no longer confined within the boundaries of teaching and learning. As the status of higher education is changing according to the needs of globalization, it goes parallel with the responsibilities of academic staff (##UREF##67##Sidek et al., 2012##).</p>", "<title>3 Literature review</title>", "<title>3.1 Excessive use of SNSs</title>", "<p>Social networking sites are frequently accessed by employees as a distraction from challenging or time-consuming work situations or to put off finishing responsibilities. Much research suggests compulsive Internet use, with some users becoming overly reliant on web-based communication applications (SNSs). This could lead to a variety of impairments, such as psychological, social, and professional impairments (##REF##8969098##Young, 1996##; ##REF##19178219##Orzack and Orzack, 1999##; ##UREF##29##Griffiths, 2000##; ##REF##19072079##Meerkerk et al., 2009##; ##UREF##13##Blackwell et al., 2017##; ##UREF##18##Carbonell and Panova, 2017##).</p>", "<title>3.2 Three dimensions of excessiveSNS use</title>", "<p>According to ##UREF##76##Yu et al. (2018)## and ##UREF##15##Cao et al. (2019)## research, when people use social media excessively, they believe that much time and effort is being wasted while at work. There are three types of excessive social media use at work: excessive hedonic, excessive social, and excessive cognitive.</p>", "<title>3.3 Excessive social use at work</title>", "<p>In order to use SNSs for social purposes, users must engage in two-way communication with friends, family, and other network users. This communication helps users establish and sustain social relationships (##UREF##52##Luqman et al., 2017##). Excessive social use at work is defined as putting too much time and effort into using social media to establish and maintain professional contacts (##UREF##6##Ali-Hassan et al., 2015##). But overuse of social media can encourage users, which can lead to compulsive behaviors like constant checking (##UREF##76##Yu et al., 2018##; ##UREF##1##Abbasi et al., 2021a##). Numerous pieces of data show that SNS social users lack effective self-control, which interferes with daily activities in the home, workplace, schools, and businesses (##UREF##16##Cao and Sun, 2018##).</p>", "<title>3.4 Excessive hedonic use at work</title>", "<p>The hedonistic usage of social media is predicated on pleasure and fun, similar to that which is had while playing games and watching entertainment (##UREF##80##Zheng and Lee, 2016##; ##UREF##52##Luqman et al., 2017##). When someone uses social networking sites (SNSs) excessively to relax, escape from, or amuse themselves while at work, it’s referred to as “extreme hedonic use at work” (##UREF##6##Ali-Hassan et al., 2015##). Extreme SNSs users use SNSs applications, play games, or watch movies for a sizable portion of their workday (##UREF##18##Carbonell and Panova, 2017##). Users of SNSs frequently use the platform to fulfill their personal demands rather than complete their work obligations, which has a detrimental impact on how successfully they accomplish their employment. Numerous studies have found a connection between the use of SNSs and poor student and employee performance in the classroom (##UREF##11##Benson et al., 2019##; ##UREF##2##Abbasi et al., 2021b##). Additionally, hedonistic use of SNSs includes compulsively rewarding behavior, which raises the danger of long-term usage and the urge to continue using it (##UREF##52##Luqman et al., 2017##).</p>", "<title>3.5 Excessive cognitive use at work</title>", "<p>The final aspect of using social networking sites, cognitive use, is involved with creating content to share and accessing content created by others, such as reviews, tales, ratings, images, and videos (##UREF##17##Cao and Yu, 2019##). When people “produce and exchange content, ideas, and opinions; build habits; participate in debates; see photographs; and consume content posted by other users,” they are using social networking sites (SNS) cognitively (##UREF##62##Papacharissi and Gibson, 2011##; ##UREF##52##Luqman et al., 2017##). According to ##UREF##23##Deryakulu and Ursavaş (2014)##, using social media (SNS) extensively while at work to create and distribute user-generated content is referred to as excessive cognitive use. Thanks to information technology, people now have greater access to information and communication than ever before, however, one study found that the harmful impacts of “over information” have also attracted increased attention from researchers (##UREF##52##Luqman et al., 2017##; ##UREF##3##Abbasi et al., 2021c##). Because of this, the employee’s capacity is impaired by excessive cognitive use, which also affects their ability to perform their daily activities at work. Due to being distracted, employees could perform worse at work (##UREF##33##Hou et al., 2017##).</p>", "<title>3.6 The influence of excessive social use at work on social media exhaustion</title>", "<p>Among other technologies, social media platforms like instant messaging (IM) can be used to improve collaboration within organizations (##UREF##22##Davison et al., 2014##). Once connected via social media, workers prefer to put everything on hold and respond to contact requests instantly (##UREF##22##Davison et al., 2014##). It normally takes a few minutes to return to previous job duties after processing a communication break that was instigated by another party (##UREF##60##Nisar and Whitehead, 2016##). Given the limitations of the human brain, unexpected encounters may lead to someone’s attention being distracted and their cognitive load increasing. People who stay in touch would therefore find it challenging to focus on their work, which would lead to them becoming weary and experiencing weary when using social media.</p>", "<p>The overuse of social media at work, which has been linked to stress and social media exhaustion (##UREF##7##Ayyagari, 2012##; ##UREF##68##Sullivan and Koh, 2019##), is the subject of this study. If someone uses social media excessively to establish and maintain business relationships, they could feel out of control (##UREF##6##Ali-Hassan et al., 2015##; ##UREF##0##Abbasi et al., 2022##). Their mental health may be negatively impacted by these circumstances, which may ultimately result in anxiety and exhaustion (##UREF##63##Ragu-Nathan et al., 2008##; ##REF##27148100##Lee S. L. et al., 2016##). According to ##UREF##16##Cao and Sun (2018)## and ##UREF##56##Moqbel and Kock (2018)## compulsive monitoring behavior brought on by excessive sharing on social networking sites (SNS) can be exhausting and readily stopped.</p>", "<p>Based on the theory and previous research above, it can be derived as follows:</p>", "<title>3.7 The influence of excessive hedonic use at work on social media exhaustion</title>", "<p>According to ##UREF##52##Luqman et al. (2017)##, social media is about having fun and enjoying oneself, especially through games and entertainment. To pass the time while viewing films and playing games, there are a variety of apps accessible (##UREF##18##Carbonell and Panova, 2017##). When participating in social media, staff members typically put everything on hold to reply to requests (##UREF##25##Eliyana et al., 2020##).</p>", "<p>Employees may feel emotions like curiosity, happiness, and enjoyment when they use the technology. As a result, it would be challenging for them to concentrate on their activity while engaged in constant contact; also, they would feel antsy and exhausted while using social media.</p>", "<p>Based on the theory and previous research above, it can be derived as follows:</p>", "<title>3.8 The influence of excessive cognitive use at work on social media exhaustion</title>", "<p>Excessive cognitive use at work focuses on producing, disseminating, and accessing content that has already been produced by others. Examples of this content include opinions, tales, ratings, photos, and videos (##UREF##17##Cao and Yu, 2019##). Employees are diverted from their tasks when using SNSs at work, though. Employees who have self-awareness and self-management skills can still offset the negative impacts of excessive SNSs usage and cause social media tiredness.</p>", "<p>The last aspect of social media use, known as cognitive use, involves producing, sharing, and accessing original content. This content includes, among other things, articles, photographs, videos, ratings, and opinions (##UREF##6##Ali-Hassan et al., 2015##). In order to influence task performance, employees typically use this function to collect and disseminate information. When they invest a lot of time learning and gathering information during working hours, employees are exposed to more extensive and in-depth information than is necessary (##UREF##46##Landers and Schmidt, 2016##). A person may experience information overload and be unable to fulfill their regular work commitments when they are unable to process a large volume of information (##UREF##25##Eliyana et al., 2020##). According to studies, a person’s ability to solve problems and make judgments may be impacted, reducing work productivity (##UREF##10##Bawden and Robinson, 2009##; ##UREF##17##Cao and Yu, 2019##; ##UREF##58##Moughal et al., 2023a##).</p>", "<p>Employees who often access and exchange information via social media at work may experience information overload as a result of a high information load and cognitive process limitations (##UREF##64##Roetzel, 2019##). Due to the limitations of the human intellect, cognitive load occurs when employees are given more knowledge than they have the time or ability to digest and use (##UREF##25##Eliyana et al., 2020##). They could make you feel powerless. Technology weariness may result from such an incident having a negative impact on their mental health (##UREF##47##Lee A. R. et al., 2016##; ##UREF##28##Fu et al., 2020##). Therefore, using social media at work in an excessively cognitive manner can lead to stressed-out employees.</p>", "<p>Thus, we propose the following hypothesis:</p>", "<title>3.9 The influence of social media exhaustion on job performance</title>", "<p>Social media can become saturated if it is used excessively. A person’s emotional reaction to difficult events is referred to as exhaustion. When under difficult and prolonged conditions, exhaustion is the loss of mental energy (##UREF##40##Kamal et al., 2020##). Up to one-third of all online time is spent on social networking sites (SNSs), and many users claim to feel worn out as a result. Social media tiredness is a relatively new concept that deserves further study, according to some (##REF##27148100##Lee S. L. et al., 2016##; ##UREF##15##Cao et al., 2019##). According to ##UREF##56##Moqbel and Kock (2018)##, Social networking users (SNSs), for instance, may submit friend invitations and post about their daily life as the first source of it. Secondly, there is business interaction, including posting about the goods and services a company provides. The use of interfaces or the addition of new features comes third. Social media exhaustion has been brought on by the rising use of social media at work. This highlights the exhaustion that comes with utilizing technology.</p>", "<p>Due to pressure to spend excessive amounts of time on social networking, employees may feel exhausted and stressed out (##REF##30821647##Zivnuska et al., 2019##; ##UREF##59##Moughal et al., 2023b##). They will thus perform less well at work (##UREF##28##Fu et al., 2020##; ##REF##33601985##Ngien and Jiang, 2022##). In light of the aforementioned theory and study, we can draw the following conclusions:</p>", "<title>3.10 The influence of self-control management on job performance</title>", "<p>Self-control management is defined as the process of having higher control over one’s self while doing work (##UREF##74##Wilson et al., 2018##). ##UREF##54##Mezo (2009)## identified three key elements of employee self-control management.</p>", "<p>Self-monitoring</p>", "<p>Self-evaluation</p>", "<p>Self-reinforcement</p>", "<p>The process of exercising greater self-control while working is known as self-control management (##UREF##74##Wilson et al., 2018##). In the current dynamic environment, self-management has evolved into a crucial quality of successful employees, according to ##REF##30601717##Allegrante et al. (2019)##. Employees who are managed with self-control remain focused on their work and are able to carry out their responsibilities more effectively (##REF##30448709##Moore and Brown, 2019##). Self-management is essentially keeping track of one’s behavior while one tries to complete a task and avoiding becoming sidetracked by other things like excessive SNS use or other distractions (##REF##26291954##Wheelock et al., 2015##). Typically, a self-managed person makes plans for themselves and works hard to fulfill those objectives (##REF##31385324##Javed et al., 2019##; ##UREF##53##Majid et al., 2019##). Self-control management practices are helpful in enhancing employees’ intended behavior at workplace and managing undesired behavior, which might effect from impulses, ingrained habits, and behavior picked up through upbringing. Consequently, implementing self-management strategies at work can help organizations deal with numerous problems they currently confront (##UREF##38##Javed et al., 2023##). Employee job performance is positively impacted by internalizing the organization’s ideals through self-control management.</p>", "<p>In essence, an employee’s capacity for self-management determines how well they perform (##REF##31385324##Javed et al., 2019##; ##REF##30448709##Moore and Brown, 2019##). Self-management will be used as a moderating variable between SNSs stressors and employees’ performance based on the hypothesis that self-managed employees perform better. It is likely to moderate the association (##REF##31385324##Javed et al., 2019##).</p>", "<title>3.11 Job performance</title>", "<p>Many various terms are used to indicate how successfully someone does the work. The ability to accomplish work-related tasks is referred to as one’s job performance (##UREF##77##Yu et al., 2022##). This displays how adeptly an individual can fulfill the demands of their employment. A definition of “job performance” also includes “conduct or behavior that promotes organizational goals” (##UREF##6##Ali-Hassan et al., 2015##). The work product itself is essentially what the employees produce. Enhancing performance for both individuals and organizations is the main goal of initiatives to improve organizational performance (##UREF##57##Moqbel et al., 2013##).</p>", "<p>According to ##UREF##25##Eliyana et al. (2020)##, performance refers to the results attained by an individual or group within a company. Performance in general refers to what employees do, and performance in particular refers to the quantity and quality of work that an employee completes by the responsibilities that have been assigned to him or her. Performance is often measured and associated with the accomplishment of certain company goals during those times. According to ##UREF##21##Darma and Supriyanto (2017)## and ##UREF##25##Eliyana et al. (2020)##, job performance is a true behavior that everyone demonstrates as work performance created by employees in accordance with their position within the firm.</p>", "<title>3.12 Stressor–strain–outcome (SSO) theory</title>", "<p>##UREF##45##Koeske and Koeske (1993)##’s explication of the SSO model clarifies the connection between stressors, strains, and outcomes. According to this theoretical framework, a stressor has an indirect impact on the result, with stress being mediated through strain as social media exhaustion and typically producing unfavorable results (reducing employee performance) (##UREF##45##Koeske and Koeske, 1993##). The SSO theoretical framework is employed in the present research to clarify how excessive social media use, cognitive use and hedonic use at work lead to stress (##UREF##45##Koeske and Koeske, 1993##). Excessive social, hedonic, and cognitive usage in the workplace, excessive communication, and information overload are all elements that make working circumstances uncomfortable for employees and reduce their productivity. This study thus adds more information to the stressor-strain-outcome paradigm. This strategy is ideal for understanding the SNS stressors, looking into excessive social media use at work and its negative impacts, and looking into how social media exhaustion causes distraction and affects employees’ job performance.</p>", "<p>This idea holds that the two fundamental elements of the stress phenomenon are stressors and strain. Stressors are external events that cause humans to experience strain, which is a psychological response. Stress is the result of an imbalance between a person’s true motivations, skills, and the need to maintain their surroundings. It includes unpleasant emotions including anxiety, fear, aggravation, pressure, and melancholy. The psychological stressors associated with technological stress that are most frequently studied in social media research are exhaustion (##UREF##48##Lee and Lee, 2018##), job fatigue (##REF##24444742##Meyer et al., 2015##), and task distractions (##REF##31385324##Javed et al., 2019##). Stress can also have a negative impact on an employee’s effectiveness at work. However, the link between stress and work performance is not fully explored from the standpoint of SNS stressors and the mediating effect of social media exhaustion to reduce performance. The performance of the company as a whole as well as that of the employee may be directly impacted by this. This can be a crucial sign to gauge how successful it was. Therefore, the stressor-strain-outcome model, which considers the user’s experience, is used in this study along with the mediating effect of social media exhaustion and the moderating effect of self-control management to improve employee performance. The model incorporates excessive social use at work, excessive hedonic use at work, and excessive cognitive use at work as a stressor, social media exhaustion as a mediator to reduce employee performance, and self-control management as a moderator to significantly improve employee job performance which is the dependent variable (see ##FIG##0##Figure 1##). The context of this study precisely identifies the process through which excessive social, hedonic and cognitive use at work impairs each employee’s capacity for performance.</p>", "<title>6 Assessment of the measurement model</title>", "<p>Using structural equation modeling with partial least squares, confirmatory factor analysis was performed (PLS-SEM). According to this research, the SmartPLS technique was used to examine a fictitious framework from a prediction perspective.</p>", "<p>In the initial stage factor loadings of identified indicators were estimated, followed by the Cronbach alpha value, and at last the average variance was examined of the constructs’ reliability and validity (AVE). The average Cronbach alpha for excessive cognitive use at work is 0.790, which is considered good for testing validity and reliability. The reliability of Cronbach’s alpha for excessive hedonic use at work is 0.811. For excessive social use at work, Cronbach alpha reliability is 0.745. The social media exhaustion scale’s Cronbach alpha reliability is 0.864. Cronbach alpha reliability for self-control management is 0.857. The job performance scale’s Cronbach alpha value is 0.796. All the values mentioned here show no reliability issue because identified constructs values are more than the threshold values.</p>", "<p>The next step is to complete confirmatory factor analysis (CFA) was executed by applying structural equation modeling based on partial least squares (PLS-SEM). The SmartPLS practice was applied by ##UREF##44##Koay et al. (2020)##, ##UREF##50##Leung et al. (2020)##, and ##UREF##19##Cheung et al. (2021)## because their research examined a hypothetical relationship framework from a prediction view.</p>", "<p>All the mentioned ways to assess construct reliability and validity are mentioned (##REF##35009967##Hair et al., 2017##). All the factor loading of all indicators was examined in an initial phase, later on, the Cronbach alpha value, and lastly the average variance extracted (AVE) was extracted (##UREF##32##Henseler et al., 2009##).</p>", "<title>6.1 Construct reliability and validity</title>", "<p>All the items of variables were embraced in the model, however after the evaluation process, items having factor loading less than 0.50 were removed (##UREF##30##Hair et al., 2011##, ##UREF##31##2013##). The recommended threshold values for outer loading are 0.60 or above, and composite reliability (CR) and values for Cronbach’s alpha (CA) should be above 0.70. To obtain the convergent validity with the AVE must be &gt; 0.50. This study results met all the identified threshold values and depicts reliable constructs in ##TAB##1##Table 2##.</p>", "<title>6.2 Discriminant validity</title>", "<p>Discriminant validity was assessed by the Heterotrait-Monotrait Ratio (HTMT) in methodology which is regarded as a significant method to examine the value of discriminant validity. Literature used the regression analysis method to evaluate the effect of the exogenous variables excessive cognitive use at work, excessive hedonic use at work, and excessive social use at work on employee job performance, with social media exhaustion serving as a mediator. The results of the study demonstrated that, when social media exhaustion taking into account, all the exogenous variables significantly affect the job performance of employees, except for excessive hedonic use at work. As recommended by ##REF##35009967##Hair et al. (2017)##, SmartPLS was used to carry out the bootstrapping technique with 5,000 resamples. All the values show that there is no discriminant validity issue (see ##TAB##2##Table 3##).</p>", "<title>6.3 Assessment of structural model</title>", "<p>The value of Q2 is 0.299, indicating the predictive significance of 30.3% shown in ##FIG##1##Figure 2## for job performance as a dependent variable. However, the <italic>R</italic><sup>2</sup> value for the mediation value is 0.341, indicating that 34.1% predict task distraction affecting job performance, and the value of Q2 is 0.534, indicating that task distraction has a predictive relevance. The structural model assessment depicted in ##FIG##2##Figure 3## was used in this work to examine the theory after the measurement model generated the necessary results. Analysis of the statistical significance, T-value, and effect size of the route coefficient was done using the bootstrapping of 5,000 samples (##UREF##39##Jeon et al., 2019##).</p>", "<p>Meanwhile, multiple regression has been employed to examine the effects of the independent variable’s excessive social use at work, excessive hedonic use at work, and excessive cognitive use at work overload leading to social media exhaustion and decrease employee job performance. The findings showed the usage of social networking sites and apps increases employee interest in excessive information, cognitive and hedonic use at work increase and leads to social media exhaustion and decrease job performance.</p>", "<p>This study depicted that employees while using excessive social media exhaust them to perform their tasks and decrease their job performance. Earlier studies also retrieved social media effective in reducing the employee performance output (##UREF##78##Zhan et al., 2016##), whereas SSO was also found effective in influencing employee efficiency on the job (##UREF##79##Zhang et al., 2015##; ##UREF##51##Liu et al., 2017##; ##UREF##5##Al Hammadi and Hussain, 2018##; ##UREF##48##Lee and Lee, 2018##; ##REF##28612930##Sherman et al., 2018##; ##REF##31385324##Javed et al., 2019##; ##UREF##77##Yu et al., 2022##). So, this study found the core related issues of excessive social media use (ESMU), excessive hedonic use and excessive cognitive use at work, social media exhaustion, and employee job performance (see ##TAB##3##Table 4##). In this study, except for excessive hedonic use at work all of the factors were found significant to influence job performance.</p>", "<p>According to ##UREF##20##Cohen (2013)## general guidelines, values of (f2) between 0.02 and 0.15, between 0.15 and 0.35, and over 0.35 indicate that the exogenous construct has, respectively, a mild, moderate, and substantial effect on the dependent variable. While results below 0.02 suggest that there is no effect, but when (##UREF##4##Aguinis et al., 2017##) examined earlier research from the previous three decades, and discovered that the average moderating impact is 0.009. In recent research, (##UREF##42##Kenny, 2018##) proposed 0.005, 0.01, and 0.025 as small, medium, and large effect sizes of moderation, respectively, which represent more practical criteria. The fact that even these figures are optimistic is also highlighted by him. The following formula can be used to compute the magnitude of the moderating effect size (f2):</p>", "<p>where,</p>", "<p>According to the Kenny propositions, the value of <italic>f</italic><sup>2</sup> was more than the large effect that is 0.025 &gt; it shows that the social media effect has large effect to influence consumer purchase intention (see ##TAB##4##Table 5##).</p>", "<title>6.5 Theoretical implications</title>", "<p>The study covered theoretical ramifications; firstly, rather than focusing on the positive side, the research can expand the body of self-control management literature to improve employee performance, which is largely ignored in the conceptualizations; drivers, and effects of SNS usage (##UREF##70##Tarafdar et al., 2020a##). To highlight the behavioral outcome as a coping mechanism for stressful situations, we looked at the antecedents of low academic performance as SNS stressors. The present research specifically analyses the potential negative impacts of problematic social networking site (SNS) usage on academic performance and broadens the theoretical and empirical understanding of the etiology of problematic SNS use. This study brought attention to the SNS stressors by using the SSO framework. It helped to provide a clearer and more thorough knowledge of inappropriate SNS use, which results in stress in the shape of low job performance. It is important to consider the causes of low academic performance, particularly when they are brought on by an unbalanced flow of SNS usage. Furthermore, to some extent, this study adds to the body of social networking sites’ self-control management and SSO theory literature. In particular, it reveals that SSO philosophy can be employed to pinpoint important factors like excessive social use at work, excessive cognitive use at work, and excessive hedonic use at work in addition to being an appropriate explanation for inconsistent usage of SNS. In comparison to planned behavior, SSO theory is more effective in explaining less rational behavior. This makes a significant contribution to the body of literature on the usage of SNSs during office hours to improve job performance. Thirdly, this research supports not only the development of SSO theory but also the comprehension of self-control management to improve employee performance.</p>", "<p>This is an important contribution to SNSs use literature that has mainly relied on planned behavior research (##UREF##66##Shin and Hall, 2011##). Thirdly, in addition to the importance of SSO theory, this research also subsidizes developing an understanding of discontinuance intention and the underlying mechanism involved. We considered different discontinuance intentions behavioral patterns such as suspending the usage, rationalizing the usage, and permanently leaving the use of SNS which is consistent with the prevailing scholars in IS (##UREF##34##Hwang et al., 2019##). This study considered factors of SNS i.e., excessive social, cognitive, and hedonic use at work, which are in line with views of distracting employees from their assigned jobs (##UREF##34##Hwang et al., 2019##), and the usage of social media exhaust employees and distract employees.</p>", "<p>Additionally, the moderating role of self-control management enhanced the employee to refrain from using SNSs during office hours who experience poor academic performance as a result of SNSs stresses, it is also considered a significant contribution to the existing literature. In other words, it suggests that employees during office timings reduce SNS usage as a coping mechanism to avoid stressful circumstances in the future (##UREF##78##Zhan et al., 2016##). Ultimately, this study contributed by evaluating all three kinds of stressors (excessive social use at work, excessive hedonic use at work, and excessive cognitive use at work) as social media-related stressors and examining their impact on academic performance.</p>", "<p>The aforementioned stressor is prevalent among employees and is covered in the social media aspect (##UREF##52##Luqman et al., 2017##). These three types are hardly ever discussed in relation to academic performance, although results show that they are all significant sources of SNSs linked to stressors that have an impact on employees’ academic performance. Additionally, our study demonstrates the significance of stimulus features in impacting academic performance with the use of SSO frameworks, such as social media exhaustion can be released with effective usage of self-control management. This discovery contributes to SNS-related research and exposes the negative effects of SNSs use by demonstrating cognitive attention spurred by SNS stimuli, which has an impact on academic performance. Particularly, our research demonstrates the clear link between SNSs affecting employee job performance and deviating their attention.</p>", "<title>6.6 Practical implications</title>", "<p>To keep an eye on the negative impacts of SNSs use, this research also makes several practical recommendations for SNSs users and educational institutions. First, our data show that an excessive usage pattern of SNSs is to blame for the link between stress and low academic performance. Making it simpler to manage and monitor their usage can reduce the stress brought on by SNSs overuse and improve performance. Second, this study has significant implications for organizations that want to increase employee performance by managing their excessive SNSs usage during office hours. Organizations can host seminars to explain why using SNSs excessively is risky, why employees should be careful and avoid using SNSs during office timings, and how to stop its negative consequences. SNS usage can be reduced even slightly to benefit organizations by improving employee performance.</p>", "<p>It is crucial to highlight the need for effective instruction regarding the harmful implications of SNSs usage and adaptation in the workplace and educational institutes as well. Ultimately, this study highlights the importance of a logical point of view, which is a relatively new paradigm of SNSs stress reduction by self-control management. The available empirical findings support the current literature, which suggests that employee should moderate their behavior while using SNS to avoid unpleasant emotions and the possibility that they may negatively impact academic performance.</p>", "<title>6.7 Limitations and future recommendations</title>", "<p>The approach this study used to collect data was online, online lagged survey data do not allow for the assessment of connection since they may be impacted by distracting variables and do not clearly indicate the direction of effect between the examined constructs. Different approach for data collection should be used in future studies to get specific direction and actual relationship between the variables. Furthermore, this study was limited to data collection of universities employees of Malaysia, future studies can target other organizations’ employees as well to confirm the generalizability. Furthermore, the effects of stressors excessive social use, excessive hedonic use, and excessive cognitive use at work, and other factors like information overload, and system feature overload can be observed in other aspects of life, such as the workplace. The “tipping point” at which individuals begin to experience stress using SNSs has to be rigorously investigated in the future.</p>", "<p>Furthermore, while we have utilized gender as well as age as controls in Malaysian context, future studies should examine these control factors in other part of world. Other characteristics that are linked to excessive SNSs usage, such as drinking and smoking, which affect the employees’ academic success can also be used. These elements may aid in identifying additional effects associated with subpar academic achievement. The framework suggested in this study should be expanded in subsequent investigations. The indirect impact of social media exhaustion can also be examined in a later study, which can also include additional variables pertaining to beliefs and actions in linked research domains.</p>", "<title>Data availability statement</title>", "<p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p>", "<title>Ethics statement</title>", "<p>Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.</p>", "<title>Author contributions</title>", "<p>WM: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Writing – original draft. SN: Funding acquisition, Supervision, Writing – review &amp; editing. RS: Funding acquisition, Resources, Supervision, Validation, Writing – review &amp; editing. HA: Formal analysis, Software, Validation, Writing – original draft.</p>" ]
[ "<title>Conflict of interest</title>", "<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>", "<title>Publisher’s note</title>", "<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>FIGURE 1</label><caption><p>Conceptual framework.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>FIGURE 2</label><caption><p>Blindfolding. *Is used in analyzing the moderation effect of ScM between SME and JP.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>FIGURE 3</label><caption><p>Structural model assessment. *Is used in analyzing the moderation effect of ScM between SME and JP.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>TABLE 1</label><caption><p>Demographics.</p></caption><table frame=\"box\" rules=\"all\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Demographic</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Frequency rate</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Percentage</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"3\" style=\"background-color: #dcdcdc;\" rowspan=\"1\">\n<bold>Sex</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">209</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">184</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46%</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" style=\"background-color: #dcdcdc;\" rowspan=\"1\">\n<bold>Age group</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">25–30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">122</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">31–40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.80%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">41–50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">126</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.10%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">51–above</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.10%</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" style=\"background-color: #dcdcdc;\" rowspan=\"1\">\n<bold>Race</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Malay</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">240</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.40%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chinese</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">138</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.30%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Indian</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.30%</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" style=\"background-color: #dcdcdc;\" rowspan=\"1\">\n<bold>Education</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PhD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">295</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75.40%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Masters</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bachelor’s</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.30%</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" style=\"background-color: #dcdcdc;\" rowspan=\"1\">\n<bold>Experience</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Less than 5 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">128</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.7%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Less than 10 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.4%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Less than 15 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">More than 15 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.9%</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>TABLE 2</label><caption><p>Construct reliability and validity.</p></caption><table frame=\"box\" rules=\"all\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Cronbach’s alpha</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">rho_A</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Composite reliability</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Average variance extracted (AVE)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ECUW</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.790</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.796</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.866</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.621</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EHUW</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.811</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.814</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.888</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.726</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ESUW</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.745</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.748</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.831</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.498</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.796</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.798</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.869</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.626</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SMEx</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.864</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.865</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.902</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.647</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>TABLE 3</label><caption><p>Discriminant validity.</p></caption><table frame=\"box\" rules=\"all\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" colspan=\"6\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\">Discriminant validity (HTMT)</td></tr><tr><td valign=\"top\" align=\"left\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">ECUW</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">EHUW</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">ESUW</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">JP</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">SMEx</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ECUW</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EHUW</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.394</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ESUW</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.611</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.684</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.643</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.360</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.640</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SMEx</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.769</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.484</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.766</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.824</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>TABLE 4</label><caption><p>Structural model assessment.</p></caption><table frame=\"box\" rules=\"all\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Original sample (O)</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Sample mean (M)</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Standard deviation (STDEV)</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">T statistics (| O/STDEV|)</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\"><italic>P</italic>-values</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ECUW &gt; SMEx</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.443</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.443</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.052</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.592</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EHUW &gt; SMEx</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.066</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.066</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.049</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.327</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.092</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ESUW &gt; SMEx</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.373</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.374</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.060</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.250</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SME<xref rid=\"t4fns1\" ref-type=\"table-fn\">*</xref>ScM &gt; JP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.124</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.158</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.059</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.117</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.017</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SMEx &gt; JP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.656</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.655</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.033</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.760</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>TABLE 5</label><caption><p><italic>F</italic><sup>2</sup> values effect.</p></caption><table frame=\"box\" rules=\"all\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">\n<italic>F</italic>\n<sup>2</sup>\n</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Weak</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Moderate</td><td valign=\"top\" align=\"center\" style=\"color:#ffffff;background-color: #7f8080;\" rowspan=\"1\" colspan=\"1\">Large</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Propositions</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.005 &gt;</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01 &gt;</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.025 &gt;</td></tr></tbody></table></table-wrap>" ]
[ "<disp-formula id=\"S6.Ex1\">\n<mml:math id=\"M1\" overflow=\"scroll\"><mml:mtable columnalign=\"left\"><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:mpadded width=\"+3.3pt\"><mml:mtext>Moderation Effect Size:</mml:mtext></mml:mpadded><mml:mo>⁢</mml:mo><mml:msup><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:mpadded width=\"+3.3pt\"><mml:mi>  </mml:mi></mml:mpadded><mml:mo rspace=\"5.8pt\">=</mml:mo><mml:mstyle displaystyle=\"true\"><mml:mfrac><mml:mrow><mml:msubsup><mml:mi>R</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>l</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>w</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>h</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup><mml:mo>-</mml:mo><mml:msubsup><mml:mi>R</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>l</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>w</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>h</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>u</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>t</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:msubsup><mml:mi>R</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>l</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>w</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>h</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:mrow></mml:mfrac></mml:mstyle></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:math>\n</disp-formula>", "<disp-formula id=\"S6.Ex2\">\n<mml:math id=\"M2\" overflow=\"scroll\"><mml:mtable columnalign=\"left\"><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:msup><mml:mtext>f</mml:mtext><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">moderation effect size (change in </mml:mi><mml:msup><mml:mi mathvariant=\"normal\">R</mml:mi><mml:mo>2</mml:mo></mml:msup><mml:mi mathvariant=\"normal\"> value of the endogenous</mml:mi></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mspace width=\"2em\"/><mml:mrow><mml:mi mathvariant=\"normal\">construct due to moderator/interaction term).</mml:mi></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:math>\n</disp-formula>", "<disp-formula id=\"S6.Ex3\">\n<mml:math id=\"M3\" overflow=\"scroll\"><mml:mtable columnalign=\"left\"><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:msubsup><mml:mi>R</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>l</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>w</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>h</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mspace width=\"1em\"/><mml:mrow><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">the value of </mml:mi><mml:msup><mml:mi mathvariant=\"normal\">R</mml:mi><mml:mo>2</mml:mo></mml:msup><mml:mi mathvariant=\"normal\"> exogenous construct when the interaction</mml:mi></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mspace width=\"2em\"/><mml:mrow><mml:mi mathvariant=\"normal\">term of the moderator model is included.</mml:mi></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:math>\n</disp-formula>", "<disp-formula id=\"S6.Ex4\">\n<mml:math id=\"M4\" overflow=\"scroll\"><mml:mtable columnalign=\"left\"><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:msubsup><mml:mi>R</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>l</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>w</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5pt\"><mml:mi>h</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>u</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mspace width=\"1em\"/><mml:mrow><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">the value of </mml:mi><mml:msup><mml:mi>R</mml:mi><mml:mo>2</mml:mo></mml:msup><mml:mi mathvariant=\"normal\"> exogenous construct when the interaction</mml:mi></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mspace width=\"2em\"/><mml:mrow><mml:mi mathvariant=\"normal\">term of the moderator model is excluded.</mml:mi></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:math>\n</disp-formula>", "<disp-formula id=\"S6.Ex5\">\n<mml:math id=\"M5\" overflow=\"scroll\"><mml:mtable columnalign=\"left\"><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:msup><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mstyle displaystyle=\"true\"><mml:mfrac><mml:mrow><mml:mn>0.519</mml:mn><mml:mo>-</mml:mo><mml:mn>0.479</mml:mn></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:mn>0.519</mml:mn></mml:mrow></mml:mfrac></mml:mstyle><mml:mo rspace=\"5.8pt\">,</mml:mo><mml:mo rspace=\"5.8pt\">=</mml:mo><mml:mpadded width=\"+3.3pt\"><mml:msup><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:mpadded><mml:mo rspace=\"5.8pt\">=</mml:mo><mml:mstyle displaystyle=\"true\"><mml:mfrac><mml:mn>0.04</mml:mn><mml:mn>0.481</mml:mn></mml:mfrac></mml:mstyle></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:msup><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mn>0.083</mml:mn></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:math>\n</disp-formula>" ]
[]
[]
[ "<disp-quote><p>H1: Excessive social use at work has a significant effect on social media exhaustion.</p></disp-quote>", "<disp-quote><p>H2: Excessive hedonic use has a significant effect on social media exhaustion.</p></disp-quote>", "<disp-quote><p>H3. Excessive cognitive use has a significant effect on social media exhaustion.</p></disp-quote>", "<disp-quote><p>H4: Social media exhaustion has a significant effect on job performance.</p></disp-quote>", "<disp-quote><p>H5: Self-control management moderates the relationship between social media exhaustion and job performance.</p></disp-quote>" ]
[]
[]
[ "<table-wrap-foot><fn id=\"t4fns1\"><p>*Values shows the moderating effect examined.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"fpsyg-14-1254707-g001\" position=\"float\"/>", "<graphic xlink:href=\"fpsyg-14-1254707-g002\" position=\"float\"/>", "<graphic xlink:href=\"fpsyg-14-1254707-g003\" position=\"float\"/>" ]
[]
[{"surname": ["Abbasi", "Johl", "Moughal", "Mazhar", "ZebAbbasi"], "given-names": ["H. A.", "S. K.", "W.", "M.", "H."], "year": ["2022"], "article-title": ["Procurement of electric vehicles to reduce transport emission: an empirical study of consumer motivation towards purchase intention."], "italic": ["J. Hunan Univ. Nat. Sci."], "volume": ["49"], "issue": ["13"], "pub-id": ["10.55463/issn.1674-2974.49.4.13"]}, {"surname": ["Abbasi", "Johl", "Shaari", "Moughal", "Mazhar", "Musarat"], "given-names": ["H. A.", "S. K.", "Z. B. H.", "W.", "M.", "M. A."], "year": ["2021a"], "article-title": ["Consumer motivation by using unified theory of acceptance and use of technology towards electric vehicles."], "italic": ["Sustainability"], "volume": ["13"], "issue": ["12177"], "pub-id": ["10.3390/su132112177"]}, {"surname": ["Abbasi", "Shaari", "Moughal", "Ismail"], "given-names": ["H. A.", "Z. B. H.", "W.", "I. N. B."], "year": ["2021b"], "italic": ["Motivation Toward Plugin Hybrid Electric Vehicles to Reduce Transport Carbon Emission in Malaysia: A Conceptual Paper."], "publisher-loc": ["Cham"], "publisher-name": ["Eurasian Business and Economics Perspectives"], "pub-id": ["10.1007/978-3-030-85304-4_10"]}, {"surname": ["Abbasi", "Shaari", "Moughal"], "given-names": ["H. A.", "Z. H.", "W."], "year": ["2021c"], "italic": ["Consumer Motivation to Enhance Purchase Intention Towards Electric Vehicles in Malaysia. SHS Web of Conferences."], "publisher-name": ["EDP Sciences"], "publisher-loc": ["Malaysia"], "pub-id": ["10.1051/shsconf/202112409003"]}, {"surname": ["Aguinis", "Edwards", "Bradley"], "given-names": ["H.", "J. R.", "K. J."], "year": ["2017"], "article-title": ["Improving our understanding of moderation and mediation in strategic management research."], "italic": ["Org. Res. Methods"], "volume": ["20"], "fpage": ["665"], "lpage": ["685"], "pub-id": ["10.1177/1094428115627498"]}, {"surname": ["Al Hammadi", "Hussain"], "given-names": ["F.", "M."], "year": ["2018"], "article-title": ["Sustainable organizational performance: A study of health-care organizations in the United Arab Emirates."], "italic": ["Int. J. Org. Anal."], "volume": ["27"], "fpage": ["169"], "lpage": ["186"], "pub-id": ["10.1108/IJOA-10-2017-1263"]}, {"surname": ["Ali-Hassan", "Nevo", "Wade"], "given-names": ["H.", "D.", "M."], "year": ["2015"], "article-title": ["Linking dimensions of social media use to job performance: The role of social capital."], "italic": ["J. Strat. Inf. Syst."], "volume": ["24"], "fpage": ["65"], "lpage": ["89"], "pub-id": ["10.1016/j.jsis.2015.03.001"]}, {"surname": ["Ayyagari"], "given-names": ["R."], "year": ["2012"], "article-title": ["Impact of information overload and task-technology fit on technostress"], "italic": ["SAIS 2012 Proceedings"], "publisher-loc": ["Washington, DC"]}, {"surname": ["Bandura"], "given-names": ["A."], "year": ["1986"], "article-title": ["Fearful expectations and avoidant actions as coeffects of perceived self-inefficacy."], "italic": ["Am. Psychol."], "volume": ["41"], "fpage": ["1389"], "lpage": ["1391"], "pub-id": ["10.1037/0003-066X.41.12.1389"]}, {"surname": ["Barrot", "Acomular"], "given-names": ["J. S.", "D. R."], "year": ["2022"], "article-title": ["How university teachers navigate social networking sites in a fully online space: Provisional views from a developing nation."], "italic": ["Int. J. Educ. Technol. Higher Educ."], "volume": ["19"], "issue": ["51"], "pub-id": ["10.1186/s41239-022-00357-3"]}, {"surname": ["Bawden", "Robinson"], "given-names": ["D.", "L."], "year": ["2009"], "article-title": ["The dark side of information: Overload anxiety and other paradoxes and pathologies."], "italic": ["J. Inf. Sci."], "volume": ["35"], "fpage": ["180"], "lpage": ["191"], "pub-id": ["10.1177/0165551508095781"]}, {"surname": ["Benson", "Hand", "Hartshorne"], "given-names": ["V.", "C.", "R."], "year": ["2019"], "article-title": ["How compulsive use of social media affects performance: insights from the UK by purpose of use."], "italic": ["Behav. Inf. Technol."], "volume": ["38"], "fpage": ["549"], "lpage": ["563"], "pub-id": ["10.1080/0144929X.2018.1539518"]}, {"surname": ["Bevan", "Gomez", "Sparks"], "given-names": ["J. L.", "R.", "L."], "year": ["2014"], "article-title": ["Disclosures about important life events on Facebook: Relationships with stress and quality of life."], "italic": ["Comput. Hum. Behav."], "volume": ["39"], "fpage": ["246"], "lpage": ["253"], "pub-id": ["10.1016/j.chb.2014.07.021"]}, {"surname": ["Blackwell", "Leaman", "Tramposch", "Osborne", "Liss"], "given-names": ["D.", "C.", "R.", "C.", "M."], "year": ["2017"], "article-title": ["Extraversion, neuroticism, attachment style and fear of missing out as predictors of social media use and addiction."], "italic": ["Pers. Individ. Dif."], "volume": ["116"], "fpage": ["69"], "lpage": ["72"], "pub-id": ["10.1016/j.paid.2017.04.039"]}, {"surname": ["Boyd", "Ellison"], "given-names": ["D. M.", "N. B."], "year": ["2007"], "article-title": ["Social network sites: Definition, history, and scholarship."], "italic": ["J. Comput.-Med. Commun."], "volume": ["13"], "fpage": ["210"], "lpage": ["230"], "pub-id": ["10.1111/j.1083-6101.2007.00393.x"]}, {"surname": ["Cao", "Khan", "Ali", "Khan"], "given-names": ["X.", "A. N.", "A.", "N. A."], "year": ["2019"], "article-title": ["Consequences of cyberbullying and social overload while using SNSs: A study of users\u2019 discontinuous usage behavior in SNSs."], "italic": ["Inf. Syst. Front."], "volume": ["46"], "fpage": ["83"], "lpage": ["92"], "pub-id": ["10.1007/s10796-019-09936-8"]}, {"surname": ["Cao", "Sun"], "given-names": ["X.", "J."], "year": ["2018"], "article-title": ["Exploring the effect of overload on the discontinuous intention of social media users: An SOR perspective."], "italic": ["Comput. Hum. Behav."], "volume": ["81"], "fpage": ["10"], "lpage": ["18"], "pub-id": ["10.1016/j.chb.2017.11.035"]}, {"surname": ["Cao", "Yu"], "given-names": ["X.", "L."], "year": ["2019"], "article-title": ["Exploring the influence of excessive social media use at work: A three-dimension usage perspective."], "italic": ["Int. J. Inf. Manage."], "volume": ["46"], "fpage": ["83"], "lpage": ["92"], "pub-id": ["10.1016/j.ijinfomgt.2018.11.019"]}, {"surname": ["Carbonell", "Panova"], "given-names": ["X.", "T."], "year": ["2017"], "article-title": ["A critical consideration of social networking sites\u2019 addiction potential."], "italic": ["Add. Res. Theory"], "volume": ["25"], "fpage": ["48"], "lpage": ["57"], "pub-id": ["10.1080/16066359.2016.1197915"]}, {"surname": ["Cheung", "Pires", "Rosenberger", "Leung", "Sharipudin"], "given-names": ["M. L.", "G. D.", "P. J.", "W. K.", "M.-N. S."], "year": ["2021"], "article-title": ["The role of consumer-consumer interaction and consumer-brand interaction in driving consumer-brand engagement and behavioral intentions."], "italic": ["J. Retail. Consum. Serv."], "volume": ["61"], "issue": ["102574"], "pub-id": ["10.1016/j.jretconser.2021.102574"]}, {"surname": ["Cohen"], "given-names": ["J."], "year": ["2013"], "italic": ["Statistical power analysis for the behavioral sciences."], "publisher-loc": ["Hoboken. NJ"], "publisher-name": ["Taylor and Francis"]}, {"surname": ["Darma", "Supriyanto"], "given-names": ["P. S.", "A. S."], "year": ["2017"], "article-title": ["The effect of compensation on satisfaction and employee performance."], "italic": ["Manage. Econ. J."], "volume": ["1"], "issue": ["10"], "pub-id": ["10.18860/mec-j.v1i1.4524"]}, {"surname": ["Davison", "Ou", "Martinsons", "Zhao", "Du"], "given-names": ["R. M.", "C. X.", "M. G.", "A. Y.", "R."], "year": ["2014"], "article-title": ["The communicative ecology of Web 2.0 at work: Social networking in the workspace."], "italic": ["J. Assoc. Inf. Sci. Technol."], "volume": ["65"], "fpage": ["2035"], "lpage": ["2047"], "pub-id": ["10.1002/asi.23112"]}, {"surname": ["Deryakulu", "Ursava\u015f"], "given-names": ["D.", "\u00d6F."], "year": ["2014"], "article-title": ["Genetic and environmental influences on problematic Internet use: A twin study."], "italic": ["Comput. Hum. Behav."], "volume": ["39"], "fpage": ["331"], "lpage": ["338"], "pub-id": ["10.1016/j.chb.2014.07.038"]}, {"surname": ["Dimock"], "given-names": ["M."], "year": ["2019"], "article-title": ["Defining generations: Where Millennials end and Generation Z begins."], "italic": ["Pew Res. Cent."], "volume": ["17"], "fpage": ["1"], "lpage": ["7"]}, {"surname": ["Eliyana", "Ajija", "Sridadi", "Setyawati", "Emur"], "given-names": ["A.", "S. R.", "A. R.", "A.", "A. P."], "year": ["2020"], "article-title": ["Information Overload and Communication Overload on Social Media Exhaustion and Job Performance."], "italic": ["Syst. Rev. Pharmacy"], "volume": ["11"]}, {"surname": ["Ellison", "Vitak", "Gray", "Lampe"], "given-names": ["N. B.", "J.", "R.", "C."], "year": ["2014"], "article-title": ["Cultivating social resources on social network sites: Facebook relationship maintenance behaviors and their role in social capital processes."], "italic": ["J. Comput.-Med. Commun."], "volume": ["19"], "fpage": ["855"], "lpage": ["870"], "pub-id": ["10.1111/jcc4.12078"]}, {"surname": ["Fox", "Moreland"], "given-names": ["J.", "J. J."], "year": ["2015"], "article-title": ["The dark side of social networking sites: An exploration of the relational and psychological stressors associated with Facebook use and affordances."], "italic": ["Comput. Hum. Behav."], "volume": ["45"], "fpage": ["168"], "lpage": ["176"], "pub-id": ["10.1016/j.chb.2014.11.083"]}, {"surname": ["Fu", "Li", "Liu", "Pirkkalainen", "Salo"], "given-names": ["S.", "H.", "Y.", "H.", "M."], "year": ["2020"], "article-title": ["Social media overload, exhaustion, and use discontinuance: Examining the effects of information overload, system feature overload, and social overload."], "italic": ["Inf. Process. Manage."], "volume": ["57"], "issue": ["102307"]}, {"surname": ["Griffiths"], "given-names": ["M."], "year": ["2000"], "article-title": ["Internet addiction-time to be taken seriously?"], "italic": ["Add. Res. Theory"], "volume": ["8"], "fpage": ["413"], "lpage": ["418"], "pub-id": ["10.3109/16066350009005587"]}, {"surname": ["Hair", "Ringle", "Sarstedt"], "given-names": ["J. F.", "C. M.", "M."], "year": ["2011"], "article-title": ["PLS-SEM: Indeed a silver bullet."], "italic": ["J. Market. Theory Pract."], "volume": ["19"], "fpage": ["139"], "lpage": ["152"], "pub-id": ["10.2753/MTP1069-6679190202"]}, {"surname": ["Hair", "Ringle", "Sarstedt"], "given-names": ["J. F.", "C. M.", "M."], "year": ["2013"], "article-title": ["Partial least squares structural equation modeling: Rigorous applications, better results and higher acceptance."], "italic": ["Long Range Plann."], "volume": ["46"], "fpage": ["1"], "lpage": ["12"], "pub-id": ["10.1016/j.lrp.2013.01.001"]}, {"surname": ["Henseler", "Ringle", "Sinkovics"], "given-names": ["J.", "C. M.", "R. R."], "year": ["2009"], "italic": ["The use of partial least squares path modeling in international marketing in new challenges to international marketing."], "publisher-loc": ["Bingley"], "publisher-name": ["Emerald Group Publishing Limited"], "pub-id": ["10.1108/S1474-7979(2009)0000020014"]}, {"surname": ["Hou", "Wang", "Guo", "Gaskin", "Rost", "Wang"], "given-names": ["X.-L.", "H.-Z.", "C.", "J.", "D. H.", "J.-L."], "year": ["2017"], "article-title": ["Psychological resilience can help combat the effect of stress on problematic social networking site usage."], "italic": ["Pers. Ind. Diff."], "volume": ["109"], "fpage": ["61"], "lpage": ["66"], "pub-id": ["10.1016/j.paid.2016.12.048"]}, {"surname": ["Hwang", "Shim", "Park"], "given-names": ["H. S.", "J. W.", "S. B."], "year": ["2019"], "article-title": ["Why we migrate in the virtual world: factors affecting switching intentions in SNS."], "italic": ["Inf. Commun. Soc."], "volume": ["22"], "fpage": ["2127"], "lpage": ["2137"], "pub-id": ["10.1080/1369118X.2018.1477970"]}, {"surname": ["Iqbal", "Baluch", "Abdullah"], "given-names": ["S.", "N.", "C."], "year": ["2017"], "article-title": ["The factors affecting employee\u2019s performance of higher education"], "italic": ["Pak. J. Humanit. Soc. Sci"], "volume": ["5"], "fpage": ["79"], "lpage": ["103"], "pub-id": ["10.52131/pjhss.2017.0502.0024"]}, {"surname": ["Isa", "Kadir Shahar"], "given-names": ["K.", "H."], "year": ["2021"], "article-title": ["Factors associated with stress among academic staff at a Malaysian public university"], "italic": ["AIP Conference Proceedings"], "publisher-loc": ["Washington, DC"], "pub-id": ["10.1063/5.0052834"]}, {"surname": ["Janssen", "Van Yperen"], "given-names": ["O.", "N. W."], "year": ["2004"], "article-title": ["Employees\u2019 goal orientations, the quality of leader-member exchange, and the outcomes of job performance and job satisfaction."], "italic": ["Acad. Manage. J."], "volume": ["47"], "fpage": ["368"], "lpage": ["384"], "pub-id": ["10.2307/20159587"]}, {"surname": ["Javed", "Khan", "Khan", "Shah"], "given-names": ["A.", "S. H.", "M. A. S.", "H. A."], "year": ["2023"], "italic": ["Impact of social media addiction on librarians\u2019 performance: mediating role of task distraction, moderating role of effective self-control."], "publisher-loc": ["Delhi"], "publisher-name": ["Library Hi Tech"], "pub-id": ["10.1108/LHT-01-2022-0078"]}, {"surname": ["Jeon", "Ali", "Lee"], "given-names": ["H.-M.", "F.", "S.-W."], "year": ["2019"], "article-title": ["Determinants of consumers\u2019 intentions to use smartphones apps for flight ticket bookings."], "italic": ["Serv. Ind. J."], "volume": ["39"], "fpage": ["385"], "lpage": ["402"], "pub-id": ["10.1080/02642069.2018.1437908"]}, {"surname": ["Kamal", "Rabbani", "Samdani", "Shujaat", "Ahmad"], "given-names": ["N.", "S.", "H.", "S.", "M."], "year": ["2020"], "article-title": ["Social media usage, overload and exhaustion: A performance perspective."], "italic": ["Int. Rev. Manage. Mark."], "volume": ["10"], "issue": ["19"], "pub-id": ["10.32479/irmm.10190"]}, {"surname": ["Karr-Wisniewski", "Lu"], "given-names": ["P.", "Y."], "year": ["2010"], "article-title": ["When more is too much: Operationalizing technology overload and exploring its impact on knowledge worker productivity."], "italic": ["Comput. Hum. Behav."], "volume": ["26"], "fpage": ["1061"], "lpage": ["1072"], "pub-id": ["10.1016/j.chb.2010.03.008"]}, {"surname": ["Kenny"], "given-names": ["A."], "year": ["2018"], "italic": ["An Illustrated Brief History of Western Philosophy."], "publisher-loc": ["Hoboken, NJ"], "publisher-name": ["John Wiley and Sons"]}, {"surname": ["Kinman", "Jones"], "given-names": ["G.", "F."], "year": ["2003"], "article-title": ["\u2018Running Up the Down Escalator\u2019: Stressors and strains in UK academics."], "italic": ["Qual. High. Educ."], "volume": ["9"], "fpage": ["21"], "lpage": ["38"], "pub-id": ["10.1080/13538320308162"]}, {"surname": ["Koay", "Ong", "Khoo", "Yeoh"], "given-names": ["K. Y.", "D. L. T.", "K. L.", "H. J."], "year": ["2020"], "article-title": ["Perceived social media marketing activities and consumer-based brand equity: Testing a moderated mediation model."], "italic": ["Asia Pac. J. Mark. Log."], "pub-id": ["10.1108/APJML-07-2019-0453"]}, {"surname": ["Koeske", "Koeske"], "given-names": ["G. F.", "R. D."], "year": ["1993"], "article-title": ["A preliminary test of a stress-strain-outcome model for reconceptualizing the burnout phenomenon."], "italic": ["J. Soc. Serv. Res."], "volume": ["17"], "fpage": ["107"], "lpage": ["135"], "pub-id": ["10.1300/J079v17n03_06"]}, {"surname": ["Landers", "Schmidt"], "given-names": ["R. N.", "G. B."], "year": ["2016"], "italic": ["Social media in employee selection and recruitment: An overview. Social media in employee selection and recruitment: Theory, practice, and current challenges."], "publisher-name": ["Springer"], "publisher-loc": ["Cham"], "fpage": ["3"], "lpage": ["11"], "pub-id": ["10.1007/978-3-319-29989-1_1"]}, {"surname": ["Lee", "Son", "Kim"], "given-names": ["A. R.", "S.-M.", "K. K."], "year": ["2016"], "article-title": ["Information and communication technology overload and social networking service fatigue: A stress perspective."], "italic": ["Comput. Hum. Behav."], "volume": ["55"], "fpage": ["51"], "lpage": ["61"], "pub-id": ["10.1016/j.chb.2015.08.011"]}, {"surname": ["Lee", "Lee"], "given-names": ["S. Y.", "S. W."], "year": ["2018"], "article-title": ["The effect of Facebook use on office workers\u2019 job performance and the moderating effects of task equivocality and interdependence."], "italic": ["Behav. Inf. Technol."], "volume": ["37"], "fpage": ["828"], "lpage": ["841"]}, {"surname": ["Leidner", "Koch", "Gonzalez"], "given-names": ["D.", "H.", "E."], "year": ["2010"], "article-title": ["Assimilating Generation Y IT New Hires into USAA\u2019s Workforce: The Role of an Enterprise 2.0 System."], "italic": ["MIS Quart. Execut."], "volume": ["9"], "issue": ["4"]}, {"surname": ["Leung", "Shi", "Chow"], "given-names": ["W. K. S.", "S.", "W. S."], "year": ["2020"], "article-title": ["Impacts of user interactions on trust development in C2C social commerce."], "italic": ["Internet Res."], "volume": ["30"], "fpage": ["335"], "lpage": ["356"], "pub-id": ["10.1108/INTR-09-2018-0413"]}, {"surname": ["Liu", "Kirschner", "Karpinski"], "given-names": ["D.", "P. A.", "A. C."], "year": ["2017"], "article-title": ["A meta-analysis of the relationship of academic performance and Social Network Site use among adolescents and young adults."], "italic": ["Comput. Hum. Behav."], "volume": ["77"], "fpage": ["148"], "lpage": ["157"], "pub-id": ["10.1016/j.chb.2017.08.039"]}, {"surname": ["Luqman", "Cao", "Ali", "Masood", "Yu"], "given-names": ["A.", "X.", "A.", "A.", "L."], "year": ["2017"], "article-title": ["Empirical investigation of Facebook discontinues usage intentions based on SOR paradigm."], "italic": ["Comput. Hum. Behav."], "volume": ["70"], "fpage": ["544"], "lpage": ["555"], "pub-id": ["10.1016/j.chb.2017.01.020"]}, {"surname": ["Majid", "Yasir", "Yousaf", "Qudratullah"], "given-names": ["A.", "M.", "Z.", "H."], "year": ["2019"], "article-title": ["Role of network capability, structural flexibility and management commitment in defining strategic performance in hospitality industry."], "italic": ["Int. J. Contemp. Hosp. Manage."], "volume": ["31"], "fpage": ["3077"], "lpage": ["3096"], "pub-id": ["10.1108/IJCHM-04-2018-0277"]}, {"surname": ["Mezo"], "given-names": ["P. G."], "year": ["2009"], "article-title": ["The self-control and self-management scale (SCMS): Development of an adaptive self-regulatory coping skills instrument."], "italic": ["J. Psychopathol. Behav. Assess."], "volume": ["31"], "fpage": ["83"], "lpage": ["93"], "pub-id": ["10.1007/s10862-008-9104-2"]}, {"surname": ["Milo\u0161evi\u0107-\u00d0or\u0111evi\u0107", "\u017de\u017eelj"], "given-names": ["J. S.", "I. L."], "year": ["2014"], "article-title": ["Psychological predictors of addictive social networking sites use: The case of Serbia."], "italic": ["Comput. Hum. Behav."], "volume": ["32"], "fpage": ["229"], "lpage": ["234"]}, {"surname": ["Moqbel", "Kock"], "given-names": ["M.", "N."], "year": ["2018"], "article-title": ["Unveiling the dark side of social networking sites: Personal and work-related consequences of social networking site addiction."], "italic": ["Inf. Manage."], "volume": ["55"], "fpage": ["109"], "lpage": ["119"], "pub-id": ["10.1016/j.im.2017.05.001"]}, {"surname": ["Moqbel", "Nevo", "Kock"], "given-names": ["M.", "S.", "N."], "year": ["2013"], "article-title": ["Organizational members\u2019 use of social networking sites and job performance: An exploratory study."], "italic": ["Inf. Technol. People"], "volume": ["26"], "fpage": ["240"], "lpage": ["264"]}, {"surname": ["Moughal", "Nordin", "Salleh", "Abbasi"], "given-names": ["W.", "S. B. T. M.", "R. B. T.", "H. A."], "year": ["2023a"], "italic": ["Role of social media to influence the environmental knowledge and awareness toward education for sustainable development in Malaysia."], "publisher-loc": ["Cham"], "publisher-name": ["Eurasian Business and Economics Perspectives"], "pub-id": ["10.1108/ITP-10-2012-0110"]}, {"surname": ["Moughal", "Nordin", "Salleh", "Abbasi"], "given-names": ["W.", "S. B. T. M.", "R. B. T.", "H. A."], "year": ["2023b"], "article-title": ["Social networking sites for success: A UTAUT-based investigation into university employee performance enhancement."], "italic": ["Bus. Manage. Strategy"], "volume": ["14"], "fpage": ["144"], "lpage": ["163"], "pub-id": ["10.5296/bms.v14i2.21369"]}, {"surname": ["Nisar", "Whitehead"], "given-names": ["T. M.", "C."], "year": ["2016"], "article-title": ["Brand interactions and social media: Enhancing user loyalty through social networking sites."], "italic": ["Comput. Hum. Behav."], "volume": ["62"], "fpage": ["743"], "lpage": ["753"], "pub-id": ["10.1016/j.chb.2016.04.042"]}, {"surname": ["Ou", "Davison"], "given-names": ["C. X.", "R. M."], "year": ["2011"], "article-title": ["Interactive or interruptive? Instant messaging at work"], "italic": ["Decis. Support Syst"], "volume": ["52"], "fpage": ["61"], "lpage": ["72"]}, {"surname": ["Papacharissi", "Gibson"], "given-names": ["Z.", "P. L."], "year": ["2011"], "italic": ["Fifteen minutes of privacy: Privacy, sociality, and publicity on social network sites. In Privacy online: Perspectives on privacy and self-disclosure in the social web."], "publisher-loc": ["Berlin"], "publisher-name": ["Springer"], "fpage": ["75"], "lpage": ["89"], "pub-id": ["10.1007/978-3-642-21521-6_7"]}, {"surname": ["Ragu-Nathan", "Tarafdar", "Ragu-Nathan", "Tu"], "given-names": ["T.", "M.", "B. S.", "Q."], "year": ["2008"], "article-title": ["The consequences of technostress for end users in organizations: Conceptual development and empirical validation."], "italic": ["Inf. Syst. Res."], "volume": ["19"], "fpage": ["417"], "lpage": ["433"]}, {"surname": ["Roetzel"], "given-names": ["P. G."], "year": ["2019"], "article-title": ["Information overload in the information age: a review of the literature from business administration, business psychology, and related disciplines with a bibliometric approach and framework development."], "italic": ["Bus. Res."], "volume": ["12"], "fpage": ["479"], "lpage": ["522"]}, {"surname": ["Sekaran"], "given-names": ["U."], "year": ["2003"], "italic": ["Research method for business: A skill building approach."], "publisher-loc": ["New York"], "publisher-name": ["John Wiley & Sons"]}, {"surname": ["Shin", "Hall"], "given-names": ["S. I.", "D."], "year": ["2011"], "article-title": ["Identifying factors affecting SNS users as a temporary or persistent user: an empirical study"], "italic": ["Proceedings of Seventeenth of Americas Conference on Information System"], "publisher-loc": ["Panama City"]}, {"surname": ["Sidek", "Dora", "Kudus", "Hassan"], "given-names": ["S.", "M. T.", "N.", "M. A."], "year": ["2012"], "article-title": ["Academic career in the Malaysian higher education: Becoming a professor."], "italic": ["J. Hum. Cap. Dev."], "volume": ["5"], "fpage": ["127"], "lpage": ["140"]}, {"surname": ["Sullivan", "Koh"], "given-names": ["Y. W.", "C. E."], "year": ["2019"], "article-title": ["Social media enablers and inhibitors: Understanding their relationships in a social networking site context."], "italic": ["Int. J. Inf. Manage."], "volume": ["49"], "fpage": ["170"], "lpage": ["189"]}, {"surname": ["Tang", "Chen", "Yang", "Chung", "Lee"], "given-names": ["J.-H.", "M.-C.", "C.-Y.", "T.-Y.", "Y.-A."], "year": ["2016"], "article-title": ["Personality traits, interpersonal relationships, online social support, and Facebook addiction."], "italic": ["Telem. Inf."], "volume": ["33"], "fpage": ["102"], "lpage": ["108"]}, {"surname": ["Tarafdar", "Maier", "Laumer", "Weitzel"], "given-names": ["M.", "C.", "S.", "T."], "year": ["2020a"], "article-title": ["Explaining the link between technostress and technology addiction for social networking sites: A study of distraction as a coping behavior."], "italic": ["Inf. Syst. J."], "volume": ["30"], "fpage": ["96"], "lpage": ["124"]}, {"surname": ["Tarafdar", "Maier", "Laumer", "Weitzel"], "given-names": ["M.", "C.", "S.", "T."], "year": ["2020b"], "article-title": ["Explaining the link between technostress and technology addiction for social networking sites: A study of distraction as a coping behavior."], "italic": ["Inf. Syst. J."], "volume": ["30"], "fpage": ["96"], "lpage": ["124"]}, {"surname": ["Tytherleigh", "Webb", "Cooper", "Ricketts"], "given-names": ["M. Y.", "C.", "C. L.", "C."], "year": ["2005"], "article-title": ["Occupational stress in UK higher education institutions: A comparative study of all staff categories."], "italic": ["High. Educ. Res. Dev."], "volume": ["24"], "fpage": ["41"], "lpage": ["61"]}, {"surname": ["Vishwanath"], "given-names": ["A."], "year": ["2015"], "article-title": ["Habitual Facebook use and its impact on getting deceived on social media."], "italic": ["J. Comput. Med. Commun."], "volume": ["20"], "fpage": ["83"], "lpage": ["98"]}, {"surname": ["Wilson", "Crowe", "Scott", "Lacey"], "given-names": ["L.", "M.", "A.", "C."], "year": ["2018"], "article-title": ["Self-management for bipolar disorder and the construction of the ethical self."], "volume": ["25"], "issue": ["e12232"]}, {"surname": ["Wu"], "given-names": ["L."], "year": ["2013"], "article-title": ["Social network effects on productivity and job security: Evidence from the adoption of a social networking tool."], "italic": ["Inf. Syst. Res."], "volume": ["24"], "fpage": ["30"], "lpage": ["51"]}, {"surname": ["Yu", "Cao", "Liu", "Wang"], "given-names": ["L.", "X.", "Z.", "J."], "year": ["2018"], "article-title": ["Excessive social media use at work: Exploring the effects of social media overload on job performance."], "italic": ["Inf. Technol. People"], "volume": ["31"], "issue": ["6"]}, {"surname": ["Yu", "Zhong", "Sun", "Qin"], "given-names": ["L.", "Y.", "Y.", "G."], "year": ["2022"], "article-title": ["The impact of excessive social media use at work: a usage experience\u2013stressor\u2013strain perspective."], "italic": ["Behav. Inf. Technol."], "volume": ["2022"], "fpage": ["1"], "lpage": ["20"]}, {"surname": ["Zhan", "Sun", "Wang", "Zhang"], "given-names": ["L.", "Y.", "N.", "X. J. A."], "year": ["2016"], "article-title": ["Understanding the influence of social media on people\u2019s life satisfaction through two competing explanatory mechanisms."], "italic": ["Aslib J. Inf. Manage."], "volume": ["68"], "fpage": ["347"], "lpage": ["361"], "pub-id": ["10.1108/AJIM-12-2015-0195"]}, {"surname": ["Zhang", "Tang", "Yang", "Pei", "Yu"], "given-names": ["Y.", "J.", "Z.", "J.", "P. S."], "year": ["2015"], "article-title": ["Cosnet: Connecting heterogeneous social networks with local and global consistency"], "italic": ["Proceedings of the 21th ACM SIGKDD international conference on knowledge discovery and data mining"], "publisher-loc": ["New York, NY"], "pub-id": ["10.1145/2783258.2783268"]}, {"surname": ["Zheng", "Lee"], "given-names": ["X.", "M. K."], "year": ["2016"], "article-title": ["Excessive use of mobile social networking sites: Negative consequences on individuals."], "italic": ["Comput. Hum. Behav."], "volume": ["65"], "fpage": ["65"], "lpage": ["76"], "pub-id": ["10.1016/j.chb.2016.08.011"]}, {"surname": ["Zwarun", "Hall"], "given-names": ["L.", "A."], "year": ["2014"], "article-title": ["What\u2019s going on? Age, distraction, and multitasking during online survey taking."], "italic": ["Comput. Hum. Behav."], "volume": ["41"], "fpage": ["236"], "lpage": ["244"], "pub-id": ["10.1016/j.chb.2014.09.041"]}]
{ "acronym": [], "definition": [] }
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Front Psychol. 2023 Dec 29; 14:1254707
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PMC10787649
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[ "<title>1. Introduction</title>", "<p>\n<italic>Aedes aegypti</italic> (Linnaeus) and <italic>Ae. albopictus</italic> are vectors of global importance for transmission of arboviruses, such as dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV), while <italic>Ae. vittatus</italic> is reported from few SEAR countries [##UREF##0##1##]. Lack of effective therapy and vaccination against these arboviruses, except for YFV, has increased the arboviral disease burden worldwide. In addition, emergence and re-emergence of these arboviruses has increased the disease prevalence. Among these, dengue has emerged as one of the fast-spreading diseases with approximately 100–400 million DENV infections/year [##UREF##1##2##]. The disease is reportedly endemic in more than 100 countries in the 5 WHO Regions with 3.5 billion people at risk of contracting dengue fever and 1.3 billion people living in dengue-endemic areas in 10 countries of South-East Asia Region (SEAR), except in DPR Korea.</p>", "<p>In the absence of effective medication and vaccines, <italic>Aedes-</italic>borne diseases are presently managed by vector control [##UREF##1##2##]. Of the different mechanical, biological, and chemical methods in use, <italic>Aedes</italic> control is still reliant on the chemical-based interventions [##REF##34487519##3##]. Unfortunately, the continued use of these chemicals over a long period of time has caused evolution of insecticide resistance in different mosquito species, including in <italic>Aedes</italic> spp. [##UREF##2##4##, ##UREF##3##5##] that retard the disease control.</p>", "<p>Use of attractive toxic sugar baits (ATSB) is a relatively new and effective strategy that consists of three components: sugar as a phago-stimulant, a toxin, and an odorant for alluring feeding [##REF##34487519##3##, ##UREF##4##6##, ##UREF##5##7##]. The mosquitoes are attracted towards the bait because of the odour of fruit/flower juice and are killed on ingesting the toxin [##UREF##6##8##]. ATSBs, initially formulated as toxic sugar baits (TSBs) without odorant, were unable to attract mosquitoes due to the absence of an attractant [##UREF##4##6##]. Thus, fermented fruit/flower juices, with potential to lure mosquitoes by the production of attractive volatiles, were added in the formulations as attractants to formulate ATSBs [##REF##20546128##9##, ##REF##20180309##10##]. Use of ATSBs is contemplated as a reliable control method for both male and female mosquitoes as both quest for sugar sources outdoors. Various fruit juices have been assessed for their attractant potential against mosquitoes, such as guava, banana, mango, orange, tomato, watermelon, and papaya juice against <italic>Anopheles arabiensis</italic> [##REF##28810866##11##], mango and guava juice against <italic>Ae. albopictus</italic> [##REF##28504432##12##], guava juice against <italic>An. gambiae</italic> [##UREF##7##13##], and guava, mango, muskmelon, orange, papaya, pineapple, plum, sweet lemon, and watermelon juice against <italic>Ae. aegypti</italic> and <italic>An. stephensi</italic> [##UREF##8##14##, ##REF##36899429##15##].</p>", "<p>Initial studies on the sugar-baiting methods against <italic>Ae. aegypti</italic> conducted in the laboratory, using malathion [##UREF##9##16##] and dinotefuran as toxicants, had effectively reduced the population of <italic>An. gambiae</italic> in Mali, West Africa [##REF##32059671##17##]. The boric acid-containing ATSB has been found effective against <italic>Ae. albopictus</italic>, <italic>Ae. taeniorhynchus</italic>, <italic>Ae. japonicus japonicus</italic>, and <italic>Culex nigripalpus</italic> [##REF##34487519##3##, ##REF##17067052##18##]. Likewise, ATSBs laced with insecticides of different classes, namely, bifenthrin, cyfluthrin, deltamethrin and permethrin (pyrethroids), fipronil (phenylpyrazole), chlorfenapyr (pyrrole), imidacloprid and thiamethoxam (neonicotinoids), and spinosad, ivermectin (macrocyclic lactones), were found effective against <italic>Cx. quinquefasciatus</italic>, <italic>An. quadrimaculatus, Ae. aegypti</italic>, and <italic>Ae. taeniorhynchus</italic> [##REF##21635642##19##, ##UREF##10##20##]. The formulations containing deltamethrin, fipronil, and imidacloprid were found the most effective followed by other pyrethroids, spinosad, and thiamethoxam, while chlorfenapyr and ivermectin containing TSBs registered least efficacy. Among the pyrethroids, deltamethrin and permethrin were reported highly toxic, bifenthrin moderately toxic while cyfluthrin as the least toxic against mosquitoes [##REF##21635642##19##]. The deltamethrin-containing ATSBs have shown good efficacy against <italic>An. stephensi</italic> in laboratory studies [##REF##36899429##15##].</p>", "<p>A comparative assessment of three toxic sugar baits against deltamethrin-resistant <italic>Cx. quinquefasciatus</italic> population showed that the bait containing deltamethrin (0.05%, 0.1%) caused a lower mortality than the bait with boric acid (0.5%, 1.0%) or dinotefuran (0.2%, 0.5%) [##UREF##11##21##]. Cage bioassays with bait containing guava juice with chlorfenapyr 0.5% v/v, boric acid 2% w/v, and tolfenpyrad 1% v/v registered &gt;90% mortality of pyrethroid-resistant <italic>An. arabiensis</italic> and <italic>Cx. quinquefasciatus</italic> [##UREF##7##13##]. The ATSB formulated with guava juice-ASB and 0.2–2% boric acid or 0.05–0.5% chlorfenapyr caused 100% mortality in the adults of susceptible (Kisumu) and resistant (M'bé) strains of <italic>An. gambiae</italic> at the maximum tested concentration [##REF##31910831##22##].</p>", "<p>ATSB formulations can be applied at the target site in a variety of forms, such as in traps, as toxin-incorporated phago-stimulants in bait stations, as baits at the entrance of storm drain system, or by spraying on plants [##UREF##4##6##, ##REF##20546128##9##, ##REF##20180309##10##, ##REF##32059671##17##]. In addition, researchers have also demonstrated that use of ATSBs in wild has shown minimal harmful impact on non-target arthropods including the beneficial ones [##REF##20546128##9##, ##REF##20180309##10##, ##REF##24331613##23##, ##REF##24122115##24##]. This novel vector control tool could be used effectively in attracting and killing a large number of mosquitoes, demonstrating its effectiveness in both indoor and outdoor environments. Despite its initial success, it is imperative to standardize the process to ensure its seamless integration and effectiveness at a larger scale.</p>", "<p>Current study is a step in this direction with nine ATSB formulations containing cane sugar (10% sucrose solution in water as a phago-stimulant), fermented guava juice (attractant), and graded concentration of deltamethrin (toxin). In this study, guava juice-ASB was selected based on our laboratory studies that showed its superior attractancy than the other eight juices prepared from the locally available fruits [##UREF##8##14##]. The objective of the study was to assess the laboratory efficacy of nine ATSB formulations containing different concentrations of deltamethrin against two laboratory strains (AND<italic>-Aedes aegypti</italic> and AND<italic>-Aedes aegypti-</italic>DL10) and two wild-caught laboratory colonized strains (GVD-Delhi) and (SHD-Delhi) of <italic>Aedes aegypti</italic> to identify the most effective ATSB formulation.</p>" ]
[ "<title>2. Materials and Methods</title>", "<title>2.1. Rearing and Maintenance of <italic>Aedes aegypti</italic> Mosquitoes in Insectary</title>", "<p>The cyclic colonies of <italic>Ae. aegypti</italic> mosquito strains were reared and cultured in the insectary in Insect Pest and Vector Control Laboratory at the Acharya Narendra Dev College, University of Delhi, India, since 2009. The rearing conditions are maintained at regulated temperature (27 ± 2°C), relative humidity (80 ± 10%), and photo-period regime (14L:10D). Adults are reared in cotton cloth fabric cages (45 × 40 × 40 cm) and fed on 10% sucrose solution soaked in a cotton swab kept on the cage roof. For egg maturation, female adults are provided with blood meals from an albino mouse. Laid eggs are collected in an ovicup placed in the cage. The eggs are hatched and cultured in plastic trays half-filled with dechlorinated water and provided a mixture of finely ground dog biscuits and yeast 3 : 2 (w/w) for larval nutrition. The pupae are held in the mosquito cage for emergence to adults.</p>", "<title>2.2. Strains of <italic>Aedes aegypti</italic> Employed in Study</title>", "<p>Two laboratory strains and two wild-caught colonized strains were used for the studies (##TAB##0##Table 1##).</p>", "<title>2.3. Laboratory Strains</title>", "<p>Insecticide susceptible strain of <italic>Ae. aegypti</italic> (AND-<italic>Aedes aegypti</italic>): the strain was procured in 2009 from ICGEB (International Centre for Genetic Engineering and Biotechnology), New Delhi, India, and established in the laboratory. Since then, it is being maintained without any insecticide selection pressure.</p>", "<p>Deltamethrin larval-selected strain of <italic>Ae. aegypti</italic> (AND-<italic>Aedes aegypti-</italic>DL10): the early fourth instars of the strain procured from ICGEB in 2009 were subjected to deltamethrin selection pressure at the LC<sub>90</sub> level for 10 successive generations. The deltamethrin susceptibility status of <italic>F</italic><sub>10</sub> was computed, and thereafter, each generation is being selected with the computed LC<sub>90</sub> value of deltamethrin in order to prevent variations in the deltamethrin susceptibility levels. The adults of the strain, however, were susceptible to deltamethrin as they developed only 1.07-fold deltamethrin resistance.</p>", "<title>2.4. Wild-Caught Colonized Strains</title>", "<p>The wild strains have been maintained in the laboratory since May-June 2021. Since then, approximately 42 generations of these wild strains have been passed.<list list-type=\"alpha-lower\"><list-item><p>Govindpuri strain of <italic>Ae. aegypti</italic> (GVD-Delhi): larvae were collected in June, 2021, from the Govindpuri locality of the Southeast Delhi, India, (28.534°N, 77.265°E) and maintained at Acharya Narendra Dev College, India, without insecticide selection pressure.</p></list-item><list-item><p>Shahdara strain of <italic>Ae. aegypti</italic> (SHD-Delhi): larvae were collected in May, 2021, from the Shahdara locality of the East Delhi, India, (28.689°N, 77.290°E) and maintained at Acharya Narendra Dev College, India, without insecticide selection pressure.</p></list-item></list></p>", "<p>The adult susceptibility data to deltamethrin of the four <italic>Aedes aegypti</italic> strains are presented in ##TAB##0##Table 1##. The adult strains were completely susceptible to deltamethrin. The LT<sub>50</sub> values were in the range of 4.43 to 8.78 min and LT<sub>90</sub> values in the range of 8.85 to 15.99 min (##TAB##0##Table 1##). The LT<sub>90</sub> values and resistance ratios in these strains, when compared to susceptible AND-<italic>Aedes aegypti</italic> strain, decreased in the order of <italic>Aedes aegypti</italic> (GVD-Delhi) [1.8] &gt; <italic>Aedes aegypti</italic> (SHD-Delhi) [1.68] &gt; AND-<italic>Aedes aegypti-</italic>DL10 [1.12] &gt; AND-<italic>Aedes aegypti</italic> [1.0] (##TAB##0##Table 1##).</p>", "<title>2.5. Formulation of Attractive Toxic Sugar Baits (ATSBs)</title>", "<p>The guava juice-ASB was prepared by mixing fermented pure juice of guava and 10% sucrose solution (w/v) in 1 : 1 ratio [##UREF##8##14##]. Deltamethrin was serially diluted in ethanol to obtain the concentrations in range of 0.03125 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 4.0 mg/mL, and 8.0 mg/mL. The ATSB solutions were prepared by mixing 9 mL of guava juice-ASB with 1 mL of deltamethrin solution at a particular concentration (9 : 1 ratio), resulting in the nine ATSB formulations containing deltamethrin in the range of 0.003125–0.8 mg/10 mL ATSB.</p>", "<title>2.6. Cage Bioassays with ATSBs</title>", "<p>The bioassay with each of the nine ATSB formulations was conducted in individual cloth cages (45 × 40 × 40 cm). A total of eighteen (nine controls and nine experimental) cotton discs (5 cm diameter, 1.5 cm thickness, 0.5 g) were prepared. The cotton discs were soaked with 5 mL of 10% sucrose solution in water (w/v), and the experimental cotton discs were soaked with 5 mL of nine (9) different deltamethrin concentration ATSB formulations.</p>", "<p>The assay was conducted in cloth cages of the dimensions (45 × 40 × 40 cm). Earlier studies have shown the high attractant and non-toxic properties of guava juice-ASB [##UREF##8##14##], and the studies with ATSBs have shown their toxic effects against mosquitoes feeding upon them (unpublished data). To assess the efficacy of an ATSB in a controlled environment, one control disc and one experimental disc of given deltamethrin-ATSB concentration were placed on the two sides of a cage (##FIG##0##Figure 1(a)##). In each cage, 2-3 days old and unfed fifty <italic>Ae. aegypti</italic> adults, 25 females and 25 males, were released for 24 h (##FIG##0##Figure 1(b)##). After 24 h exposure, a number of mosquitoes, landed on ATSB and either knocked down (unable to fly but alive) or dead, were recorded and analysed. Four (4) replicates were tested for each concentration of ATSB. Thus, for each strain, a total of thirty-six cages were set-up for ATSB bioassays (9 doses × 4 replicates). Concurrently, control assays were held with attractive sugar baits (ASB) containing fermented pure juice of guava and 10% sucrose solution (w/v) in 1 : 1 ratio.</p>", "<title>2.7. Statistical Analysis</title>", "<p>The percent mortality in adults was calculated in each bioassay by using the following formula (equation (##FORMU##0##1##)):</p>", "<p>The control bioassays resulting in &gt;20% mortality were discarded, and experiments with control mortality in the range of 5–20% were corrected using Abbott's formula given in the following equation (##FORMU##0##2##) [##UREF##12##25##]:where T is the percent mortality of <italic>Ae. aegypti</italic> on the guava juice-deltamethrin-ATSB and C is the percent control mortality of <italic>Ae. aegypti</italic>.</p>", "<p>The mortality data were analyzed and interpreted using one-way ANOVA and Tukey's all pairwise multiple comparison test using Predictive Analytics Soft Ware (PASW) 19.0 program.</p>" ]
[ "<title>3. Results</title>", "<p>The cage bioassays were carried out with ATSB prepared with guava juice-ASB and deltamethrin insecticide (9 : 1) using nine doses of deltamethrin in the range of 0.003125–0.8 mg/10 mL ATSB. Each strain showed a dose-dependent mortality response to deltamethrin-ATSBs with respect to the deltamethrin dosage in the ATSB. After 24 hours of assay, the % mortality in AND-<italic>Aedes aegypti</italic> strain on the ATSBs ranged from 8.33% to 97.44%, while the % mortality in AND-<italic>Aedes aegypti</italic>-DL10 adults was in the range of 5.15% to 96.91% (##TAB##1##Table 2##). No mortality was recorded in the mosquitoes fed on ASB.</p>", "<p>The guava juice-ATSB with the lowest dose of deltamethrin (0.003125 mg/10 mL ATSB) resulted in 8.33% and 5.15% adult mortality in the AND-<italic>Aedes aegypti</italic> and AND-<italic>Aedes aegypti</italic>-DL10 strains, respectively (##TAB##1##Table 2##). After 24 h of feeding on 0.0625 mg/10 mL and 0.0125 mg/10 mL deltamethrin-ATSB, the observed mortality in AND-<italic>Aedes aegypti</italic> strain was 14.74% and 19.79%, respectively (<italic>P</italic> &lt; 0.05). Likewise, the ATSB formulations containing higher doses of deltamethrin, 0.025 mg/10 mL, 0.05 mg/10 mL, and 0.1 mg/10 mL ATSB, enhanced the adult mortality to 26.53%, 38.38%, and 49.48% in AND-<italic>Aedes aegypti</italic> adults. Similarly, 24 h provision of 0.2, 0.4, and 0.8 mg deltamethrin/10 mL ATSB increased mortality further by 1.35, 1.19, and 1.21-fold in AND-<italic>Aedes aegypti</italic> adults (##TAB##1##Table 2##). The results showed &gt;80% mortality caused by 0.4 mg deltamethrin-ATSB against AND<italic>-Aedes aegypti</italic> strain.</p>", "<p>In comparison, relatively lower mortality was recorded in the adults of deltamethrin larval-selected AND-<italic>Aedes aegypti</italic>-DL10 strain (##TAB##1##Table 2##). Using 0.0625 mg/10 mL and 0.0125 mg/10 mL deltamethrin-ATSB as baits caused 10.53% and 17.71% adult deaths (<italic>P</italic> &lt; 0.05) which enhanced to 23.71%, 34.02%, and 44.21% on providing 0.025, 0.05 and 0.1 mg deltamethrin/10 mL ATSB baits (<italic>P</italic> &lt; 0.05), respectively (##TAB##1##Table 2##). Similar trend was noticed with 0.2, 0.4, and 0.8 mg/10 mL deltamethrin-ATSB resulting in further increased mortality (1.37, 1.29, and 1.23-fold) in AND-<italic>Aedes aegypti</italic>-DL10 strain, respectively (##TAB##1##Table 2##, ##FIG##1##Figure 2##), with &gt;80% mortality obtained with 0.8 mg deltamethrin/10 mL ATSB.</p>", "<p>The attract and kill potential of nine ATSBs containing 0.003125 to 0.8 mg deltamethrin/10 mL ATSB was also investigated against two wild-caught strains of <italic>Ae. aegypti,</italic> the <italic>Aedes aegypti</italic> (GVD-Delhi), and <italic>Aedes aegypti</italic> (SHD-Delhi) strains which were colonized in the laboratory (##TAB##2##Table 3##). During 24 h exposure, the ATSB formulations induced 2.04% to 95.83% mortality in GVD-Delhi strain, while comparatively higher mortality of 5.10% to 97.96% was observed in the SHD-Delhi strain of <italic>Ae. aegypti</italic>. The lowest adult mortality rates of 2.04% and 5.10% in <italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi), respectively, were observed with ATSB containing the lowest dose of deltamethrin (0.003125 mg/10 mL ATSB). The increase in the concentrations of deltamethrin in the ATSB formulations increased the adult mortality in both the strains indicating a dose-mortality response correlation.</p>", "<p>The 24 h ATSB exposure with 0.0625 mg and 0.0125 mg deltamethrin/10 mL ATSB resulted in 5.21% and 10.47% (<italic>P</italic> &lt; 0.05) adult mortality in GVD-Delhi strain, whereas relatively higher mortality of 9.28% and 13.40% (<italic>P</italic> &lt; 0.05) was obtained in SHD-Delhi strain (##TAB##2##Table 3##). When exposed to the ATSB formulations with higher doses of deltamethrin (0.025, 0.05, and 0.1 mg/10 mL ATSB), the mortality in <italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi) increased to 22.45–40.21% (<italic>P</italic> &lt; 0.05) and 23.47–39.58% (<italic>P</italic> &gt; 0.05), respectively (##TAB##2##Table 3##). Mortality increased further in the adults of GVD-Delhi and SHD-Delhi strains by 1.28 and 1.21-folds when exposed to the 0.4 mg deltamethrin/10 mL ATSB with respect to the 0.2 mg deltamethrin/10 mL ATSB; and by 1.36 and 1.41-folds with 0.8 mg deltamethrin/10 mL ATSB in comparison to 0.4 mg deltamethrin/10 mL ATSB, respectively (##TAB##2##Table 3##, ##FIG##1##Figure 2##). Both the strains registered &gt;80% mortality with 0.8 mg deltamethrin/10 mL ATSB.</p>", "<p>The dose mortality response lines obtained on providing nine deltamethrin-ATSBs to AND-<italic>Aedes aegypti</italic> and AND-<italic>Aedes aegypti</italic>-DL10 showed <italic>R</italic><sup>2</sup> values of 0.7983 and 0.8012, respectively (Figures ##FIG##2##3(a)## and ##FIG##2##3(b)##) while <italic>R</italic><sup>2</sup> values obtained with <italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi) strains were 0.8488 and 0.9358 (Figures ##FIG##2##3(c)## and ##FIG##2##3(d)##).</p>" ]
[ "<title>4. Discussion</title>", "<p>The ATSB is a mixture of three components; fruit juice, a toxin, and sugar solution; to attract for feeding and kill the adults by toxin feed. It is based on the fact that mosquitoes require a sugar diet throughout their life for energy, growth, development, mating, and egg production [##UREF##6##8##, ##UREF##13##26##]. Since mosquitoes search for sugar sources in the environment, the ATSB with table sugar competes with the available sources of plant sugar and provides nourishment for survival [##UREF##4##6##].</p>", "<p>The first toxic sugar bait (TSB) was developed against <italic>Ae. aegypti</italic> using malathion and 20% sucrose solution combination [##UREF##9##16##]. Malathion was added to sucrose in different concentrations (1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, and 0.1 mg/mL) resulting in up to 85.2% adult mortality. However, TSBs, though effective in laboratory evaluation, could not register comparable mortalities in the field due to the presence of the competing environment's natural sugar sources and attractants. Consequently, addition of odour attractants, in the form of fruit juices, flower nectar, or bug honeydew, resulted in the formulation of ATSBs [##REF##20546128##9##, ##REF##20180309##10##, ##REF##21366782##27##]. The laboratory or field trials with different ATSBs have showed varied efficacy which may be not only because of the toxin used but also due to the attractant used, type and prevalence of mosquito species, level of resistance in the mosquitoes to the toxin, and ecological factors. Thereafter, several toxins have been used in the ATSB formulations such as deltamethrin, boric acid, dinotefuran and spinosad [##UREF##4##6##, ##UREF##8##14##, ##REF##36899429##15##, ##REF##32059671##17##, ##UREF##11##21##], fipronil [##REF##31910831##22##, ##REF##18939695##28##, ##REF##28095875##29##], chlorfenapyr and tolfenpyrad [##UREF##7##13##], eugenol [##REF##24361724##30##], ivermectin [##REF##28810866##11##], sodium ascorbate [##REF##31134270##31##], and microencapsulated garlic oil in beta-cyclodextrin [##UREF##5##7##, ##REF##26403337##32##–##REF##28206859##34##].</p>", "<p>Earlier studies with nine ASBs prepared by combining nine different fermented pure fruits juices with 10% sucrose solution in water in 1 : 1 ratio revealed guava juice-ASB as the most effective attractant for <italic>Ae. aegypti</italic> [##UREF##8##14##]. The present study is to validate these laboratory results on wild-caught laboratory colonized <italic>Ae. aegypti</italic> strains with nine ATSBs formulated by adding 9 parts of guava juice-ASB to 1 part of various dosages of a contact pyrethroid insecticide, deltamethrin, in the range of 0.003125 to 0.8 mg/10 mL ATSB. These ATSBs were evaluated for their toxic potential against the two laboratory strains (AND<italic>-Aedes aegypti</italic> and AND<italic>-Aedes aegypti-</italic>DL10) and two wild-caught laboratory colonized strains (<italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi)) of <italic>Ae. aegypti.</italic> The studies revealed a dose-dependent mortality response in adult <italic>Ae. aegypti</italic> of ATSBs after 24 h exposure. The recorded mortality in AND-<italic>Aedes aegypti</italic> and AND-<italic>Aedes aegypti</italic>-DL10 ranged from 8.33 to 97.44% and 5.15–96.91%, respectively, whereas these formulations induced relatively less mortality in the laboratory colonized wild-caught deltamethrin susceptible GVD-Delhi strain (2.04–95.83%) and SHD-Delhi strain (5.10–97.96%). The formulations with 0.4 mg deltamethrin/10 mL ATSB caused &gt;80% mortality in the adults of AND-<italic>Aedes aegypti,</italic> while rest of the three strains registered &gt;80% mortality in the adults with 0.8 mg deltamethrin/10 mL ATSB.</p>", "<p>The observed mortality in cage bioassays was found to be correlated with the LT<sub>50</sub> values of deltamethrin obtained against these strains. Increased adult mortality was observed in the AND-<italic>Aedes aegypti</italic> strain (LT<sub>50</sub> = 4.431 min) followed by AND-<italic>Aedes aegypti</italic>-DL10 (LT<sub>50</sub> = 4.766 min) and <italic>Aedes aegypti</italic> (SHD-Delhi) (LT<sub>50</sub> = 8.382 min), while <italic>Aedes aegypti</italic> (GVD-Delhi) (LT<sub>50</sub> = 8.787 min) showed lowest mortality relative to the other strains. These results are encouraging and need to be validated with the field studies as to date, most of the research in the field of ATSB has been carried out with oral toxins: dinotefuran, spinosad, chlorfenapyr, and boric acid. Contact insecticides-TSBs, though investigated, have been in limited focus.</p>", "<p>A few studies have assessed ATSBs formulated using different pyrethroids such as deltamethrin, permethrin, cyfluthrin, and bifenthrin and found them effective against different species of mosquitoes, <italic>Cx. quinquefasciatus, An. quadrimaculatus, An. stephensi, Ae. aegypti,</italic> and <italic>Ae. taeniorhynchus</italic> [##REF##36899429##15##, ##REF##21635642##19##–##UREF##11##21##]. It was reported that formulations were generally more effective against pyrethroid-susceptible populations than the pyrethroid-resistant populations [##UREF##11##21##]. Thus, ATSBs containing insecticides with modes of action different from that of pyrethroids were formulated and found effective against pyrethroid-resistant mosquitoes [##UREF##7##13##]. It has been thus recommended that use of insecticides with an alternate mode of action to the existing insecticide in use should be preferred as a strategy for effective vector management [##UREF##0##1##].</p>", "<p>Current laboratory investigations revealed the efficacy of deltamethrin as a toxin component in the ATSB to control <italic>Ae. aegypti</italic> population. The dosage of 0.8 mg deltamethrin in 10 mL ATSB was found to be highly effective resulting in 96% to 98% mortality in adult mosquitoes. However, reports have suggested the reduced efficacy of ATSBs in the fields in comparison to the laboratory assays probably due to the development of resistant strains [##UREF##7##13##, ##REF##23388730##35##]. Moreover, it can be due to the availability of natural sugar sources in the natural environment which compete with bait stations.</p>", "<p>Based on the encouraging results from this study, it is pertinent to assess the efficacy of the developed deltamethrin-ATSB formulation for use in the field against wild mosquitoes. Further, supplementary studies are recommended on the impact of ATSBs on the environment and non-target organisms, which would help in ascertaining the safe use of ATSBs.</p>" ]
[ "<title>5. Conclusions</title>", "<p>The study was conducted using nine ATSB formulations with fermented guava juice (100%), sucrose solution (10% w/v), and nine doses of pyrethroid deltamethrin (0.003125–0.8 mg/10 mL ATSB) to assess their toxic potential against two laboratory strains and two wild-caught colonized strains of <italic>Ae. aegypti.</italic> The studies revealed a deltamethrin dose-dependent impact of ATSBs on the mortality in adult mosquitoes. The recorded mortality in laboratory strains, AND-<italic>Aedes aegypti</italic> and adults derived from AND-<italic>Aedes aegypti-</italic>DL10 strain selected with deltamethrin at the larval stage for 10 generations, ranged from 8.33–97.44% to 5.15–96.91%, respectively, whereas these formulations induced 2.04–95.83% and 5.10–97.96% mortality in laboratory colonized wild-caught GVD-Delhi and SHD-Delhi strains of <italic>Ae. aegypti</italic>, respectively. The investigations indicating a positive correlation between the % mortality in the adults and the deltamethrin susceptibility demonstrated the efficacy of these ATSB formulations against <italic>Ae. aegypti</italic> with deltamethrin. This study highlighted the need to conduct structured field trials and investigation of the impact on non-target organisms.</p>" ]
[ "<p>Academic Editor: Rajib Chowdhury</p>", "<title>Background</title>", "<p> The attractive toxic sugar bait (ATSB) is a promising strategy for controlling mosquitoes at the adult stage. The strategy is based on the use of a combination of fruit juice, sugar, and a toxin in order to attract and kill the adult mosquitoes. The selection of the components and optimization of their concentrations is significant for the formulation of an effective ATSB. </p>", "<title>Methods</title>", "<p> The present study formulated nine ATSBs and evaluated their efficacy against two laboratory strains (AND-<italic>Aedes aegypti</italic> and AND-<italic>Aedes aegypti-</italic>DL10) and two wildcaught colonized strains of <italic>Aedes aegypti</italic> (GVD-Delhi and SHD-Delhi). Initially, nine attractive sugar baits (ASBs) were prepared using a mixture of 100% fermented guava juice (attractant) with 10% sucrose solution (w/v) in 1 : 1 ratio. ATSBs were formulated by mixing each ASB with different concentrations of deltamethrin in the ratio of 9 : 1 to obtain final deltamethrin concentration of 0.003125–0.8 mg/10 mL ATSB. Cage bioassays were conducted with 50 mosquitoes for 24 h in order to evaluate the efficacy of each ATSB against the four strains of <italic>Ae. aegypti</italic>. The data were statistically analyzed using PASW software 19.0 program and 2-way ANOVA. </p>", "<title>Results</title>", "<p> The ATSB formulations registered 8.33–97.44% mortality against AND-<italic>Aedes aegypti</italic> and 5.15–96.91% mortality against AND-<italic>Aedes aegypti</italic>-DL10 strains of <italic>Ae. aegypti</italic>, while GVD-Delhi strain registered 2.04–95.83% mortality and SHD-Delhi strain showed 5.10–97.96% mortality. The administration of 0.8 mg of deltamethrin within 10 mL of attractive toxic sugar bait (ATSB) has led to the maximum mortality rate in adult mosquitoes. </p>", "<title>Conclusions</title>", "<p> The ATSBs formulated with guava juice-ASB and deltamethrin (9 : 1) showed toxin dose-dependent toxicity by all the four strains of <italic>Ae. aegypti.</italic> Most effective dosage was found as 0.8 mg deltamethrin/10 mL ATSB which imparted 96% to 98% mortality in adult mosquitoes. The investigations demonstrated the efficacy of deltamethrin-laced ATSB formulations against <italic>Ae. aegypti</italic> and highlighted the need for conduct of structured field trials and investigating the impact on disease vectors and nontarget organisms.</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>The authors thank Director, ICMR-NIMR, Delhi, India, for continuous support for the conduction of research. They sincerely thank the principal, Acharya Narendra Dev College, University of Delhi, for providing facilities and infrastructure. This work was supported by the Indian Council of Medical Research-MERA India (Grant number: MERA/3/2020-ECD-II).</p>", "<title>Data Availability</title>", "<p>All data generated or analyzed during this study are included in the article.</p>", "<title>Ethical Approval</title>", "<p>The study does not involve humans.</p>", "<title>Disclosure</title>", "<p>Kamaraju Raghavendra's present address is H. No. 28 B, Block ED, Pitampura, Delhi-110 088, India.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no conflicts of interest.</p>", "<title>Authors' Contributions</title>", "<p>AS and RRS conducted the experiments and wrote the manuscript. AS, VV, and RRS designed the experiments supervised by KR, SK, and SPS. The statistical analysis of the results was done by AS, RRS, and RKS. All authors were involved in the preparation of the manuscript. The manuscript was reviewed and agreed by all.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Cage bioassay with <italic>Aedes aegypti</italic> adults: (a) screening cage setup with 50 unfed adult mosquitoes (25 males and 25 females) and (b) screening cage setup with ATSB (guava juice-ASB + deltamethrin) and control (10% sucrose solution) bait placed at two sides.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>Percent mortality in the adults of laboratory strains (AND-<italic>Aedes aegypti</italic> and AND-<italic>Aedes aegypti-</italic>DL10) and wild-caught laboratory colonized strains (<italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi)) of <italic>Aedes aegypti</italic> exposed to guava juice-deltamethrin-ATSB for 24 h in cage bioassay.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>Dosage-mortality regression lines on providing deltamethrin-ATSBs to the laboratory strains (AND<italic>-Aedes aegypti</italic> and AND-<italic>Aedes aegypti</italic>-DL10) and wild-caught colonized strains (<italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi)) of <italic>Aedes aegypti</italic>.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Deltamethrin susceptibility status of the four strains of <italic>Aedes aegypti</italic>: laboratory strains (AND-<italic>Aedes aegypti</italic> and AND-<italic>Aedes aegypti</italic>-DL10) and wild-caught colonized strains (GVD-Delhi and SHD-Delhi) of <italic>Aedes aegypti</italic> after 24 h exposure to 0.05% deltamethrin-impregnated papers.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Strains</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">LT<sub>50</sub> (min.)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">RR LT<sub>50</sub> (min.)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">LT<sub>90</sub> (min.)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">RR LT<sub>90</sub> (min.)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">% Mortality after 24 h</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">AND-<italic>Aedes aegypti</italic></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.85</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">AND-<italic>Aedes aegypti-</italic>DL10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.07 (S)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.98</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.12 (S)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Aedes aegypti</italic> (GVD-Delhi)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.98 (S)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.99</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.80 (S)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Aedes aegypti</italic> (SHD-Delhi)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.89 (S)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.91</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.68 (S)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Number of adults of laboratory strain (AND<italic>-Aedes aegypti</italic> and AND<italic>-Aedes aegypti-</italic>DL10) of <italic>Aedes aegypti</italic> attracted and killed in ATSB cage bioassays.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">ATSB (guava juice-ASB + mg deltamethrin/10 mL)</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">No. of dead adults ± SE (mortality)</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">AND<italic>-Aedes aegypti</italic><sup><italic>∗</italic></sup></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">AND<italic>-Aedes aegypti-</italic>DL10<sup><italic>∗</italic></sup></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Control (ASB)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.003125</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.16 ± 1.00<sup>a</sup> (8.33%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.57 ± 1.50<sup>a</sup> (5.15%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.00625</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.36 ± 1.00<sup>b</sup> (14.74%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.26 ± 1.00<sup>a</sup> (10.53%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.0125</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.89 ± 1.50<sup>b</sup> (19.79%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.85 ± 0.50<sup>b</sup> (17.71%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.025</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.26 ± 1.00<sup>c</sup> (26.53%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.85 ± 0.50<sup>c</sup> (23.71%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.19 ± 1.00<sup>d</sup> (38.38%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.01 ± 0.50<sup>d</sup> (34.02%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.74 ± 2.00<sup>e</sup> (49.48%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.10 ± 3.00<sup>e</sup> (44.21%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33.50 ± 1.50<sup>f</sup> (67.02%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30.36 ± 1.50<sup>f</sup> (60.73%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40.20 ± 2.00<sup>g</sup> (80.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39.17 ± 1.00<sup>g</sup> (78.35%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.71 ± 0.00<sup>h</sup> (97.44%)<sup><italic>∗∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.45 ± 0.00<sup>h</sup> (96.91%)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Number of adults of wild-caught colonized population of <italic>Aedes aegypti</italic> (GVD-Delhi) and <italic>Aedes aegypti</italic> (SHD-Delhi) strains of <italic>Aedes aegypti</italic> attracted and killed towards ATSB formulation during ATSB cage bioassays.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">ATSB (guava juice-ASB + mg deltamethrin/10 mL)</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">No. of dead adults ± SE (mortality)</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Aedes aegypti</italic> (GVD-Delhi)<sup><italic>∗</italic></sup></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Aedes aegypti</italic> (SHD-Delhi)<sup><italic>∗</italic></sup></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Control (ASB)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.003125</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.02 ± 0.00<sup>a</sup> (2.04%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.55 ± 0.50<sup>a</sup> (5.10%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.00625</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.60 ± 0.50<sup>a</sup> (5.21%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.63 ± 0.50<sup>b</sup> (9.28%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.0125</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.23 ± 0.50<sup>b</sup> (10.47%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.70 ± 1.50<sup>b</sup> (13.40%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.025</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.22 ± 1.00<sup>c</sup> (22.45%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.73 ± 1.50<sup>c</sup> (23.47%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.46 ± 1.00<sup>d</sup> (30.93%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.01 ± 0.50<sup>d</sup> (34.02%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.10 ± 1.50<sup>e</sup> (40.21%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.79 ± 1.00<sup>d</sup> (39.58%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.36 ± 1.00<sup>f</sup> (54.74%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.57 ± 1.00<sup>e</sup> (57.14%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.05 ± 1.00<sup>g</sup> (70.10%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34.69 ± 1.00<sup>f</sup> (69.39%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">47.91 ± 0.00<sup>h</sup> (95.83%)<sup><italic>∗∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.97 ± 0.00<sup>g</sup> (97.96%)</td></tr></tbody></table></table-wrap>" ]
[ "<disp-formula id=\"EEq1\"><label>(1)</label><mml:math id=\"M1\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>percent mortality </mml:mtext><mml:mrow><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mo>%</mml:mo></mml:mrow></mml:mfenced></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>total number of dead adults </mml:mtext></mml:mrow><mml:mrow><mml:mtext>total number of exposed adults</mml:mtext></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq2\"><label>(2)</label><mml:math id=\"M2\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mi mathvariant=\"normal\">p</mml:mi><mml:mtext>ercent test mortality </mml:mtext><mml:mrow><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mo>%</mml:mo></mml:mrow></mml:mfenced></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">T</mml:mi><mml:mo>−</mml:mo><mml:mi mathvariant=\"normal\">C</mml:mi><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow><mml:mrow><mml:mn>100</mml:mn><mml:mtext> </mml:mtext><mml:mo>−</mml:mo><mml:mtext> </mml:mtext><mml:mi mathvariant=\"normal\">C</mml:mi></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>" ]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><fn><p>LT<sub>50 or 90</sub>: lethal time for killing 50% or 90% mosquitoes exposed; RR: resistance ratio; S: susceptible, based on the WHO protocol (WHO, 2022).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>\n<sup>\n<italic>∗</italic>\n</sup>Four replicates each with <italic>n</italic> = 50, 25 males and 25 females (24 h), total <italic>n</italic> = 200. <sup><italic>∗∗</italic></sup>Corrected percent mortality; values in the table represent the number of mosquitoes dead; ATSBs with different letters (column-wise) are significantly different (<italic>P</italic> &lt; 0.05) computed by one-way ANOVA followed by Tukey's all pair wise multiple comparison test.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>\n<sup>\n<italic>∗</italic>\n</sup>Four replicates each with <italic>n</italic> = 50, 25 males and 25 females (24 h), total <italic>n</italic> = 200. <sup><italic>∗∗</italic></sup>Corrected percent mortality; values in the table represent the number of mosquitoes dead; ATSBs with different letters (column-wise) are significantly different (<italic>P</italic> &lt; 0.05) computed by one-way ANOVA followed by Tukey's all pair wise multiple comparison test.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"JTM2024-6966205.001\" position=\"float\"/>", "<graphic xlink:href=\"JTM2024-6966205.002\" position=\"float\"/>", "<graphic xlink:href=\"JTM2024-6966205.003\" position=\"float\"/>" ]
[]
[{"label": ["1"], "collab": ["WHO (World Health Organization)"], "article-title": ["Report on insecticide resistance in "], "italic": ["Aedes", "Aedes aegypti", "Ae. albopictus, Ae. vittatus"], "year": ["2022"], "comment": ["\n"], "ext-link": ["https://www.who.int/publications/i/item/sea-cd-334"]}, {"label": ["2"], "collab": ["WHO (World Health Organization)"], "article-title": ["Dengue and severe dengue"], "year": ["2023"], "comment": ["\n"], "ext-link": ["https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue"]}, {"label": ["4"], "person-group": ["\n"], "surname": ["Rivero", "V\u00e9zilier", "Weill", "Read", "Gandon"], "given-names": ["A.", "J.", "M.", "A. F.", "S."], "article-title": ["Insecticide control of vector-borne diseases: when is insecticide resistance a problem?"], "source": ["\n"], "italic": ["PLoS Pathogens"], "year": ["2010"], "volume": ["6"], "issue": ["8"], "pub-id": ["e1001000", "10.1371/journal.ppat.1001000", "2-s2.0-77958147136"]}, {"label": ["5"], "person-group": ["\n"], "surname": ["Hemingway", "Ranson", "Magill"], "given-names": ["J.", "H.", "A."], "article-title": ["Averting a malaria disaster: will insecticide resistance derail malaria control?"], "source": ["\n"], "italic": ["The Lancet"], "year": ["2016"], "volume": ["387"], "issue": ["10029"], "fpage": ["1785"], "lpage": ["1788"], "pub-id": ["10.1016/s0140-6736(15)00417-1", "2-s2.0-84957682262"]}, {"label": ["6"], "person-group": ["\n"], "surname": ["Fiorenzano", "Koehler", "Xue"], "given-names": ["J. M.", "P. G.", "R. D."], "article-title": ["Attractive toxic sugar bait (ATSB) for control of mosquitoes and its impact on non-target organisms: a review"], "source": ["\n"], "italic": ["International Journal of Environmental Research and Public Health"], "year": ["2017"], "volume": ["14"], "issue": ["4"], "fpage": ["p. 398"], "pub-id": ["10.3390/ijerph14040398", "2-s2.0-85017429040"]}, {"label": ["7"], "person-group": ["\n"], "surname": ["Sissoko", "Junnila", "Traore"], "given-names": ["F.", "A.", "M. M."], "article-title": ["Frequent sugar feeding behavior by "], "italic": ["Aedes aegypti", "PLoS One"], "source": ["\n"], "year": ["2019"], "volume": ["14"], "issue": ["6"], "pub-id": ["e0214170", "10.1371/journal.pone.0214170", "2-s2.0-85067315696"]}, {"label": ["8"], "person-group": ["\n"], "surname": ["Foster"], "given-names": ["W. A."], "article-title": ["Mosquito sugar feeding and reproductive energetics"], "source": ["\n"], "italic": ["Annual Review of Entomology"], "year": ["1995"], "volume": ["40"], "issue": ["1"], "fpage": ["443"], "lpage": ["474"], "pub-id": ["10.1146/annurev.en.40.010195.002303", "2-s2.0-0029106658"]}, {"label": ["13"], "person-group": ["\n"], "surname": ["Stewart", "Oxborough", "Tungu", "Kirby", "Rowland", "Irish"], "given-names": ["Z. P.", "R. M.", "P. K.", "M. J.", "M. W.", "S. R."], "article-title": ["Indoor application of attractive toxic sugar bait (ATSB) in combination with mosquito nets for control of pyrethroid-resistant mosquitoes"], "source": ["\n"], "italic": ["PLoS One"], "year": ["2013"], "volume": ["8"], "issue": ["12"], "pub-id": ["e84168", "10.1371/journal.pone.0084168", "2-s2.0-84893170847"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Kumar", "Sharma", "Samal"], "given-names": ["S.", "A.", "R. R."], "article-title": ["Attractive sugar bait formulation for development of attractive toxic sugar bait for control of "], "italic": ["Aedes aegypti", "Journal of Tropical Medicine"], "source": ["\n"], "year": ["2022"], "volume": ["2022"], "fpage": ["10"], "pub-id": ["2977454", "10.1155/2022/2977454"]}, {"label": ["16"], "person-group": ["\n"], "surname": ["Lea"], "given-names": ["A. O."], "article-title": ["Sugar-baited insecticide residues against mosquitoes"], "source": ["\n"], "italic": ["Mosquito News"], "year": ["1965"], "volume": ["25"], "fpage": ["65"], "lpage": ["66"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Tenywa", "Musa", "Musiba"], "given-names": ["F. S.", "J. J.", "R. M."], "article-title": ["Evaluation of an ivermectin-based attractive targeted sugar bait (ATSB) against "], "italic": ["Aedes aegypti", "Wellcome Open Research"], "source": ["\n"], "year": ["2022"], "volume": ["7"], "fpage": ["p. 4"], "pub-id": ["10.12688/wellcomeopenres.17442.1"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Gu", "He", "Teng"], "given-names": ["Z. Y.", "J.", "X. D."], "article-title": ["Efficacy of orally toxic sugar baits against contact-insecticide resistant "], "italic": ["Culex quinquefasciatus", "Acta Tropica"], "source": ["\n"], "year": ["2020"], "volume": ["202"], "pub-id": ["105256", "10.1016/j.actatropica.2019.105256"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Abbott"], "given-names": ["W. S."], "article-title": ["A method of computing the effectiveness of an insecticide"], "source": ["\n"], "italic": ["Journal of Economic Entomology"], "year": ["1925"], "volume": ["18"], "issue": ["2"], "fpage": ["265"], "lpage": ["267"], "pub-id": ["10.1093/jee/18.2.265a"]}, {"label": ["26"], "person-group": ["\n"], "surname": ["Clements"], "given-names": ["A. N."], "article-title": ["The biology of mosquitoes"], "source": ["\n"], "italic": ["Sensory Reception and Behaviour"], "year": ["1999"], "publisher-loc": ["Oxford, England"], "publisher-name": ["CABI publishing"]}]
{ "acronym": [ "AND:", "ASB:", "ATSB:", "GVD:", "SHD:", "LC:", "NIMR:", "TSBs:", "WHO:" ], "definition": [ "Acharya Narendra Dev", "Attractive Sugar Bait", "Attractive Toxic Sugar Bait", "Govindpuri", "Shahdara", "Lethal Concentration", "National Institute of Malaria Research", "Toxic Sugar Baits", "World Health Organization" ] }
35
CC BY
no
2024-01-14 23:43:50
J Trop Med. 2024 Jan 6; 2024:6966205
oa_package/e6/b1/PMC10787649.tar.gz
PMC10787650
0
[ "<title>1. Introduction</title>", "<p>The use of irrigating solutions during endodontic procedures aims to promote debridement, cleaning, and disinfection of the root canal system [##REF##24651335##1##]. No substance has all the ideal characteristics, therefore, the combined use of solutions in irrigation protocols has been suggested [##REF##24651335##1##]. For example, the final irrigation using alternate applications of ethylenediaminetetraacetic acid (EDTA), a calcium-chelating agent, and sodium hypochlorite solution (NaOCl), an organic tissue solvent, was recommended after instrumentation to remove the smear layer from root canal walls [##REF##20002799##2##]. Another protocol suggested the use of chlorhexidine gluconate (CHX) as the last irrigating solution to complement and maintain disinfection [##REF##29884999##3##], due to its substantivity [##REF##27129588##4##].</p>", "<p>However, when more than one irrigant is used in sequence, they may still remain and come into contact with each other within the root canal system [##UREF##0##5##]. The chemical interaction between NaOCl and CHX may result in the brown–orange precipitate that causes staining of the dentinal structure [##REF##17878084##6##]. It behaves like a chemical smear layer, which covers the dentinal tubules and interferes in the obturation sealing [##REF##22419366##7##]. Also, the precipitate can contain para-chloroaniline (PCA), a potentially toxic substance [##REF##20113798##8##].</p>", "<p>Passive ultrasonic irrigation (PUI) and continuous ultrasonic irrigation (CUI) have also been recommended to improve the effects of irrigants on the root canal system [##UREF##1##9##, ##REF##30264624##10##]. Both improves the dispersion of root canal irrigants via cavitation bubble implosions and/or acoustic streaming [##REF##25234972##11##]. They have advantages over conventional irrigation (CI) with a syringe and needle, improving the penetration of solutions in isthmus and lateral canals [##REF##36817024##12##].</p>", "<p>Debridement of difficult-to-access canal areas may also be accomplished by reducing the surface tension and the viscosity of an irrigant by raising its temperature [##REF##26604955##13##, ##REF##36879595##14##]. The use of heated irrigants enhances their distribution in the root canal system and smear layer removal [##UREF##1##9##]. Dos Santos et al. [##UREF##1##9##] concluded that the use of heated distilled water at 65°C (HDW) as a final irrigant after the initial use of EDTA is as efficient in removing canal wall smear layer as NaOCl irrespective of whether CUI is used for simultaneous irrigant activation, with less deleterious effects on dentin microstructure. Knowing the cleansing power provided by HDW, it has been hypothesized that its use as an intermediate irrigant between the association of NaOCl and CHX it is beneficial to avoid the formation of the brown–orange precipitate.</p>", "<p>Accordingly, this study aimed to evaluate the efficacy of the HDW as intermediate irrigation protocol between NaOCl and CHX, with and without ultrasonic activation (PUI or CUI), in preventing the formation of the brown–orange precipitate. The null hypothesis tested was that there would be no differences on the formation of the brown–orange precipitate after using HDW as intermediate irrigation protocol between NaOCl and CHX, regardless of the PUI or CUI use.</p>" ]
[ "<title>2. Materials and Methods</title>", "<title>2.1. Sample Preparation</title>", "<p>This research was approved by the local Human Research Ethics Committee (no. 3.413.317) and was conducted in full accordance with the ethical principals (World Medical Association Declaration of Helsinki, 2008). The sample size calculation was conducted (<ext-link xlink:href=\"http://estatistica.bauru.usp.br/calculoamostral/ta_comparacao_multipla_independentes.php\" ext-link-type=\"uri\">http://estatistica.bauru.usp.br/calculoamostral/ta_comparacao_multipla_independentes.php</ext-link>) considering a previous study with similar methodology [##REF##29160396##15##]. The statistical power was set in 80%, alpha level of 5%, and the estimated standard deviation of 0.8. With a total of four groups, and based on the sample calculation results, 10 specimens were evaluated per group. Forty human canines with fully formed roots, a single straight root canal, and without previous endodontic treatment were selected. The reasons for the extraction of human teeth were unrelated to this research (such as periodontal disease). They were kept stored in distilled water until the experiments begin.</p>", "<p>After access, the tooth length was obtained through the direct method by introducing a K#10 file (Dentsply Maillefer, Ballaigues, Switzerland) into the canal until its tip was seen in the apical foramen. The root of each tooth was surrounded by silicone (HydroXtreme, Swisstec, Coltene, Switzerland), in order to avoid the overflow of irrigating solutions. The working length (WL) was established 1-mm short from tooth length.</p>", "<p>The root canal chemical–mechanical preparation was performed by a single operator, with a Reciproc R40 (40/.06) instrument (VDW, Munich, Germany), according to the manufacturer's instructions. The root canals were irrigated with 2.5% NaOCl (Rioquimica, SP, Brazil) through the CI, with a 5-mL syringe (Ultradent Products Inc., UT, USA) and calibrated needle 2-mm below the WL, with back-and-forth movements and simultaneous aspiration. Apical patency was maintained with a K#10 file taken to the apical foramen.</p>", "<p>Then, grooves were made on the buccal and lingual faces of each tooth using double-sided diamond discs with 22-mm in diameter and 0.1-mm thick (ref. 7020, KG Sorensen), without penetrating the root canal. Jets of air cleared the debris. Each tooth was cleaved into two halves, mesial and distal, with the help of a hammer and chisel respecting the orientation of the grooves. In the middle of each third, a small circle was made, external to the canal, to be a reference point in obtaining the images. The samples were kept in an incubator at 37°C until they were taken for microscopic analysis.</p>", "<title>2.2. Stereomicroscope Analysis</title>", "<p>Two images were obtained with 16x and 40x magnifications from each root canal third, using a stereomicroscope (SteREO Discovery.V12, Carl Zeiss, Jena, Germany). These initial images were taken to verify the condition of the root canal walls, concerning their color, before the final irrigation protocols were carried out. The two tooth halves of each tooth were reassembled. The grooves previously created for cleavage were filled with a light-cured gingival barrier (Top Dam; FGM, SC, Brazil) to stabilize the parts. The reassembled tooth root was inserted into heavy condensation silicone impression material to increase stability and prevent leakage of the solutions used in the final irrigation protocols.</p>", "<title>2.3. Final Irrigation Protocols</title>", "<p>The 40 teeth were randomly assigned to four groups (<italic>n</italic> = 10), according to the different final irrigation protocols, that are summarized in ##TAB##0##Table 1##.</p>", "<p>\n<italic>Group 1—control (no HDW):</italic> The root canals were irrigated with 5 mL of 17% EDTA, for 60 s, (CI). Then, the irrigation proceeded with 5 mL of 2.5% NaOCl for 60 s (CI) and, finally, 5 mL of 2% CHX for 60 s (CI).</p>", "<p>\n<italic>Group 2 (HDW + CI):</italic> The canals were irrigated with 2.5 mL of 17% EDTA for 30 s (CI), then activated by PUI for 30 s, and irrigated again with 2.5 mL of 17% EDTA for 30 s (CI). Then, the same irrigation protocol was performed with 2.5% NaOCl. After, were irrigated with 5 mL of HDW at 65°C for 60 s (CI) and, finally, with 2.5 mL of 2% CHX for 30 s + PUI for 30 s + 2.5 mL 2% CHX for 30 s (CI).</p>", "<p>\n<italic>Group 3 (HDW + PUI):</italic> The irrigation technique used was the same as described in Group 2, except irrigation with HDW at 65°C, in which 2.5 mL was poured for 30 s (CI) + PUI for 30 s + 2.5 mL of HDW at 65°C for 30 s (CI).</p>", "<p>\n<italic>Group 4 (HDW + CUI):</italic> The irrigation technique used was the same as described in Group 2, except irrigation with HDW at 65°C, which was poured into the canal using the CUI technique for 60 s.</p>", "<p>During and at the end of each solution irrigation, the canals of all groups were aspirated through a metallic cannula positioned in the coronary access. PUI and CUI were performed using a specific insert (Irrisonic E1, Helse, SP, Brazil) positioned 1-mm below the WL, activated by ultrasound (JetSonic, Gnatus, SP, Brazil) at the 20% power indicated by the manufacturer, avoiding contact with the root canal walls.</p>", "<p>The distilled water was heated on a hot plate (Thelga, Minas Gerais, Brazil). The water temperature remained constant (95°C) and monitored at all times using a thermometer immersed in the solution. The CUI technique followed the methodology described by Dos Santos et al. [##UREF##1##9##]. The HDW was poured into the root canal through a peristaltic pump. Through the path taken to the insert, the water lost temperature, reaching 65°C. The rate flow (3 mL/30 s) and the temperature of the poured water were verified and standardized according to a pilot study. For CI and PUI, water was deposited inside the root canal at 65°C.</p>", "<title>2.4. Stereomicroscope and Scanning Electron Microscope (SEM) Analysis</title>", "<p>After drying the root canals, new images of the same areas already photographed were obtained. The final images taken after irrigation were analyzed blindly by two previously calibrated examiners at two different moments with an interval of 1 week. The initial and final images were organized side by side for comparison purposes and the amount of brown–orange precipitate from each third was classified by scores [##REF##25557836##16##]: 0—root canal third without precipitate; 1—precipitate present in less than half of the root canal third; 2—precipitate covering more than half of the root canal third; 3—root canal third completely covered by the precipitate (##FIG##0##Figure 1##). One specimen correspondent of each score was qualitatively evaluated under SEM, for illustration purpose, at ×500 magnification (##FIG##0##Figure 1##). The selected specimens were dried and sputter coated with a gold layer of 300 A° (Bal-Tec SCD 005, Bal-Tec Co., USA). The analysis was performed with the SEM (JEOL JSM 6390 LV, Akishima, Japan) set at 10.0 kV.</p>", "<title>2.5. Statistical Analysis</title>", "<p>The Kappa test was used to analyze the intra- and inter-examiner agreement. Data normality was checked with Shapiro–Wilk test. As the data shown nonnormality, for comparison between groups, the Kruskal–Wallis test was used. Once a statistical difference was detected, Dunn multiple comparison test was used to indicate between which groups these differences were. For the intragroup comparison, the Friedman test was used. Statistical analysis was performed using Jamovi software 1.6 (public domain) and BioEstat 5.0 (Mamirau Foundation, PA, Brazil). The level of significance adopted was 5%.</p>" ]
[ "<title>3. Results</title>", "<p>The Kappa test indicated excellent intra and inter-examiner agreement, with values above 0.86 and 0.90, respectively.</p>", "<p>The average rank, the median of the scores, and the results obtained from the comparison of the root canal thirds in the four groups are summarized in ##TAB##1##Table 2##. In general, the G1 (no HDW) had significantly higher scores for precipitate formation (<italic>p</italic> &lt; 0.05). The other experimental groups (HDW + CI, HDW + PUI and HDW + CUI) showed no differences between them. In the intragroup analysis, G1 (no HDW) had higher scores in the cervical and middle thirds when compared to the apical third (<italic>p</italic> &lt; 0.05). No significant differences were observed between the root thirds in the other experimental groups (##TAB##1##Table 2##).</p>", "<p>The qualitative analysis in SEM of the selected specimens showed correspondence with the patterns of precipitate formation, previously observed under a stereomicroscope (##FIG##0##Figure 1##). The specimens irrigated with HCW exhibited a cleaner dentin surface free of precipitates compared to G1 (no HDW).</p>" ]
[ "<title>4. Discussion</title>", "<p>The brown–orange precipitate formation is due to the acid–base reaction between NaOCl and CHX [##REF##17878084##6##]. For a safe and effective irrigation protocol using both solutions, it is imperative that the precipitate is avoided [##UREF##2##17##, ##REF##22414823##18##]. Its formation implies dentinal tubules obliteration since it is deposited over the root canal walls, which can compromise the diffusion of intracanal medication and the obturation sealing [##REF##22419366##7##], and stains root dentin [##REF##24279655##19##]. Besides, the possibility of this precipitate diffusing into the periapical tissues should be considered since it may contain irritating and toxic components to the periapical tissues [##REF##20113798##8##, ##REF##26255964##20##].</p>", "<p>Therefore, to avoid the formation of precipitate resulting from the interaction between NaOCl and CHX, the present study evaluated the effect of HDW to 65°C used as a neutralizing intermediate solution, with and without ultrasonic activation. The stereomicroscope results showed that the use of HDW, regardless of the applied irrigation technique, was able to prevent the precipitate formation.</p>", "<p>In order to show that the root dentin staining did occur through the precipitate, a longitudinal evaluation of the specimens was performed, since the same area of interest was analyzed before and after the final irrigation protocols [##UREF##1##9##]. In addition, the analysis was made in stereomicroscope images which enabled the observers to see the staining clearly, without the need to perform any additional preparation on the samples surface [##REF##32134039##21##].</p>", "<p>The choice of a temperature of 65°C for heating the water was based on previous studies [##UREF##1##9##, ##REF##28168241##22##, ##REF##28283825##23##]. Furthermore, one of these studies by Sonntag et al. [##REF##28168241##22##] observed that, after root canal irrigation, the preheated solution returns to body temperature (37°C) in an average time of 60 s, thus making it safe for use during endodontic treatment.</p>", "<p>The distilled water was heated to reduce its surface tension and viscosity [##REF##26604955##13##], to improve its reach, dispersion, and flow, especially in the anatomical complexities of the root canal system. Based on the results obtained, it is assumed that the heating has increased the water's capacity to remove the residual NaOCl deposited on root canal walls, so that the reaction with CHX is not observed. And together with the increase in temperature, the activation of irrigating solutions, through PUI and CUI, improves penetration and increases the flow of the solution, which consequently favors the contact of irrigating solutions with complex anatomical areas [##REF##30264624##10##–##REF##36817024##12##].</p>", "<p>After the comparison between initial and final images, it was possible to confirm that the dentin staining occurred after the final irrigation. From the results, it was observed that in all thirds analyzed the specimens of the control group obtained the highest scores when compared to the experimental groups. Such result was expected, because no intermediate solution was used neither an activation method. There was no statistical difference between the other experimental groups, which shows that there is a similarity between the stirring techniques tested, when used together with heated distilled water.</p>", "<p>A previous study showed that the use of different intermediate solutions failed to prevent the formation of byproducts [##REF##24962548##24##]. Other studies also concluded that the distilled water, used as an intermediate irrigant with conventional irrigation, was inefficient in preventing the formation of the orangebrown precipitate on the root canal walls [##UREF##3##25##, ##REF##23225234##26##]. One of those studies by Do Prado et al. [##REF##23225234##26##] tested various protocols, including an intermediate flush with citric acid or phosphoric acid. After evaluation under SEM, the authors concluded that only the protocol using phosphoric acid as an intermediate irrigation completely prevented the formation of the chemical smear layer [##REF##23225234##26##]. In none of these investigations [##REF##24962548##24##, ##REF##23225234##26##] did the authors use additional methods of agitating the solutions, which may have contributed to the results.</p>", "<p>Considering the activation of the solutions, Keles et al. [##REF##32134039##21##] observed successful results with different solutions, with and without activation, concerning the ability to remove the precipitate already formed from the surface of the root dentin. However, our goal was to avoid the formation of this byproduct, and not to remove it, ensuring greater predictability and safety to the procedure.</p>", "<p>Regarding the thirds, the deposition of precipitate in the control group was concentrated in the cervical and middle thirds. It is assumed that the larger diameter of the dentinal tubules may have influenced these results, as it supposedly serves as a reservoir of irrigating solution remnants after aspiration. As there was no intermediate irrigation, this reservoir was not diluted by water, resulting in a greater amount of precipitate in these regions.</p>", "<p>The results of the present study showed that HDW can be considered advantageous and useful, enabling the establishment of a final precipitate-free irrigation protocol, safely combining the studied irrigation solutions, enabling the use of chlorhexidine as the last irrigating solution.</p>", "<p>Previous studies used the stereomicroscope to evaluate the presence of orange–brown precipitate [##REF##25557836##16##, ##REF##32134039##21##, ##REF##23225234##26##] because it allows an accurate evaluation of color change in dentin walls, without covering or modifying the samples [##REF##23225234##26##]. In this study, we also used the stereomicroscope for evaluation, however, it would be interesting to realize a further evaluation in SEMe in future studies, to evaluate the presence of chemical smear layer in a greater magnification, and the formation of precipitate inside the dentinal tubules [##UREF##3##25##].</p>" ]
[ "<title>5. Conclusion</title>", "<p>The results of the current study indicate that distilled water at 65°C, when used as intermediate irrigation protocol between NaOCl and chlorhexidine solutions, was effective in preventing the formation of the brown–orange precipitate, regardless of the use of ultrasonic activation (PUI or CUI).</p>" ]
[ "<p>Academic Editor: Murilo Baena Lopes</p>", "<title>Introduction</title>", "<p> The present study aimed to investigate the capacity of different irrigation protocols using heated distilled water at 65°C (HDW), in preventing the formation of the brown–orange precipitate observed after the interaction between sodium hypochlorite (NaOCl) and chlorhexidine (CHX). </p>", "<title>Methods</title>", "<p> Forty human canines were selected, prepared, and cleaved in two halves. Images of delimited areas in each root canal thirds were obtained through a stereomicroscope (16x and 40x). After reassembly, the teeth were distributed into four groups (<italic>n</italic> = 10) according to the final irrigation protocol: G1 (no HDW): EDTA + NaOCl + CHX with conventional irrigation (CI); G2 (HDW + CI): EDTA with passive ultrasonic irrigation (PUI) + NaOCl (PUI) + HDW (CI) + CHX (PUI); G3 (HDW + PUI): EDTA + NaOCl + HDW + CHX with PUI; G4 (HDW + CUI): EDTA (PUI) + NaOCl (PUI) + HDW with continuous ultrasonic irrigation (CUI) + CHX (PUI). After irrigation, the teeth were re-separated and images of the same delimited areas were obtained again. Scores were assigned according to the amount of precipitate observed, comparing the initial and final images. The data were submitted to Kruskal–Wallis, Dunn and Friedman statistical tests (<italic>α</italic> = 5%). </p>", "<title>Results</title>", "<p> G1(no HDW) showed the highest scores in the analysis between groups (<italic>p</italic> &lt; 0.001), with a greater amount of precipitate in the cervical and medium thirds (<italic>p</italic> &lt; 0.001). The thirds of the other experimental groups did not differ from each other (<italic>p</italic> &gt; 0.05). </p>", "<title>Conclusion</title>", "<p> The intermediate irrigation with heated distilled water at 65°C prevented the formation of brown–orange precipitate, regardless of the use of ultrasonic activation (PUI or CUI).</p>" ]
[]
[ "<title>Data Availability</title>", "<p>The corresponding author can provide access to the datasets generated and/or analyzed during the current study upon a reasonable request.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no conflicts of interest.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Representative images of scores under stereomicroscope in ×16 magnification (a, c, e, g) and scanning electron microscope in ×500 magnification (b, d, f, h). (a, b) Score 0: root canal third free of precipitate and with wide-open dentinal tubules. (c, d) Score 1: precipitate in less than half of the root canal third—arrows indicate the presence of the precipitate on the root dentin. (e, f) Score 2: precipitate covering more than half of the root canal third. (g, h) Score 3: root canal third completely covered by the precipitate.  <sup><italic>∗</italic></sup>: indicates the presence of the precipitate.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Groups distribution according to the final irrigation protocol.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">Groups</th><th rowspan=\"1\" colspan=\"1\"/><th align=\"center\" colspan=\"3\" rowspan=\"1\">17% EDTA</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">2.5% NaOCl</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">HDW</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">2% CHX</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>n</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>v</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>t</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">tUA</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>v</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>t</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">tUA</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>v</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>t</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">tUA</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>v</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>T</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">tUA</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G1 (no HDW)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G2 (HDW + CI)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G3 (HDW + PUI)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G4 (HDW + CUI)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Mean rank scores, median of scores (in parentheses), and the results after statistical analysis using the Kruskal–Wallis test and the Dunn multiple comparison test by comparing root thirds in the four groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">Groups</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">Root canal thirds</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Cervical</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Middle</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Apical</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G1 (no HDW)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">56.5 (3.0)<sup>Aa</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">56.1 (3.0)<sup>Aa</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">55.2 (1.0)<sup>Ab</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G2 (HDW + IC)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.5 (0)<sup>Ba</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.5 (0)<sup>Ba</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.0 (0)<sup>Ba</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G3 (HDW + PUI)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.0 (0)<sup>Ba</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.0 (0)<sup>Ba</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.0 (0)<sup>Ba</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">G4 (HDW + CUI)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.0 (0)<sup>Ba</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.2 (0)<sup>Ba</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.7 (0)<sup>Ba</sup></td></tr></tbody></table></table-wrap>" ]
[]
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[]
[]
[]
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[ "<table-wrap-foot><fn><p>HDW, heated distilled water; CI, conventional irrigation; PUI, passive ultrasonic irrigation; CUI, continuous ultrasonic irrigation; <italic>v</italic>, solution volume (mL); <italic>t</italic>, application time (s); tUA, PUI and CUI application time (s).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>HDW, heated distilled water; CI, conventional irrigation; PUI, passive ultrasonic irrigation; CUI, continuous ultrasonic irrigation. Lowercase superscript letters indicate a significant difference (Dunn test, <italic>p</italic> &gt; 0.001) within a group among root thirds (row). Uppercase superscript letters indicate a significant difference (Dunn test, <italic>p</italic> &gt; 0.001) among groups within a root third (column).</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"IJD2024-6612675.001\" position=\"float\"/>" ]
[]
[{"label": ["5"], "person-group": ["\n"], "surname": ["Wright", "Kahler", "Walsh"], "given-names": ["P. P.", "B.", "L. J."], "article-title": ["Alkaline sodium hypochlorite irrigant and its chemical interactions"], "source": ["\n"], "italic": ["Materials"], "year": ["2017"], "volume": ["10"], "issue": ["10"], "pub-id": ["10.3390/ma10101147", "2-s2.0-85030646397", "1147"]}, {"label": ["9"], "person-group": ["\n"], "surname": ["Dos Santos", "da Silveira Teixeira", "Garcia", "Henriques", "Tay", "Bortoluzzi"], "given-names": ["M. C.", "C.", "L. D.", "B.", "F. R.", "E. A."], "article-title": ["Heated distilled water with or without continuous ultrasonic irrigation improves final irrigation efficacy and reduces dentine erosion"], "source": ["\n"], "italic": ["Journal of Dentistry"], "year": ["2020"], "volume": ["103"], "pub-id": ["10.1016/j.jdent.2020.103507", "103507"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Drews", "Nguyen", "Diederich", "Gernhardt"], "given-names": ["D.-J.", "A. D.", "A.", "C. R."], "article-title": ["The interaction of two widely used endodontic irrigants, chlorhexidine and sodium hypochlorite, and its impact on the disinfection protocol during root canal treatment"], "source": ["\n"], "italic": ["Antibiotics"], "year": ["2023"], "volume": ["12"], "issue": ["3"], "pub-id": ["10.3390/antibiotics12030589", "589"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Maiola", "Boppr\u00e9", "Savaris"], "given-names": ["E. C.", "L. D.", "J. M."], "article-title": ["Did in-between rinsing and agitating with distilled water prevents precipitate formation by the interaction between sodium hypochlorite and chlorhexidine canal irrigants?"], "source": ["\n"], "italic": ["Microscopy Research and Technique"], "year": ["2023"], "pub-id": ["10.1002/jemt.24435"]}]
{ "acronym": [], "definition": [] }
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2024-01-14 23:43:50
Int J Dent. 2024 Jan 6; 2024:6612675
oa_package/84/89/PMC10787650.tar.gz
PMC10787651
0
[ "<title>1. Introduction</title>", "<p>Microbial infections and antimicrobial resistance have been recognized as a critical issue worldwide, affecting public health, therefore considering the most important causes of mortality and morbidity [##UREF##0##1##]. According to INEbase death statistics, in the year 2020, infectious diseases were the third most common cause of death, accounting for 16.4% of the total, which includes identified and suspected COVID-19 cases [##UREF##1##2##]. Although intensive care units (ICUs) account for fewer than ten percent of total beds, they serve as a factory for the development and spread of microbial infections [##REF##9187957##3##]. In countries where routine infection control measures are implemented extensively, ICUs are still potential sources of nosocomial infections [##UREF##2##4##]. Accommodation of seriously ill patients who are usually immunocompromised, and undergoing invasive procedures in ICUs, results in a five to seven-fold higher risk of nosocomial infections than other patients [##UREF##3##5##]. Other factors involved are increased duration of stay, use of immunosuppressive drugs, and prolonged or inappropriate use of broad-spectrum antibiotics [##UREF##3##5##]. This leads to a huge economic burden on the health system of developing countries. Therefore, we hypothesized to investigate the antimicrobial resistance among bacterial pathogens isolated from patients admitted in ICUs.</p>", "<p>The development of antibiotics has been acknowledged as the greatest medical advance in human history. However, antimicrobial resistance (AMR) has been rising due to misuse of these valuable compounds which has ultimately resulted in some infections becoming effectively untreatable [##REF##31733401##6##]. According to a report by the UK government health department, ten million people will die in a year from drug-resistant infections by 2050, if urgent action is not taken. Currently, at least 700,000 people lose their lives each year globally, because of drug resistance in illnesses such as bacterial infections, malaria, HIV/AIDS, or tuberculosis [##UREF##4##7##]. In addition, the emergence of highly resistant microorganisms in ICUs has become a major threat to patients, leading to worse outcomes and demand for the last line of antimicrobials [##REF##25968288##8##].</p>", "<p>Surveillance of AMR is the first and foremost essential step towards curtailing the spread of antimicrobial resistance, forming policies, and for infection prevention and control interventions. Importantly, it is the cornerstone for monitoring the impact of local, national, and global strategies. In 2015, WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS), the first global collaborative effort to standardize AMR surveillance. Similarly, in 2021, the Indian Council of Medical Research (ICMR) also started the Antimicrobial Resistance Surveillance System (i-AMRSS), a promising tool for the collection, management, and analysis of AMR data [##UREF##5##9##].</p>", "<p>AMR surveillance helps to generate baseline data on the pattern of microorganisms in the hospital and their susceptibility profile, which in turn helps in deciding effective and rational empirical treatment. These data vary from country to country, hospital to hospital, and even among different wards of the same hospital. Therefore, the objective of this study was to determine the bacteriological profiles and their drug resistance pattern among different infections in ICU patients of a tertiary care hospital.</p>" ]
[ "<title>2. Material and Methods</title>", "<title>2.1. Settings</title>", "<p>This retrospective observational study was carried out in the department of microbiology of a tertiary care hospital in North India from January 2020 to May 2022.</p>", "<title>2.2. Subjects</title>", "<p>The study was undertaken based on reports of bacterial isolates of various clinical specimens from different ICUs, such as medical ICUs (MICUs), surgical ICUs (SICUs), and paediatric ICUs (PICUs), which were submitted to the microbiology laboratory for culture and sensitivity during the study period.</p>", "<title>2.3. Inclusion Criteria</title>", "<p>All the patients who were admitted to various ICUs (medical, surgical, and paediatric ICUs) during the study period and whose reports were retrieved from the laboratory were included in the study. Various sources of clinical specimens included blood, urine, pus, cerebrospinal fluid (CSF), catheter tips, endotracheal tips, drainage fluids (trauma pleural and ascitic), bronchial aspirates (BALs), central venous catheters (CVCs), sputum, and gastric aspirate. Only bacterial isolates were included.</p>", "<title>2.4. Exclusion Criteria</title>", "<p>Mixed growths (three or more isolates) per specimen in urine culture only</p>", "<p>Unsatisfactory sputum samples in accordance with the Bartlett scoring system</p>", "<p>Any leaked or incorrectly labelled samples</p>", "<p>Multiple samples of the same type of specimen from a single patient</p>", "<title>2.5. Ethical Approval</title>", "<p>This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the institutional ethics committee (LHMC/IEC/2022/03/78). Consent was taken from study subjects for inclusion in the study.</p>", "<title>2.6. Methodology</title>", "<p>A total of 2308 patients were admitted to the ICUs during the study period, from which 3056 samples were received. All the organisms were identified morphologically and biochemically by a standard laboratory procedure. The received specimens were streaked on the blood agar and MacConkey agar and incubated aerobically overnight at 37°C. Growths isolated from all the samples were identified by observing the colony characteristics and biochemical reactions using standard microbiological methods. Unidentified isolates were subjected to further identification using the VITEK 2 ID system. Antimicrobial susceptibility testing was conducted by the disk diffusion method as per the Clinical and Laboratory Standard Institute (CLSI). The zone of diameter was measured and interpreted as susceptible (S), intermediate (I), and resistant (R) as per the CLSI (2020–22). The control strains used were <italic>E. coli</italic> ATCC 25922, <italic>Pseudomonas aeruginosa</italic> ATCC 27853, and <italic>Staphylococcus aureus</italic> ATCC 29213.</p>", "<p>Colistin susceptibility testing was performed using the microbroth dilution test as per CLSI guidelines. The test for extended-spectrum beta-lactamase (ESBL) production was performed using the combined disk diffusion method with antibiotic disks of ceftazidime/clavulanic acid (30/10 mcg) and ceftazidime (30 mcg) (as per CLSI). Cefoxitin disk diffusion (30 <italic>μ</italic>g) was used to detect MRSA. Vancomycin susceptibility testing for <italic>Staphylococcus aureus</italic> was done using vancomycin screen agar method (BHI agar with 6<italic>μ</italic>g/mL of vancomycin).</p>", "<p>Statistical analysis: data were analyzed statistically with categorical variables like the proportion of bacterial infections across different ICUs, sample type, age groups, and gender. Patterns of microorganisms, their susceptibility profiles, and sites of infections were also analyzed and expressed as percentages. SPSS software was used for statistical analysis.</p>" ]
[ "<title>3. Results</title>", "<p>In the present study, a total of 3056 samples were received from 2308 patients admitted to ICUs and were used for data analysis. Of 3056 samples, bacterial pathogens were isolated from 1632 samples (53.40%).</p>", "<title>3.1. Clinical Specimen and Demographic Profile of Culture-Positive Patients</title>", "<p>Of 1632, the majority of isolates were from 1 to 11 years old children (31.21%) followed by adults from 18 to 45 years of age (28.03%). Percentage of culture positivity was highest in PICU (41%), followed by MICUs (30.88%), and SICUs (27.57%). Culture isolates belonged equally to males (50.86%) and females (49.14%). The most common culture-positive clinical specimen received was blood (39.08%), followed by respiratory samples (29.45%), exudates (7.32%), and body fluids (6.71%). The distribution of specimens and demographic details of culture-positive isolates are shown in ##TAB##0##Table 1##.</p>", "<title>3.2. Distribution of Bacteriological Isolates</title>", "<p>\n<italic>Acinetobacter</italic> sp. (33.02%) was the most common organism isolated from various clinical specimens, followed by <italic>Klebsiella pneumoniae</italic> (20.89%), and <italic>Escherichia coli</italic> (13.8%). Among the Gram-positive organisms, <italic>Staphylococcus aureus</italic> (16.78%) was the most common organism followed by <italic>Enterococcus</italic> sp. (3.73%). Details of the distribution of bacterial isolates are shown in ##TAB##1##Table 2##.</p>", "<title>3.3. Pattern of Antimicrobial Resistance in Detected Predominant Organisms</title>", "<p>The antimicrobial sensitivity pattern of the different major bacterial isolates to different antimicrobials is shown in graphs 1-2. The majority of Gram-negative bacteria (GNB) were resistant to <italic>β</italic>-lactam antimicrobials and <italic>β</italic>-lactam/<italic>β</italic>-lactamase inhibitor combination. High resistance was also shown to quinolone, cotrimoxazole, and to some extent to carbapenem groups.</p>", "<p>The susceptibility pattern of <italic>Acinetobacter</italic> spp. showed that almost all the isolates were resistant to all drugs (up to 93% resistant) except doxycycline (53.91% R) and minocycline (27.33% R), as shown in ##FIG##0##Figure 1##. <italic>Escherichia coli (E.coli)</italic> was moderately resistant to tetracycline (51.45%), meropenem (55.5%), piperacillin tazobactam (62.2%), ertapenem (62.8%), and imipenem (64.71%) and least resistant to chloramphenicol (33.3%), amikacin (40.74%), and gentamicin (42.14%). Similarly, <italic>Klebsiella</italic> sp. was found to be least resistant to tetracycline (43.97%) and doxycycline (55.84%) with high resistance varying from 60 to 97% to all other drugs (##FIG##0##Figure 1##). ESBL production was similar in both the organisms (16%).</p>", "<p>Almost half of the isolates of <italic>Pseudomonas aeruginosa</italic> (<italic>P. aeruginosa</italic>) were resistant to <italic>β</italic>-lactam antimicrobials, quinolones, and carbapenems. Least resistance was seen in aztreonam (21.82%) and piperacillin-tazobactam (29.89%) as shown in ##FIG##1##Figure 2##. All the Gram-negative isolates were susceptible to colistin (100% S). Among GNB isolated from urine specimens, <italic>E.coli</italic> was most susceptible to nitrofurantoin followed by <italic>Klebsiella</italic> sp. <italic>Acinetobacter</italic> spp. and <italic>Pseudomonas</italic> spp. were extremely resistant to nitrofurantoin.</p>", "<p>\n<italic>Staphylococcus aureus (S. aureus)</italic> showed the highest percentage of resistance towards penicillin (96.34%) followed by erythromycin (77.01%). On the other hand, linezolid, teicoplanin, and vancomycin displayed absolutely no resistance. Eighty-two percent strains were methicillin-resistant (MRSA). <italic>Enterococci</italic> spp. expressed a high level of resistance to all beta-lactams and norfloxacin (93.75%), erythromycin (81.97%), ciprofloxacin (79.63%), tetracycline (74.58%), and high-level gentamicin (65.57%). Vancomycin-resistant <italic>Enterococci</italic> (VRE) spp. accounted for 16.67% of isolates. No resistance was seen against linezolid (##TAB##2##Table 3##).</p>" ]
[ "<title>4. Discussion</title>", "<p>The rapid development and spread of antimicrobial resistance among bacteria are threatening public health worldwide. Multidrug-resistant infections are one of the major causes of mortality and morbidity among patients admitted to hospitals. According to the World Health Organization (WHO), people living in a low-income country are far more likely to die of a communicable disease than of a noncommunicable disease. Despite the global decline, six of the top ten causes of death in low-income countries are communicable diseases [##UREF##6##10##]. Hence, this study was undertaken to provide insight into the extent of antimicrobial resistance among bacteria isolated from patients admitted to ICUs.</p>", "<p>A total of 2308 patients were admitted to ICUs in the study period, from which 3056 samples were taken and sent to the microbiology lab for the bacterial culture. The infection rate among ICU patients was found to be 53.40%.</p>", "<p>The demographic variables of culture-positive patients in this study revealed that the number of males and females developing infection inside the ICU was almost equal. In many studies, the infection rate in men was found to be higher than the one in women [##REF##34683344##11##, ##UREF##7##12##].</p>", "<p>The majority of the isolates were from 1 to 11 years old children followed by adults from 18 to 45 years of age (28.03%). Bloodstream infections (BSIs) accounted for the most common infection in the ICU setting (39.08%) followed by respiratory infections (29.45%). This finding is similar to that of the study performed by Fahim in 2021 in Egypt, where the highest number of pathogens was isolated from blood cultures (44.84%), followed by urine (41.41%), and then wound swabs (13.75%) [##REF##33779849##13##]. However, studies performed by Satyajeet et al. in 2016 and Moolchandani et al. in 2017 in different parts of India showed that pneumonia was the most common ICU infection [##UREF##7##12##, ##UREF##8##14##].</p>", "<p>Nonfermenting Gram-negative bacilli (NF-GNB) have emerged as important hospital-acquired pathogens because of an increasingly unreasonable and irrational use of broad-spectrum antimicrobials. Usually, these pathogens are inhabitants of nature, particularly in soil and water. In the hospital environment, they may be isolated from instruments such as ventilators, tubing, and from the skin of healthcare workers [##UREF##7##12##]. Also, in the present study, <italic>Acinetobacter</italic> sp. (33.02%) was the most common organism isolated from ICUs. This finding is in concordance with the study conducted by Mehta et al. in 2015 in Ahmedabad, where <italic>Acinetobacter</italic> sp. (30.92%) was the commonest organism [##UREF##0##1##]. Next to <italic>Acinetobacter</italic> spp., Enterobacteriaceae GNB such as <italic>Klebsiella</italic> sp. (20.89%) were the second most common organism in this study. In a study from the Dominican Republic in 2020, <italic>E.coli</italic> represented 17.7% of the total isolated microorganisms from ICUs, <italic>Pseudomonas</italic> sp. and <italic>Acinetobacter</italic> sp. represented 12.6% and 8.0% of the total, respectively, while <italic>S. aureus</italic> accounted only for 10% [##UREF##9##15##].</p>", "<p>Other NF-GNB isolated less frequently in this study include <italic>Salmonella Typhi, Proteus</italic> sp. (0.30%), <italic>Morganella morganii</italic> (0.24%), <italic>Burkholderia cepacia</italic> (0.06%), <italic>Stenotrophomonas</italic> sp. (0.06%), and <italic>Sphingomonas</italic> sp. (0.06%).</p>", "<p>NF-GNB were also the most common organism in the study conducted by Moolchandani et al. in 2017 in Puducherry; however, the predominating organisms were <italic>Pseudomonas</italic> sp. (19.1%) followed by <italic>Acinetobacter</italic> spp. (17.5%) [##UREF##7##12##]. In Asian countries like India, <italic>Pseudomonas</italic> spp. have been the most common organism isolated from ICUs [##UREF##2##4##, ##UREF##3##5##, ##UREF##7##12##, ##REF##29963410##16##].</p>", "<p>Gram-positive cocci such as <italic>Staphylococcus aureus</italic> (16.78%) and <italic>Enterococcus</italic> spp. (3.73%) were also seen to cause infections in the ICU settings in this study. The global scenario shows that Gram-positive organisms are more common in the western world (North America and Europe) than in Asian countries [##REF##35070914##17##–##REF##19952319##19##].</p>", "<p>Finally, the resistance patterns of various microorganisms were analyzed in this study. More than 80% of <italic>Acinetobacter</italic> spp. were found to be resistant to third-generation cephalosporins, aminoglycosides, and carbapenems. Minocycline (56.31% S) and colistin (100% S) were the most effective drugs for <italic>Acinetobacter</italic> sp. The results were similar to those of the study performed by Said et al. in 2021 in Saudi Arabia, where <italic>A. baumannii</italic> was found to be the most resistant pathogen isolated from clinical specimens and the isolates were fully resistant to almost all antibiotics tested, except for amikacin (61.25%), colistin (5%), and ertapenem (0%) [##REF##34683344##11##]. Given the noteworthy prevalence of <italic>Acinetobacter species</italic>, empirical therapy in the ICU setting may need to include agents effective against this organism depending on local resistance patterns. Consideration may be given to newer combination agents such as imipenem-cilastatin, ceftazidime-avibactam, or ceftolozane-tazobactam. Colistin in combination with carbapenem would be a potential option for the management of such drug-resistant bugs [##UREF##11##20##].</p>", "<p>A wide range of antibiotics were ineffective in the treatment of Enterobacteriaceae GNB. <italic>E. coli</italic> was highly resistant to <italic>β</italic>-lactam antibiotics including 3rd generation cephalosporins and fluoroquinolones. Least resistance was recorded in chloramphenicol (33.3%), amikacin (40.74%), and gentamicin (42.14%). <italic>Klebsiella</italic> sp. was found to be more resistant than <italic>E.coli</italic>, where least resistance was observed in tetracycline (43.97%) and doxycycline (55.84%). Colistin was again found to be 100% susceptible to both organisms. For this reason, it is imperative to reserve colistin until antimicrobial susceptibility patterns mandate its use. Similar results were seen in a study conducted by Fahim in 2021 in Egypt, where Gram-negative isolates showed the least frequency of resistance against nitrofurantoin (52.5%), amikacin (58.01%), followed by imipenem (59.78%), and meropenem (61.82%) [##REF##33779849##13##].</p>", "<p>\n<italic>P. aeruginosa</italic> was found to be moderately resistant to anti-pseudomonal cephalosporins (41.5%) and carbapenems (55.2%). Least resistant drugs included piperacillin-tazobactam (29.89%), aztreonam (21.82%), and colistin (0%). Moolchandani et al. had reported a similar resistance pattern of <italic>Pseudomonas</italic> sp. to various classes of drugs ranging from 25 to 70% [##UREF##7##12##]. In many studies, <italic>P. aeruginosa</italic> has been recorded as one of the organisms showing high levels of resistance (over 80% R) to routine drugs used for treatment, including carbapenems [##REF##34683344##11##].</p>", "<p>In this study, the production of extended-spectrum <italic>β</italic>-lactamases (ESBLs) was seen to be 16.0% in <italic>E. coli</italic> and <italic>Klebsiella</italic> sp., whereas only 4.10% was seen in <italic>Acinetobacter</italic> sp. Chakraverti in 2015 in Bihar reported a higher rate of ESBL production (30–50%) [##UREF##3##5##]. While the prevalence of ESBL-producing isolates was reported extremely high (67–84%) in a study conducted by Uc-Cachon in Mexico in 2019 [##UREF##12##21##].</p>", "<p>The analysis of the antibiotic susceptibility profile of <italic>S. aureus</italic> revealed that 82.78% strains were methicillin-resistant (MRSA). MRSA has been reported to vary from 40 to 57% in various studies [##UREF##7##12##, ##UREF##9##15##]. Methicillin resistance was observed to be higher in coagulase-negative <italic>Staphylococcus</italic> spp. (CONS) accounting for 82.05%. This result is frightening as this might lead to an increase in use of reserve antibiotics such as vancomycin, which increases the possibility of development of vancomycin resistance. However, <italic>S. aureus</italic> was found to be totally susceptible to vancomycin in this study. Similar results were seen in a study conducted by Faim in 2021 in Egypt, where <italic>S. aureus</italic> exhibited high resistance rates to many antibiotics including penicillin (97.1%), gentamicin (73.91%), and all beta-lactams. Also, CONS showed comparable <italic>β</italic>-lactam resistance rates to <italic>S. aureus</italic> with a slightly higher level of methicillin resistance (77.6%), as well as 100% susceptibility to linezolid and vancomycin [##REF##33779849##13##].</p>", "<p>In this study, <italic>Enterococci</italic> spp. expressed a high level of resistance to erythromycin (81.97%), ciprofloxacin (79.63%), tetracycline (74.58%), penicillin (74.51%), and high-level gentamicin (65.57%). This pattern of resistance obviates the synergistic action of <italic>β</italic>-lactam and aminoglycoside agents. Also, the level of VRE was seen to be 16.67% of total <italic>Enterococci</italic> spp. isolates. In a study conducted by Pawar et al. in 2016 in Maharashtra, VRE was reported in 12% of isolates [##UREF##8##14##]. VRE has been reported to be as high as 63% by Despotovic et al. in 2020 [##REF##32093978##22##]. In developed countries, such as in Europe, the proportions of vancomycin-resistant <italic>E. faecium</italic> were reported to increase from 8.1% in 2012 to 19.0% in 2018 [##REF##32498615##23##]. The limitations of the study included a lack of adequate data on clinical information and an inability to analyze the rate of coresistance among different pathogens. Also, the treatment choices and outcomes of the patients with infections could not be analyzed as it was a retrospective study.</p>", "<p>Clinicians could use this evidence-based knowledge directly to tailor antibiotic regimens to the specific pathogens identified, optimizing the chances of effective treatment [##REF##21985810##24##]. The findings can be incorporated into local antibiotic guidelines and protocols for the ICU. The study results may stimulate further research into new treatment modalities or preventive measures for infections caused by these prevalent organisms. The study encourages regular surveillance within the ICU and broader healthcare environments to track changes in resistance patterns, allowing for timely adjustments in empirical therapy and infection prevention strategies.</p>" ]
[ "<title>5. Conclusion</title>", "<p>Bacteriological profiles and antimicrobial susceptibility data are important to identify emerging drug-resistant pathogens, provide opportunities for new drug development, and form local antimicrobial policy, which further helps in having national data. Our study has shown 100% colistin susceptibility for <italic>E. coli, Acinetobacter</italic> sp., <italic>Klebsiella pneumoniae</italic>, and <italic>Staphylococcus aureus</italic>; therefore, it should be considered as the most effective drugs. Evidence-based knowledge regarding the local bacterial organisms and their antimicrobial resistance pattern is pivotal in deciding empirical drug therapy, ultimately leading to the management of antimicrobial resistance.</p>", "<p>The Global Antibiotic Resistance Partnership (GARP) guidelines recommend a multipronged strategy in low- and middle-income countries to optimize the use of antibiotics and reduce antibiotic resistance. The priority actions recommended national surveillance of antibiotic use and antibiotic resistance, as well as strengthening of infection control committees in hospitals. Hence, studies like this are particularly important in countries like India, where infection control practices and antimicrobial policies need to be strengthened to boost antibiotic stewardship and help in the reduction of antibiotic resistance and patient morbidity and mortality in the long run.</p>" ]
[ "<p>Academic Editor: Amit Singh</p>", "<p>Intensive care unit (ICU) patients are prone to develop infections by hospital prevalent organisms. The aim of the study was to determine the bacteriological profiles and their drug resistance pattern among different infections in ICU patients of a tertiary care hospital. The record-based retrospective data of culture reports of the patients admitted to all the ICUs of a tertiary care hospital during the period from January 2020 to May 2022 were analyzed. A total of 3,056 samples were obtained from 2308 patients. The infection rate among ICU patients was found to be 53.40%. Isolates belonged equally to males (50.86%) and females (49.14%). The most common culture-positive clinical specimen received was blood (39.08%) followed by respiratory samples (29.45%). <italic>Acinetobacter</italic> sp. (33.02%) was the most common organism isolated from various clinical specimens, followed by <italic>Klebsiella pneumoniae</italic> (20.89%), and <italic>Escherichia coli</italic> (13.8%). More than 80% of <italic>Acinetobacter species</italic> were found to be resistant to third-generation cephalosporins, aminoglycosides, and carbapenems, whereas minocycline (56.31% S) and colistin (100% S) were the most effective drugs. <italic>Klebsiella</italic> sp. was found to be more resistant than <italic>E.coli</italic>, and the least resistance was observed to be tetracycline (43.97%) and doxycycline (55.84%). Among <italic>Staphylococcus aureus</italic>, 82.78% of strains were methicillin-resistant (MRSA). Vancomycin-resistant <italic>Enterococci</italic> (VRE) sp. accounted for 16.67% of the isolates. Evidence-based knowledge regarding the local bacterial organisms and their antimicrobial resistance pattern is pivotal in deciding empirical drug therapy, ultimately leading to the management of antimicrobial resistance (AMR).</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>We thank all our colleagues at the Department of Microbiology, Lady Hardinge Medical College and Associated Hospitals, for providing assistance in this research.</p>", "<title>Data Availability</title>", "<p>The data used to support the findings of this study are included within the article.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that there are no conflicts of interest.</p>", "<title>Authors' Contributions</title>", "<p>GG, SS, RK, DS, and MSD conceptualized, designed, and supervised the study. GG and AK performed sample collection, testing, and data acquisition. GG, SS, DS, and MSD analyzed the data and drafted tables and figures and the manuscript. All the authors contributed to the manuscript, verified scientific editing, and approved the manuscript version.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Percentage resistance to various antimicrobials among major Gram-negative bacilli isolated from the ICU patients at LHMC and its associated hospitals.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>Percentage resistance to various antimicrobials among <italic>Pseudomonas aeruginosa</italic> isolates isolated from the ICU patients at LHMC and its associated hospitals.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Clinical specimen distribution and demographic profile of culture-positive patients (<italic>n</italic> = 1632).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Number</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Percentage of positivity (%)</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Gender</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">830</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">50.86</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">802</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49.14</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Age group</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 0-1 year</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">207</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.71</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> &gt;1–11 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">510</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.21</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> &gt;11–18 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">132</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> &gt;18–45 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">457</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> &gt;45 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">326</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.95</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Distribution among ICUs</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> MICU</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">504</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30.88</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> SICU</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">450</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.57</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> PICU</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">678</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41.55</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Sample type</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Blood</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">650</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Respiratory samples (BAL, tracheal aspirate, sputum, ET tube, nasal swabs)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">480</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.45</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Exudates (pus, wound swabs, stitch line swabs, liver abscess)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">119</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.32</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Body fluids (peritoneal, pleural, pericardial fluids)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">109</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.71</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Urine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> CSF</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.35</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> HVS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.40</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tissue</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Others (oral swabs, catheter tips, GA, throat swab, bile)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.75</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Not mentioned</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.75</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Distribution of bacterial isolates in culture-positive patients (<italic>n</italic> = 1632).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Organisms</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Number</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Percentage (%)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Acinetobacter</italic> sp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">539</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klebsiella pneumoniae ss. pneumoniae</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">341</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.89</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Staphylococcus aureus ss. aureus</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">274</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.78</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Escherichia coli</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">226</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Pseudomonas aeruginosa</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">118</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.23</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Enterococcus</italic> spp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.73</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Staphylococcus, coagulase negative</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.38</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Salmonella typhi</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klebsiella oxytoca</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Proteus mirabilis</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Morganella morganii ss. morganii</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Citrobacter</italic> spp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Aeromonas</italic> spp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Burkholderia cepacia</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Burkholderia</italic> spp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Enterobacter</italic> spp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Proteus vulgaris</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Providencia stuartii</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Salmonella Paratyphi</italic> A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Sphingomonas</italic> sp.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Stenotrophomonas maltophilia</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Streptococcus pyogenes</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Vibrio vulnificus</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1632</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Antimicrobial susceptibility pattern of predominant Gram-positive cocci.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">Antibiotic name</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">\n<italic>Staphylococcus aureus</italic>\n</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">\n<italic>Enterococcus</italic> sp</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>R</italic> (%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>I</italic> (%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>S</italic> (%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>R</italic> (%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>I</italic> (%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>S</italic> (%)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ampicillin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">74.51</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.49</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Penicillin G</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">96.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Amoxicillin/clavulanic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">71.43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gentamicin-high level</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">65.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34.43</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gentamicin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.93</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ciprofloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58.30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79.63</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.96</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Norfloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.25</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Erythromycin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">77.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">81.97</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.92</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nitrofurantoin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42.86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.52</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">47.62</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linezolid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vancomycin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.67</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">83.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Teicoplanin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">74.58</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chloramphenicol</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79.66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">85.71</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Doxycycline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">85.71</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">36.21</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Minocycline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">69.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.85</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.92</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tetracycline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">74.55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">74.58</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.98</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cefoxitin (MRSA)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">82.78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr></tbody></table></table-wrap>" ]
[]
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[]
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[ "<table-wrap-foot><fn><p>\n<italic>Note.</italic> ICUs = intensive care units, MICU = medical ICU, SICU= surgical ICU, PICU = paediatric ICU, BAL = bronchoalveolar lavage, CSF = cerebrospinal fluid, HVS = high vaginal swab, ET = endotracheal, and GA = gastric lavage.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>\n<sup>\n<italic>∗</italic>\n</sup>\n<italic> sp</italic>: species.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"CJIDMM2024-7403044.001\" position=\"float\"/>", "<graphic xlink:href=\"CJIDMM2024-7403044.002\" position=\"float\"/>" ]
[]
[{"label": ["1"], "person-group": ["\n"], "surname": ["Mehta", "Chauhan", "Rathod", "Pethani", "Shah"], "given-names": ["T.", "B.", "S.", "J.", "P. D."], "article-title": ["Bacterilogical profile and drug resistance pattern of isolates of the patients admitted in medical intensive care unit of a tertiary care hospital in Ahmedabad"], "source": ["\n"], "italic": ["International Journal of Scientific Research"], "year": ["2015"], "volume": ["4"], "fpage": ["222"], "lpage": ["225"]}, {"label": ["2"], "collab": ["Ine"], "article-title": ["INEbase/Society/Health/Death statistics according to cause of death/Latest data"], "year": ["2023"], "comment": ["\n"], "ext-link": ["https://www.ine.es/dyngs/INEbase/en/operacion.htm?c=Estadistica_C&cid=1254736176780&menu=ultiDatos&idp=1254735573175"]}, {"label": ["4"], "person-group": ["\n"], "surname": ["Singh", "Kaur", "Singh"], "given-names": ["A. A.", "M.", "A."], "article-title": ["Prevalence of microbial infection and strategic pattern of antimicrobial resistance among intensive care unit patients in a tertiary care teaching hospital from rural Northern India"], "source": ["\n"], "italic": ["International Archives of Integrated Medicine"], "year": ["2015"], "volume": ["2"], "fpage": ["14"], "lpage": ["20"]}, {"label": ["5"], "person-group": ["\n"], "surname": ["Chakraverti", "Tripathi"], "given-names": ["T. K.", "P. C."], "article-title": ["Pattern of antibiotic susceptibility of common isolates in ICU of a tertiary care hospital: 2 years study"], "source": ["\n"], "italic": ["International Journal of Clinical Biochemistry and Research"], "year": ["2015"], "volume": ["1"], "fpage": ["80"], "lpage": ["87"]}, {"label": ["7"], "person-group": ["\n"], "surname": ["O\u2019Neill"], "given-names": ["J."], "article-title": ["Tackling drug-resistant infections globally: final report and recommendations"], "year": ["2016"], "publisher-loc": ["Victoria Street London, UK"], "publisher-name": ["UK Department of Health"], "comment": ["Review on Antimicrobial Resistance"]}, {"label": ["9"], "person-group": ["\n"], "surname": ["Kaur", "Dhama", "Buttolia"], "given-names": ["J.", "A. S.", "H."], "article-title": ["ICMR\u2019s Antimicrobial Resistance Surveillance system (i-AMRSS): a promising tool for global antimicrobial resistance surveillance"], "source": ["\n"], "italic": ["JAC-Antimicrobial Resistance"], "year": ["2021"], "volume": ["3"], "issue": ["1"], "pub-id": ["10.1093/jacamr/dlab023"]}, {"label": ["10"], "collab": ["Who World Health Organization"], "article-title": ["The top 10 causes of death"], "year": ["2020"], "comment": ["\n"], "ext-link": ["https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Moolchandani", "Sastry", "Deepashree", "Sistla", "Harish", "Mandal"], "given-names": ["K.", "A. S.", "R.", "S.", "B. N.", "J."], "article-title": ["Antimicrobial resistance surveillance among intensive care units of a tertiary care hospital in Southern India"], "source": ["\n"], "italic": ["Journal of Clinical and Diagnostic Research: Journal of Clinical and Diagnostic Research"], "year": ["2017"], "volume": ["11"], "issue": ["2"], "fpage": ["7"], "lpage": ["DC01"], "pub-id": ["10.7860/JCDR/2017/23717.9247", "2-s2.0-85011629907"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Pawar", "Patil", "Karande", "Mohite", "Pawar"], "given-names": ["S. K.", "S. R.", "G. S.", "S. T.", "V. S."], "article-title": ["Antimicrobial sensitivity pattern of clinical isolates in intensive care unit in a tertiary care hospital from Western India"], "source": ["\n"], "italic": ["International Journal of Scientific Study"], "year": ["2016"], "volume": ["4"], "fpage": ["108"], "lpage": ["113"]}, {"label": ["15"], "person-group": ["\n"], "surname": ["de Luna", "S\u00e1nchez", "Peguero"], "given-names": ["D.", "J. J.", "M."], "article-title": ["Antimicrobial resistance profiles of microorganisms isolated from hospitalized patients in Dominican Republic"], "source": ["\n"], "italic": ["Revista Panamericana de Salud P\u00fablic"], "year": ["2020"], "volume": ["44"], "fpage": ["p. 1"], "pub-id": ["10.26633/rpsp.2020.36"]}, {"label": ["18"], "person-group": ["\n"], "surname": ["Chaudhry", "Prajapat"], "given-names": ["D.", "B."], "article-title": ["Intensive care unit bugs in India: how do they differ from the western world?"], "source": ["\n"], "italic": ["The Journal of Association of Chest Physicians"], "year": ["2017"], "volume": ["5"], "issue": ["1"], "fpage": ["10"], "lpage": ["17"], "pub-id": ["10.4103/2320-8775.196645"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Sharma", "Sharma", "Singh", "Sunita"], "given-names": ["J.", "D.", "A.", "K."], "article-title": ["Colistin resistance and management of drug resistant infections"], "source": ["\n"], "italic": ["The Canadian Journal of Infectious Diseases & Medical Microbiology"], "year": ["2022"], "volume": ["2022"], "fpage": ["1"], "lpage": ["10"], "pub-id": ["10.1155/2022/4315030"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Uc-Cach\u00f3n", "Gracida-Osorno", "Luna-Chi", "Jim\u00e9nez-Guillermo", "Molina-Salinas"], "given-names": ["A. H.", "C.", "I. G.", "J. G.", "G. M."], "article-title": ["High prevalence of antimicrobial resistance among gram-negative isolated bacilli in intensive care units at a tertiary-care hospital in Yucat\u00e1n Mexico"], "source": ["\n"], "italic": ["Medicina"], "year": ["2019"], "volume": ["55"], "issue": ["9"], "fpage": ["p. 588"], "pub-id": ["10.3390/medicina55090588", "2-s2.0-85072529444"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2024-01-14 23:43:50
Can J Infect Dis Med Microbiol. 2024 Jan 6; 2024:7403044
oa_package/28/70/PMC10787651.tar.gz
PMC10787652
0
[ "<title>1. Introduction</title>", "<p>In places where the building operability certificate (SLF) implementation is subject to different performance limits that are not in line with the rules' aims, the building operability certificate (SLF)-implemented districts face these limits [##UREF##0##1##]. Probolinggo City, which has implemented SLF since 2008, has not followed local regulations when implementing the building operability certificate 2008–2019: 45 SLF issued. Investment Board and One-Stop Licensing Services issue 400 IMBs per year. The functional worthiness certificate is just 5 per year [##UREF##1##2##, ##UREF##2##3##]. The performance of SKPD buildings that could have been more optimal in implementing and issuing SLF is required to find a solution by paying attention to and continuing to improve human resources, communication, bureaucratic structure, disposition, and external factors starting from public support and legislative support.</p>", "<p>Several policy models utilize top-down policies in policy implementation, including George Edward III's success and failure factor method. Four factors contribute to successful implementation. Communication, resources, disposition, and bureaucratic structure affect policy execution [##UREF##3##4##].</p>", "<p>According to Edward III, policy decisions and instructions must be applied to the right people and communicated to people who are clear and accurate so that they can be understood quickly by the executor. Effective policy implementation according to Edward III (1984) is that policy implementers must know what they have to do, policy decisions must be transmitted (transmission) to the right people so that the communication must be accurately received clearly (clarity), and also order policies must be consistent [##UREF##4##5##, ##UREF##5##6##].</p>", "<p>Stoner et al. (1997: 47) argued that in implementing a program to successfully achieve policy objectives, the bureaucratic structure factor holds an important position. In this bureaucratic structure, there are divisions of tasks and functions as well as a delegation of authority. With the delegation of authority, the organization can function efficiently [##UREF##6##7##, ##UREF##7##8##].</p>", "<p>The bureaucratic organization or building organizer that handles the building operability certificate (SLF) since 2008 in the field of Urban Arrangement and Building Supervision has changed along with the institutional structure of the Probolinggo City Public Works Department. In 2012, the Cipta Karya and Spatial Planning sector implemented two sections: the Spatial Planning section and the Building and Environmental Structuring Section, which handled building permit recommendations [##UREF##8##9##].</p>", "<p>Edwards III (1980: 11) noted that effective implementation requires staff of the right size and expertise, relevant and adequate information on how to implement policies, and on the compliance of others involved in implementation, the authority to ensure that policies are carried out as intended, and facilities (including buildings, land, supplies, and equipment) [##UREF##4##5##].</p>", "<p>The apparatus resources that handle building implementation, especially the Building Approval (PBG) recommendation and the building operability certificate, are inadequate. Six technical and nontechnical devices handle the building operability certificate (SLF). Existing staff in building management demands an improvement in HR competences, for example, in the Building Approval (PBG) and building operability certificate (SLF) services through SIMBG [##UREF##9##10##, ##UREF##10##11##], which is one way to grow employees through employee involvement (Herrenkohl et al., 1999) [##UREF##2##3##].</p>", "<p>Implementer's disposition/attitude is vital in building-worthy certificate policy execution. If implementers are well disposed towards a policy, they are more likely to carry it out as the original decision-makers intended. However, when implementers' attitudes or perspectives differ from the decision-makers, the process of implementing a policy becomes infinitely more complicated.</p>", "<p>Commitment of the bureaucracy in realizing the vision and mission is the extent to which a Regional Government Work Unit leader, employees, or staff sides with a particular organization and realizes its goals or vision and mission, and intends to maintain its membership in the organization (Arfan Ikhsan et al., 2000). Mowday et al. (1979) state that commitment is “The strength of an individual's identification with and involvement in a particular organization.” Organizational commitment is a personal value that refers to a loyal attitude toward an organization.</p>", "<p>Edward III in Widodo (2010: 104-105) [##UREF##5##6##] noted that for policy implementation to be effective and efficient, implementers must know what to do and have the will to do it.</p>", "<p>Edward III in Widodo (2010: 100) says fiscal limitations and citizen opposition impede facility acquisition. The implementer's service quality is next. The low budget limits the quality of community services.</p>", "<p>Bureaucratic support for building usage policies does not meet municipal rules, which require a building-worthy certificate. The Budget of the Public Works Department only budgets enough money for the Secretariat of the Building Expert Team (TABG) and the TABG Team Honor to operate. In the inspection phase of the building, an inspection tool is needed for reinforcing steel in concrete; it just has a hammer test and no infrared technology was used to observe concrete reinforcement. This needs buying equipment.</p>", "<p>In addition to internal bureaucratic factors in the implementation of building policies for an operability certificate, there are external factors, notably, public support for these rules. In [##UREF##11##12##], the policy considers public participation in policy implementation. Implementation will run relatively more smoothly if the public is allowed to access the policy process, or at least in one of the processes such as setting an agenda or evaluating policies.</p>", "<p>Public support for building legislation is explained in Government Regulation No. 36/2005 concerning the Implementation of Building Law No. 28/2002 Chapter V [##UREF##12##13##]. In the administration of buildings, community participation can play a role in monitoring and maintaining order, both in the activities of construction, utilization, preservation, and demolition of buildings. Monitoring is carried out objectively with full responsibility, with no disruption and/or loss for building owners and/or users, the community, and the environment. Based on its monitoring, the community reports in writing to the city government the indications of improper functioning of buildings and buildings whose construction, utilization, preservation, and demolition have the potential to cause disruption and/or danger to users, the community, and the environment.</p>", "<p>Community support for the provision that buildings must have a Certificate of Appropriate Building Function before being occupied must be improved through public outreach and consultation. For residential function buildings, the community does not pay for building supervisors/technical service providers, but for business functions and select buildings, it does. Implementation of building-worthy certificates for building functions, notably the construction industry's willingness to work with the bureaucracy, is to examine building reliability.</p>", "<p>Implementation of Article 68 of Regional Regulation No. 4 of 2008 Probolinggo City concerning Buildings [##UREF##13##14##] is that after 5 years, the existing buildings must have a building operability certificate that has not been fulfilled. Local regulation implementation is one of the stages of a public policy process that must be well planned. Mazmanian and Sabatier (1983: 4) say the foundation of policy implementation is understanding what should happen when a program is certified legal and basic policy in the form of local laws, implementing regulations, programs, and activities. During the 5 years of local regulation implementation, there were no mayor regulations, only the Building Expert Team Mayor Decree (TABG). The Building Function Eligibility Certificate Mayor's Regulation No. 13/2013 was issued in 2013, but not enforced until 2014.</p>", "<p>Internal bureaucratic or Regional Apparatus Work Unit (SKPD) management factors (communication, bureaucratic structure, resources, and dispositions) and external factors of public support for building feasibility certificate policies on building functions must be resolved so that every building with a building permit also has the building operability certificate (SLF) so that it is safe to be inhabited and utilized.</p>", "<p>The given description can generate the following research questions:<list list-type=\"order\"><list-item><p>How does Regional Apparatus Work Unit (SKPD) implement the building operability certificate (SLF) in Probolinggo?</p></list-item><list-item><p>How can the building operability certificate (SLF) help Probolinggo achieve building-worthy performance certificates?</p></list-item></list></p>", "<p>By problem formulation, this study's aims are as follows:<list list-type=\"order\"><list-item><p>Review Technical Service building management to improve the building operability certificate</p></list-item><list-item><p>Review and analyze the implementation strategy of building operability certificate in achieving the goal of building function-worthy performance certificates in the city of Probolinggo</p></list-item></list></p>" ]
[ "<title>3. Methodology</title>", "<p>This study uses mixed methods with a survey approach. The descriptive qualitative/quasiqualitative method aims to make a systematic, factual, and accurate description of a social phenomenon or natural phenomenon [##UREF##22##23##]. Primary data collection is carried out through building institutions and secondary data collection is carried out through journals, websites, and city government institutional data. Alternative Strategy Questionnaire data are collected by using ten building policy decision officials as respondents.</p>", "<p>The descriptive quantitative approach explains a phenomenon by using numbers describing its characteristics (QSPM).</p>", "<p>First, qualitative data analysis utilizing SWOT determines the organization's control plan. Second, ranking strategies are analyzed to identify QSPM's priorities (David, 2011) [##UREF##23##24##].</p>" ]
[]
[ "<title>4. Discussion</title>", "<p>Of the building function-worthy certificate in improving the performance of the building function feasibility policy certificate and the results of in-depth interviews with key actors implementing the certificate policy strategies can be implemented to improve its performance. The strategy was prepared based on a SWOT analysis by looking at internal factors, namely, the strengths and weaknesses of the building implementing service, the strength factor of the office, which already has regional building regulations, bureaucratic structure, and SLF implementation, which has been implemented since 2008. The Service's weakness factor is the need for more communication with internal institutions and the external lack of human resources to handle SLF implementation and disposition. External factors based on opportunities, namely, legislative support, SLF as an insurance guarantee, plan to replace PBG, and Technical Service Awards for SLF implementation. While the external threat factor is the lack of public participation in owning the building operability certificate (SLF), SLF has yet to become the basis for building insurance guarantees, and the lack of solid professional teams of experts and individuals in implementing the building operability certificate (SLF).</p>", "<p>Strategy determination was performed through the first stage of carrying out internal and external quantitative approaches to strategy analysis through the IFAS and EFAS scores. The second stage is conducting a SWOT analysis, and the third stage is prioritizing strategies by performing calculations by using a Quantitative Strategic Planning Matrix (QSPM) tool.</p>", "<title>4.1. Internal and External Quantitative Approaches to Strategy Factor Analysis Summary</title>", "<p>Feasibility of buildings according to the strengths, weakness, opportunities, and treatment matrices through a quantitative approach according to Tables ##TAB##0##1## and ##TAB##1##2##of the qualitative approach SWOT through the SLF policy implementation strategy as follows [##UREF##24##25##, ##UREF##25##26##].</p>", "<p>\n##FIG##0##Figure 1## shows the organization's position (<italic>x</italic>; <italic>y</italic>) is (2.80; 3.00) in quadrant II. This indicates that SKPD subbuilding affairs can grow and develop. The strategies used in this quadrant are product or service development strategy or forward, backward, and horizontal integration strategy.</p>", "<title>4.2. SWOT Strategy</title>", "<p>The strategy for developing bureaucratic services forms the basis or strategy for forward, backward, and horizontal integration in the formulation of the strategy in ##TAB##1##Table 2##.</p>", "<title>4.3. Priority Strategy</title>", "<p>Based on ##TAB##1##Table 2##, SWOT strategy for implementing the building operability certificate (SLF) policy and the results of the choice of priority strategies for respondents, the priority for organizational development strategy with the following strategic priorities is shown in ##TAB##2##Table 3##.</p>", "<p>The QSPM tool is used in the determination of ranking strategies to obtain priority lists (David, 2011). By the results of the calculation of the Quantitative Strategic Planning Matrix (QSPM), the priority ranking of the implementation of the building operability certificate (SLF) policy and SWOT strategy for implementing SLF policy. In QSPM analysis, the value of attractive score (AS) is obtained from the opinion of the researcher. Strategies that have been obtained using SWOT analysis utilizing total attractive score (TAS) are obtained by multiplying AS and weight. The value of sum of total attractive scores (STAS) indicates the rank of the priorities. The results of QSPM analysis priority strategies based on the determination of policies from respondents are shown in ##TAB##3##Table 4## [##UREF##24##25##]:</p>", "<p>From the priority results in ##TAB##3##Table 4##, metrics of QSPM with the following strategy:<list list-type=\"order\"><list-item><p>Improvement of the building operability certificate (SLF) implementation of the Laws and Regulations: Implementation of SLF is based on the latest laws and regulations following Law 11 of 2020 concerning Job Creation, Law 28 of 2002, and Government Regulation No. 16 of 2021 concerning Buildings and Adjustments to Regional Regulations of Probolinggo City No. 4 of 2008 concerning Buildings with Government regulations.</p></list-item><list-item><p>Increasing public awareness of the building operability certificate (SLF): Increased socialization of SLF management to the Building Owners' community</p></list-item><list-item><p>Strengthening the commitment of SKPD leaders in implementing the building operability certificate (SLF): SKPD leaders prioritize the implementation of SLF by instructing staff to prioritize the performance of SLF services starting from the completeness of data, technical recommendations, and issuance of SLF.</p></list-item><list-item><p>Renewal of SKPD's coordination in the inspection of building functions: Coordination was improved in the implementation of the building operability certificate (SLF), especially for the implementing agencies, namely, the coordination and communication of building services and investment and one-stop licensing services.</p></list-item><list-item><p>Increasing resources (human resources, budget, management information system (SIMBG), authority and facilities, and infrastructure) gradually.</p></list-item></list></p>", "<p>Resources are increased by adjusting and expanding the building operability certificate (SLF) budget every year, starting from the human resource development skills of SIMBG service officers, the authority of SLF officers, and improving SLF facilities and infrastructure.</p>" ]
[ "<title>5. Conclusion</title>", "<p>Based on the results of the discussion above, the following conclusions can be drawn:<list list-type=\"order\"><list-item><p>First, highlighting the key factors that affect the implementation of building feasibility certificate policies, such as a bureaucratic structure that must focus on managing SLF implementation, does not take care of project implementation. No standard operating procedures (SOP) for a building-worthy certificate are regularly implemented, so they function according to the present conditions. Resources, namely, the budget lacking in SLF implementation, need to be added to fulfill the development of human resources, SIMBG facilities, and infrastructure. Communication needs to be improved by issuing SLF between the building office, the licensing services office, and the community that owns the building. And dispositions, namely, leadership and staff authority in implementing SLF, as well as social, economic, and political conditions needs to be improved by considering building owners' social and economic conditions in managing SLF and legislative support in implementing SLF.</p></list-item><list-item><p>Second, priority plan strategies will be implemented by building implementers in improving the implementation of building feasibility certificate policies, namely, (a) improvement of SLF implementation by the laws and regulations; (b) increasing public awareness of The SLF; (c) strengthening the commitment of SKPD leaders in implementing SLF; (d) renewal of SKPD's coordination in the inspection of building functions; and (e) increasing resources (human resources, budget, management information system (SIMBG), authority, and facilities and infrastructure) gradually.</p></list-item><list-item><p>Third, the challenge in the process of drafting building regulations that takes a long time is that the building administrator's SKPD must prepare building regulations by revising regional rules following government regulations, starting from coordination between institutions and legislative processes in the regions, provinces, and the central government and preparing regional head regulations regarding the implementation of SLF that are sufficient at the regional level. The performance of the SLF implementation strategy requires the readiness of resources (HR, budget, equipment, information systems, and authority) in stages before issuing the SLF. The increase in the limited SLF implementation budget each year is intended for developing human resources in SLF services, procurement of equipment skills of SMBG officers, and increasing the authority of SLF service officers. Intense communication between the building office and the licensing service is required in issuing the SLF.</p></list-item></list></p>" ]
[ "<p>Academic Editor: Cornelis H. Pameijer</p>", "<p>An operability certificate is required before using a building. The building operability certificate concerns occupants' safety before the building is used and is directed by Law Government Regulation 16 of 2021 for building security. This article discusses implementing a building operability certificate underperforming and the strategy for implementing the building operability certificate (SLF) policy. This research uses a descriptive qualitative method. First, qualitative data analysis utilizing SWOT determined the building operability certificate implementation strategy. Second, ranking strategies are analyzed to identify (QSPM's) priorities. According to the regulation that every building must have SLF, the building operability certificate was not appropriately implemented. With the current building information system legislation, each region can issue Building Approval (PBG) and building operability certificate (SLF). Research findings based on SWOT and QSPM are used to generate seven strategies: Regional Apparatus Work Units (SKPD) commitment, increased socialization, regulation implementation, capacity building, optimal communication, SIMBG implementation, and increasing resources.</p>" ]
[ "<title>2. Literature Review</title>", "<p>Based on the results of the discussion on the implementation, George Edward III suggests four implementation considerations. Communication, resources, disposition, and bureaucratic structure determine the success or failure of policy implementation (1980: 148). This model assumes that public policy implementers and policy performance are linear. According to Van Meter and Van Horn (Winarno, 2014: 159), six variables influence the implementation of policies: “(1) basic measures and policy objectives (standard and objectives of the policy); (2) resources of policy; (3) communication between organizations and implementation activities (Inter-Organizational Communication Enforcement Activities); (4) characteristics of the implementing agencies; (5) economic, social, and political conditions and (6) The disposition of Implementors” [##UREF##14##15##].</p>", "<p>The use of the theory of the model of George C. Edward III (1980) and Van Horn and Van Meter (1975) in this article is intended that the implementation of the building operability certificate policy is influenced by internal factors, namely, communication, organizational structure, resources, and disposition, and external factors, namely, economic, social, and political conditions as well as the public, such as the financial strength of the building owner community in fulfilling the building operability certificate (SLF) requirements, namely, the as-built drawing requirements for buildings that do not yet exist and other conditions, the awareness of building owners in having the building operability certificate (SLF), and the legislature in supervising the performance of the city government in the field of SLF implementation.</p>", "<p>Strategy formulation is carried out using gap analysis, general matrix strategy approach, Boston Consulting Groups (BSG) matrix, and SWOT matrix strategy. From this approach, what will be discussed in this study is a strategy with a SWOT matrix strategy formulation approach [##UREF##15##16##].</p>", "<p>Sianipar and Entang in Management Analysis Techniques (2003: 68) say that the SWOT matrix can be used to develop several main strategies in the four interrelated quadrants and focus on the goals formulated according to the strengths of each agency.</p>", "<p>The IE Matrix is a strategic management tool which is used to analyze the organization's position with the total IFAS score weighted as the <italic>X</italic>-axis and the total EFAS score weighted as the <italic>Y</italic>-axis. IE Matrix is divided into nine cells with the following provisions [##UREF##16##17##, ##UREF##17##18##]:<list list-type=\"order\"><list-item><p>Cells I, II, and III describe the organization conditions as grow and build. Intensive, namely, market penetration, market development, and product development, or integrative, namely, forward, backward, and horizontal, can be more appropriate for these divisions.</p></list-item><list-item><p>Cells IV, V, and VI describe the organization conditions as hold and maintain. The strategies used are market penetration and product development.</p></list-item><list-item><p>Cells VII, VIII, and IX are the harvest and divest strategies. The strategies used are divestment, conglomerate diversification, and liquidation.</p></list-item></list></p>", "<p>The stage of strategic priorities using the QSPM (Quantitative Strategic Planning Matrix) tool. According to David (2009), QSPM analysis guides organizations to objectively evaluate alternative strategies. The stages were carried out in conducting the QSPM [##UREF##17##18##].</p>", "<p>Purnamasari H (2020) researched the effectiveness of public services by providing recommendations for function-worthy certificates (SLF) at the Banjarbaru City Housing and Settlement Service, where there are still convoluted service procedures that are difficult to understand by the public and a lack of awareness from employees of their duties and responsibilities as state servants and public servants to provide fair and equitable services.</p>", "<p>The study shows that government employees deliver good services to the community, more people manage the building operability certificate (SLF), and operating costs are insufficient [##UREF##18##19##].</p>", "<p>Zenith Navigati evaluated the Application of Functional Eligibility Certificates in Regional Regulation Number 1 of 2012 concerning Buildings in Malang City. The research results show that the application of the certificate of function of Malang City has yet to be maximized [##UREF##19##20##].</p>", "<p>In the research on the Implementation of the Sidoarjo Regent's Regulation Policy Number 72 of 2017 concerning Procedures for Granting and Expansion of the building operability certificate (SLF), Octafialdo Shandy found that the implementation of the Sidoarjo Regent's regulatory policy is going quite well according to four implementation models and one implementation variable, but one implementation model has not gone well, namely, communication and resources. Information power is also included in the model so that business actors still need to fully obtain information on the procedures for granting and extending the Certificate of Building Function Worthiness [##UREF##20##21##].</p>", "<p>The research of Octafialdo and Hartono determined the implementation of the Sidoarjo Regent's policy on granting and extending the Certificate of Building Functionality. The researchers used five informants and qualitative data analysis to support this research [##UREF##21##22##].</p>", "<p>In this study, the building operability certificate (SLF) is discussed, namely, the certificate granted by the Regional Government to declare the feasibility of building functions before being able to be beneficial. However, the performance of the building management before construction is required to have a Building Approval (PBG) and the building operability certificate (SLF). One of the criteria for building performance in an area is the extent to which the building operability certificate (SLF) has been issued.</p>", "<p>This research is expected to produce a strategy for the structure of the Technical Service, communication between Technical Service Units and the One-stop Investment and Licensing Service Office (DPMPTSP) that issues the building operability certificate (SLF) and improving human resources, and increasing human resources and the budget for procuring building inspection equipment to support the issuance of the building operability certificate (SLF).</p>" ]
[ "<title>Acknowledgments</title>", "<p>We want to thank the Probolinggo City Government and the Open University Research and Community Service Institute for providing research data and the opportunity to research so that this article can be published.</p>", "<title>Data Availability</title>", "<p>The data used in this study (Respondent Questionnaire Data for strategic priorities and strategic priority choices from respondents) are provided in the supplementary materials.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no conflicts of interest.</p>", "<title>Supplementary Materials</title>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>IE Matrix.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Internal and external quantitative approaches to strategy factor analysis summary (IFAS and EFAS).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">No.</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Internal strategic factor</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Weight</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Weighting</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Rating score</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"4\" colspan=\"1\">I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Strength</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) There is a legal basis for BG regional regulations of buildings</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.95</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Bureaucratic structure exists</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) The implementation of the building operability certificate (SLF) has been a long time</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.51</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Subtotal I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.18</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"4\" colspan=\"1\">II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Weaknesses</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) External communication is lacking</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Resources (HR, funds, building information system, authority, and equipment) are lacking</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.32</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Disposition/tendency of Regional Work Units (SKPD) leadership attitude is lacking</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Subtotal II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.62</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total weight (I + II); difference in weighting score (I−II) = <italic>x</italic></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.80</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">External strategic factors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"4\" colspan=\"1\">III</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Opportunities</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Strong legislative oversight</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.68</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) As collateral for BG assets (insurance, ILO, hotel, and hospital certificates)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.90</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) PKPD-PU Building Award</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Subtotal I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.22</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"4\" colspan=\"1\">IV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Threats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Public awareness (community and private) lack of care for SLF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.34</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) SLF is not a mandatory requirement for bank guarantees (still IMB)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.32</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Technical assessors of individual BGs and agencies do not yet exist</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.32</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Subtotal II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.98</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total weight (I + II); difference in weighting score (I−II) = </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.00</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>SWOT strategy for implementing the building operability certificate (SLF) policy.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"1\">External bureaucratic factors</th><th align=\"left\" colspan=\"2\" rowspan=\"1\">Internal bureaucratic factors</th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Strengths</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Weakness (weakness)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) The existence of a legal basis for building regulations, mayor regulations, and building laws (SI1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Lack of external communication (WI1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Bureaucratic structure exists (SI2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Resources are lacking (WI2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) The implementation of SLF has been a long time (SI3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Disposition/tendency of SKPD leadership attitude is lacking (WI3)</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Opportunities (opportunities)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SO strategy</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WO strategy</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Strong legislative oversight (OE1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Improvement of SLF implementation based on local regulations, trustee, and PUPR regulation</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Increased communication optimally to building owners and construction service providers</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) As collateral for BG assets (insurance, ILO, hotel, and hospital certificates) (OE2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Strengthening SKPD's SLF tasks and functions</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Gradually increasing resources</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) PKPD-PU Building Award (OE3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Improving the implementation of SLF services every year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Strengthening the commitment of SKPD leaders in implementing SLF</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Threats (threat)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ST strategy</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">WT strategy</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Public awareness (community and private) lack of care for SLF (TE1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Increasing intensification of local regulations and building trustees</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) Increasing public awareness about SLF</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) SLF is not a mandatory requirement for bank guarantees (TE2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Renewal of SKPD's coordination in the inspection of building functions</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2) Increased resources in increasing SLF issuance</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Technical assessors of individual BGs and agencies do not yet exist (TE3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) Facilitating the role of construction services in building SLF inspections</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(3) The commitment of SKPD heads in empowering and increasing the capacity of construction service providers</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Prioritas strategy (PS).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">PS no.</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Prioritas strategy</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PS1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Improvement of the building operability certificate (SLF) implementation based on local regulations, trustee, and PUPR regulation</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PS2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increasing public awareness about building operability certificate (SLF)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PS3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Strengthening the commitment of SKPD leaders in implementing the building operability certificate (SLF)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PS4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Renewal of SKPD's coordination in the inspection of building functions</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PS5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increased resources in increasing the building operability certificate (SLF) issuance</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>QSPM.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"3\" colspan=\"1\">Key factor</th><th align=\"center\" rowspan=\"3\" colspan=\"1\">Weight</th><th align=\"center\" colspan=\"10\" rowspan=\"1\">Alternative strategy</th></tr><tr><th align=\"center\" colspan=\"2\" rowspan=\"1\">PS1</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">PS2</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">PS3</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">PS4</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">PS5</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">AS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">TAS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">AS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">TAS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">AS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">TAS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">AS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">TAS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">AS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">TAS</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<italic>Strengths</italic>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SI1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SI2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SI3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<italic>Weakness</italic>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">WI1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">WI2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">WI3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<italic>Opportunity</italic>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">OE1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">OE2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">OE3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<italic>Threatness</italic>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TE1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TE2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TE3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">STAS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.56</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.340</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.16</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Priority</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material id=\"supp-1\" position=\"float\" content-type=\"local-data\"><label>Supplementary Materials</label><caption><p>Supplementary File 1: –Respondent Questionnaire data for strategic priorities and strategic priority choices from respondents.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn><p>Source: results of analysis, 2020. <italic>Note.</italic> Rating (Likert scale: 1–5); 5 indicates very high; 4 indicates high; 3 indicates quite high; 2 indicates lacking; and 1 indicates very less.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Source: analysis results, 2020.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Source: analysis results, 2020.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Source: analysis results, 2020.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"TSWJ2024-8749628.001\" position=\"float\"/>" ]
[ "<media xlink:href=\"8749628.f1.pdf\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["1"], "person-group": ["\n"], "surname": ["Mubyarsah"], "given-names": ["L. R."], "article-title": ["Surabaya city government records 2,740 buildings do not have SLF"], "year": ["2022"], "comment": ["\n"], "ext-link": ["https://www.jawapos.com/surabaya-raya/01394519/pemkot-surabaya-catat-2740-bangunan-gedung-belum-miliki-slf"]}, {"label": ["2"], "person-group": ["\n"], "surname": ["Juliardi", "Puri", "Aziza"], "given-names": ["R. D.", "E. R.", "M. I."], "article-title": ["Temporary occupancy permit (SLF) for building sustainable strategy"], "source": ["\n"], "italic": ["IOP Conference Series: Earth and Environmental Science"], "year": ["2021"], "volume": ["738"], "issue": ["1"], "fpage": ["012079"], "lpage": ["12114"], "pub-id": ["10.1088/1755-1315/738/1/012079"]}, {"label": ["3"], "person-group": ["\n"], "surname": ["Riau", "Yuswadi"], "given-names": ["D. P.", "H."], "article-title": ["Improvement strategy policy implementation the certificate of building operability in Probolinggo city"], "source": ["\n"], "italic": ["International Journal of Management Science and Business Administration"], "year": ["2014"], "volume": ["3"], "issue": ["09"], "fpage": ["37"], "lpage": ["47"]}, {"label": ["4"], "person-group": ["\n"], "surname": ["Edward"], "given-names": ["G. C."], "source": ["\n"], "italic": ["Implementing Public Policy"], "year": ["2011"], "publisher-loc": ["Washington, DC, USA"], "publisher-name": ["Congressional Quarterly Press"]}, {"label": ["5"], "person-group": ["\n"], "surname": ["Edward"], "given-names": ["G. C."], "suffix": ["III"], "source": ["\n"], "italic": ["Implementing Public Policy"], "year": ["1980"], "publisher-loc": ["Washington, DC, USA"], "publisher-name": ["Congressional Quarterly Press"]}, {"label": ["6"], "person-group": ["\n"], "surname": ["Widodo"], "given-names": ["J."], "source": ["\n"], "italic": ["Building Performance-Based Bureaucracy"], "year": ["2006"], "edition": ["1st"], "publisher-loc": ["Malang, Indonesia"], "publisher-name": ["Bayumedia Publishing Publisher"]}, {"label": ["7"], "person-group": ["\n"], "surname": ["Istikhoroh", "Sukamdani"], "given-names": ["S.", "Y."], "article-title": ["Optimize the role of commitment of employees in improving accountability office report on local government budget"], "source": ["\n"], "italic": ["International Journal of Economics and Finance"], "year": ["2017"], "volume": ["9"], "issue": ["12"], "fpage": ["p. 116"], "pub-id": ["10.5539/ijef.v9n12p116"]}, {"label": ["8"], "person-group": ["\n"], "surname": ["Paper", "Bieli"], "given-names": ["C.", "E."], "article-title": ["Essence and significance of management control in public sector in Poland"], "conf-name": ["Proceedings of the Scientific Conference \u201cContemporary Issues in Business, Management and Education"], "conf-date": ["January, 2016"], "conf-loc": ["Poland"], "pub-id": ["10.3846/cibme.2011.04"]}, {"label": ["9"], "person-group": ["\n"], "surname": ["Riau", "Akadira", "Diana"], "given-names": ["D. P.", "T.", "B. A."], "article-title": ["Improvement licensing services based digital on building management information system (SIMBG)"], "source": ["\n"], "italic": ["Kne Social Sciences"], "year": ["2023"], "volume": ["2023"], "fpage": ["414"], "lpage": ["429"], "pub-id": ["10.18502/kss.v8i5.13014"]}, {"label": ["10"], "person-group": ["\n"], "surname": ["Firdaus", "Ristiawati"], "given-names": ["M. A.", "R."], "article-title": ["Implementing building permits in wetland environmental areas in banjarmasin city"], "source": ["\n"], "italic": ["The Innovation of Social Studies Journal"], "year": ["2022"], "volume": ["4"], "issue": ["1"], "fpage": ["p. 53"], "pub-id": ["10.20527/iis.v4i1.5687"]}, {"label": ["11"], "person-group": ["\n"], "surname": ["Rizky", "Darma", "Astana", "Dewi"], "given-names": ["I. P.", "A.", "I. N. Y.", "A. A. D. P."], "article-title": ["Melalui aplikasi SIMBG di kota denpasar the analysis of public satisfacation level of imb service through SIMBG application"], "source": ["\n"], "italic": ["Denpasar City"], "year": ["2022"], "volume": ["10"], "issue": ["1"], "fpage": ["44"], "lpage": ["51"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Mazmanian", "Daniel", "dan Paul"], "given-names": ["S.", "H.", "A."], "source": ["\n"], "italic": ["Implementation and Public Policy"], "year": ["1983"], "publisher-loc": ["New York, NY, USA"], "publisher-name": ["Harper Collins"]}, {"label": ["13"], "collab": ["Government regulation"], "source": ["\n"], "italic": ["Republic of Indonesia Government Regulation Number 36 of 2005 Concerning the Implementation of Law No. 28 of 2002 Concerning Buildings"], "year": ["2021"], "publisher-loc": ["Indonesia"], "publisher-name": ["Government regulation"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Regulations"], "given-names": ["P. C. R."], "source": ["\n"], "italic": ["Probolinggo City Government"], "year": ["2006"], "publisher-loc": ["Probolinggo, Indonesia"], "publisher-name": ["PCR Regulations"]}, {"label": ["15"], "person-group": ["\n"], "surname": ["Van Meter", "Van Horn"], "given-names": ["D. S.", "C. E."], "article-title": ["The Policy implementation process: A. Conceptual Framework"], "source": ["\n"], "italic": ["Administration and Society"], "year": ["1975"], "volume": ["6"], "issue": ["4"], "fpage": ["445"], "lpage": ["488"], "pub-id": ["10.1177/009539977500600404", "2-s2.0-84965736672"]}, {"label": ["16"], "person-group": ["\n"], "surname": ["Rangkuti"], "given-names": ["F."], "source": ["\n"], "italic": ["SWOT Analysis: Techniques for Dissecting Business Cases"], "year": ["2006"], "publisher-loc": ["Jakarta, Indonesia"], "publisher-name": ["PT Gramedia Pustaka Utama"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Qanita"], "given-names": ["A."], "article-title": ["Analisis strategi dengan metode swot dan qspm (quantitative strategic planning matrix): studi kasus pada D\u2019gruz caffe di kecamatan bluto sumenep"], "source": ["\n"], "italic": ["Komitmen: Jurnal Ilmiah Manajemen"], "year": ["2020"], "volume": ["1"], "issue": ["2"], "fpage": ["11"], "lpage": ["24"], "pub-id": ["10.15575/jim.v1i2.10309"]}, {"label": ["18"], "person-group": ["\n"], "surname": ["Kusumah", "Suryana"], "given-names": ["R. T.", "H."], "article-title": ["Model analisis swot dan qspm dalam pemilihan strategi pemasaran distro"], "volume": ["6"], "conf-name": ["Proceedings of the Industrial Engineering National Conference (IENACO)"], "conf-date": ["May, 2018"], "conf-loc": ["Surakarta, Indonesia"], "fpage": ["433"], "lpage": ["440"]}, {"label": ["19"], "person-group": ["\n"], "surname": ["Purnamasari"], "given-names": ["H."], "article-title": ["Efektivitas pelayanan publik pemberian rekomendasi sertifikat laik fungsi (slf) pada dinas perumahan dan permukiman kota Banjarbaru provinsi kalimantan selatan"], "year": ["2020"], "comment": ["\n"], "ext-link": ["http://eprints.uniska-bjm.ac.id/2946/"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Nafigati"], "given-names": ["Z."], "source": ["\n"], "italic": ["Evaluasi Penerapan Sertifikat Laik Fungsi dalam Peraturan Daerah Nomor 1 Tahun 2012 Tentang Bangunan Gedung di Kota Malang"], "year": ["2018"], "publisher-loc": ["Malang, Indonesia"], "publisher-name": ["Universitas Brawijaya"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Ildiri", "Bazille", "Lou", "Hinkelman", "Gray", "Zuo"], "given-names": ["N.", "H.", "Y.", "K.", "W. A.", "W."], "article-title": ["Impact of WELL certification on occupant satisfaction and perceived health, well-being, and productivity: a multi-office pre- versus post-occupancy evaluation"], "source": ["\n"], "italic": ["Building and Environment"], "year": ["2022"], "volume": ["224"], "issue": ["April"], "pub-id": ["109539", "10.1016/j.buildenv.2022.109539"]}, {"label": ["22"], "person-group": ["\n"], "surname": ["Octafialdo"], "given-names": ["H. S."], "article-title": ["Implementation of the peraturan bupati Sidoarjo nomor 72 tahun 2017policy concerning procedures for the provision of and renewal of certificates of appropriate functionality study at the dinas perumahan permukiman Cipta Karya dan tata ruang bidang tata ban"], "source": ["\n"], "italic": ["Journal of Computer Science"], "year": ["2020"], "volume": ["5"], "issue": ["3"], "fpage": ["248"], "lpage": ["253"]}, {"label": ["23"], "person-group": ["\n"], "surname": ["Bungin"], "given-names": ["B."], "source": ["\n"], "italic": ["Post Kualitatif Sosial Research Methods Kuantitatif-Kualitatif-Mixed Methods Positivisme-Postposititivisme-Phenomenology-Postmodern"], "year": ["2020"], "edition": ["1st"], "publisher-loc": ["Jakarta, Indonesia"], "publisher-name": ["KENCANA"]}, {"label": ["24"], "person-group": ["\n"], "surname": ["Wardhani", "Dini"], "given-names": ["F. K.", "A."], "article-title": ["Strategy formulation using SWOT analysis, space matrix and QSPM: a conceptual framework"], "source": ["\n"], "italic": ["International Journal of Innovative Science and Research Technology"], "year": ["2020"], "volume": ["5"], "issue": ["5"], "fpage": ["1520"], "lpage": ["1527"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Sumiarsih", "Legono", "Kodoatie"], "given-names": ["N. M.", "D.", "R. J."], "article-title": ["Strategic sustainable management for water transmission system: a SWOT-QSPM analysis"], "source": ["\n"], "italic": ["Journal of the Civil Engineering Forum"], "year": ["2018"], "volume": ["4"], "issue": ["1"], "fpage": ["p. 29"], "pub-id": ["10.22146/jcef.30234"]}, {"label": ["26"], "person-group": ["\n"], "surname": ["Setyorini", "Effendi", "Santoso"], "given-names": ["H.", "M.", "I."], "article-title": ["Marketing strategy analysis using SWOT matrix and QSPM (case study: WS restaurant soekarno hatta Malang)"], "source": ["\n"], "italic": ["Industria: Jurnal Teknologi dan Manajemen Agroindustri"], "year": ["2016"], "volume": ["5"], "issue": ["1"], "fpage": ["46"], "lpage": ["53"], "pub-id": ["10.21776/ub.industria.2016.005.01.6"]}]
{ "acronym": [], "definition": [] }
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2024-01-14 23:43:50
ScientificWorldJournal. 2024 Jan 6; 2024:8749628
oa_package/f1/35/PMC10787652.tar.gz
PMC10787653
0
[ "<title>1. Introduction</title>", "<p>Important strides have been made in the understanding of causes of work stress [##UREF##0##1##] and in developing risk assessment methodologies to ameliorate the psychological harm associated poor work design, organization, management, and social contexts [##UREF##1##2##, ##REF##22953229##3##]. Nevertheless, the prevalence of stress, anxiety, and depression in the workforce remains high, and it has become clear that while risk assessment approaches are absolutely necessary [##UREF##0##1##–##REF##22953229##3##], they are not sufficient to eliminate stress-related sickness absence and presenteeism. Critically, there are external factors and individual differences involved in mental health outcomes [##UREF##2##4##]. That is, resilience to positively adapt and successfully cope with necessary work demands is another important part of good mental health in the working population.</p>", "<p>Resilience, as a construct, has its roots in historical investigations of children exposed to stressful life circumstances to identify and understand protective factors for these at-risk populations [##UREF##3##5##]. Based on their substantial study of adolescent adjustment to challenging life events, Masten et al. argued that resilience is a dynamic process and not a personality trait and—noting differences in the literature regarding the conceptualization of resilience—that the term resilience should be confined to representing consistent positive adjustment under challenging life conditions [##UREF##4##6##]. Working conditions, such as those seen for healthcare workers during the COVID-19 pandemic, can certainly fall into the category of challenging life conditions [##REF##33334335##7##], and prevalence figures indicate that work-related mental health problems among a wide spectrum of employees are high [##REF##22496705##8##]. If resilience can be conceptualized as having the insight and capability to cope in conditions of high risk or adversity, then educational interventions that can strengthen an employee's resilience will allow them to maintain or recover good mental health while facing challenging working conditions. To date, there are no evidence-based recommendations to improve resilience in the workplace, even though there is some understanding of predictors of resilience [##UREF##3##5##, ##UREF##5##9##–##UREF##7##11##].</p>", "<p>The interest in resilience training is not new. In 2015, drawing on a decade of research into employee well-being and performance, Robertson et al. [##UREF##8##12##] reviewed fourteen intervention studies which suggested that resilience training could provide a number of benefits for both individuals and organizations. Their critique of the research methodologies, however, led to their conclusion that “there is no definitive evidence for the most effective training content or format” (p.533), because evidence of the skills involved in employee resilience was poor. In fact, only three of the fourteen studies provided a definition of resilience in line with the measure they used for evaluating their intervention. Ultimately, an effective training program to develop employee resilience must be founded on resilience skills. This starts with using an appropriate Resilience Skills Questionnaire to ascertain improvements in resilience and the efficacy of the intervention program.</p>", "<p>A subsequent review of the literature concerned with individual resilience in the workplace identified 27 measures used to measure resilience [##UREF##9##13##]. This review described various shortcomings in the tools used in intervention studies to measure resilience. Briefly, some provided no information relating to the validity or reliability of the resilience scale used; most tools were unidimensional measures that conceptualized resilience as a stable trait for the purpose of identifying resilient individuals to understand a particular outcome (e.g. burnout and job satisfaction); and the focus of the studies that used a multidimensional resilience measure was on positive outcomes associated with resilient individuals, and thus, they do not provide information about determinants of resilience [##UREF##9##13##].</p>", "<p>The social cognitive theory (SCT) [##UREF##10##14##] has been nominated as a means of improving understanding of resilience in the workplace [##UREF##11##15##]. The SCT has previously been used to provide a framework to interpret human behaviors in occupational settings [##REF##28322660##16##] based on its premise that learning can occur in a social context through a dynamic, reciprocal interaction of the biological, personal, and environmental factors that contribute to the manifestation of resilience [##REF##31178098##17##–##UREF##13##20##]. The SCT proposes that behavior, behavior change, and maintenance of behavior change are all a function of beliefs that the person has the resources to perform the behavior. The SCT accepts that the upkeep of behaviors over time needs environmental reinforcement and personal self-regulation, incorporating the notion of reciprocal determinism. These components of reciprocal determinism are influenced by various constructs of SCT of which self-efficacy, self-regulation, observational learning, and reinforcement through social support have been the most frequently utilized to explain and alter behavior [##UREF##14##21##]. Self-efficacy includes a person's confidence in their ability to pursue resilient behaviors, and thus, self-efficacy plays a central role in changing behaviors. Self-regulation includes setting goals and creating plans to perform resilient behaviors. Social support includes an individual's perception of the availability of emotional, informational, evaluative, financial, or instrumental support if needed [##UREF##10##14##, ##UREF##11##15##].</p>", "<p>It has been suggested that resilience is a skill that can be learned and that providing knowledge and coaching can help improve coping styles, even in frontline healthcare workers during the COVID-19 pandemic [##REF##22429170##22##, ##REF##33378141##23##]. Although not directly rebutting the findings, it has been pointed out that evidence to answer the question of whether resilience can be taught is weak, because there is a need for robust tools to evaluate employees' resilience skills [##UREF##7##11##, ##REF##34320697##24##, ##UREF##15##25##]. If this suggestion is to be examined, then such a tool needs to be developed. To our knowledge, this study is the first to develop a measure of resilience skills. This is the first step to develop an intervention to improve resilience skills and reduce work-related stress and associated mental health problems. The literature indicates that the SCT can be used as a suitable framework in developing and designing a tool to measure key determinants of resilience, and in view of the ongoing mental health challenges of those working to manage the COVID-19 pandemic [##UREF##16##26##], the health sector provides a suitable dynamic setting to validating a new tool. Therefore, the present study is aimed at developing and validating a new questionnaire to measure determinants of resilience in health sector employees based on the SCT.</p>" ]
[ "<title>2. Methods</title>", "<title>2.1. Design</title>", "<p>The study used a sequential exploratory mixed methods design following the DeVellis' guidelines for scale development [##UREF##17##27##], and the ten general recommendations of the COnsensus Study for the Selection of health status Measurement INstruments (COSMIN) were applied to the study [##UREF##18##28##]. First, a qualitative phase identified resilience items based on SCT constructs using an analysis of relevant literature and an expert panel to guide the development of dimensions. Then, a quantitative phase tested the psychometric properties of the developed questionnaire.</p>", "<title>2.2. Item Development and Participants</title>", "<p>To develop a Resilience Skills Questionnaire, first, we clearly defined what we wanted to measure. The content of the scale would be evidence-based and guided by the SCT as this theory was nominated as a useful framework for understanding resilience skills [##UREF##9##13##]. Initially, 38 items for the questionnaire were drawn from the academic literature on resilience skills, features of existing resilience scales, theoretical discussions about resilience, and the opinion of the Expert Panel. As shown in ##TAB##0##Table 1##, the sample items (translated from Persian to English) were aligned with the three SCT constructs associated with resilience: self-efficacy (14 items), self-regulation (14 items), and social support (10 items).</p>", "<p>We recruited an expert panel of thirteen professors in health promotion, psychology, and public health to support the item development phase. They were a targeted convenience sample recruited from universities. All participants were volunteers, and no payment was made to any participant. All gave written informed consent, and all understood their right to withdraw at any time.</p>", "<p>Participants for the remainder of the questionnaire development were health professionals working in urban healthcare centers in Shiraz, Iran, involved in the planning and monitoring of the frontline COVID-19 healthcare services, the planning and monitoring of new COVID-19 vaccination centers, and evaluating service delivery during the pandemic. Urban health centers in Iran are provided as public services by government systems and provide a holistic model for healthcare [##REF##29259949##29##].</p>", "<p>The study was advertised in the health centers in Shiraz for three weeks mid-2021. The inclusion criterion was having at least one year of postqualification experience in the current place of work for the main study. To maximize recruitment, we posed no exclusion criteria on the presumption that the study was relevant to all employed in this context. There are many recommendations regarding the number of participants required for adequate statistical power in confirmatory factor analysis [##UREF##19##30##], as well as software which can support determination of sufficiency by demonstrating the minimum sample size required for 80% power. This is generally considered the minimum sample size for drawing accurate conclusions and avoiding type II errors [##UREF##20##31##]. Using the recommending settings [##UREF##20##31##], a G<sup>∗</sup>Power calculation (power 0.80, effect size 0.15, and alpha 0.05) for the initial 38 predictor items yielded a minimum sample size of 209 participants.</p>", "<p>Altogether, 400 employees expressed an interest in the study, and full information sheets were sent to them. A total of 346 health sector workers who met the inclusion criterion, as confirmed by providing their job tenure on the written informed consent form, joined the study. From this sample, we randomly selected ten healthcare workers who identified that they have at least five years of work experience to support the face validity phase in the development of the items for the tool. The remaining 336 completed the developing tool to measure the psychometric properties of the developed Resilience Skills Questionnaire (RSQ). Based in the discussion of sample sizes for CFA by Kyriazos [##UREF##19##30##] and the G<sup>∗</sup>Power calculation to provide a sample with a minimum of 80% power, our sample size of 336 participants was sufficient to proceed.</p>", "<title>2.3. Procedure</title>", "<p>A face-to-face meeting of the thirteen members of the expert panel was instrumental in determining the format for item measurement. That is, for all items, the direction would be positive and the scoring should use a 5-point Likert agreement scale, strongly disagree = 1, agree = 2, neutral = 3, agree = 4, and strongly agree = 5, and higher scores would indicate better resilience skill status in each of the three SCT constructs.</p>", "<p>The expert panel met a second time to examine the content validity of the tool and to ensure that the developed questionnaire was parsimonious and included only functional items. The initial item reduction used content validity ratio (CVR) and content validity index (CVI). Each expert was asked to independently classify each the sample items according to whether it was unnecessary = 1, somewhat necessary = 2, or necessary = 3. The formula (Ne − <italic>N</italic>/2)/(<italic>N</italic>/2), where Ne is the number of panelists who stated that the item was necessary and <italic>N</italic> is the number of panelists, was used to compute CVR [##UREF##21##32##]. According to Lawshe's table, the acceptable numeric value for CVR is 0.54 for thirteen experts [##UREF##21##32##]. Items that did not meet this threshold were removed from the item pool of the developing questionnaire.</p>", "<p>CVI was then calculated by asking the expert panel to rate the relevance, clarity, and simplicity of the remaining items based on 4-point Likert scales. (Experts determined the relevance of each item in their opinions where not relevant = 1, relatively relevant = 2, relevant = 3, or completely relevant = 4; the simplicity of each item where not simple = 1, relatively simple = 2, simple = 3, and very simple = 4; and the clarity of each item where unclear = 1, relatively clear = 2, clear = 3, and very clearly = 4.) CVI was calculated by adding the relevance, clarity, and simplicity rating scores from each expert for each item, which were then divided by the total number of experts. Polit and Beck suggested CVI scores above 0.79 as appropriate [##REF##16977646##33##].</p>", "<p>The next step was to evaluate the scale to ascertain face validity using a sample of the target population [##REF##29942800##34##]. For this purpose, the ten experienced health workers were asked to rate the importance of the remaining items to provide an impact score for each item, using a 5-point Likert scale (not important = 1, slightly important = 2, moderately important = 3, important = 4, and very important = 5), and the following formula: impact score = frequency × importance (frequency = the number of people who gave the item a score of 4 or 5; importance = mean score for each item).</p>", "<title>2.3.1. Quantitative Steps</title>", "<p>The data collection phase took place in November 2021. The 38 sample items for the new questionnaire, and the 20-item Resilience at Work (RAW) scale [##REF##24064782##35##], were made available to the participants through an online platform. The RAW scale was included to examine convergent validity. These are two tools that measure a form of employee resilience and while their purpose is not the same, they should share enough of the underlying general factor associated with resilience to yield a moderate to high correlation (i.e., <italic>r</italic> ≥ 0.05) when given to the same population [##UREF##22##36##]. The link to the questionnaires was distributed through a central email. A total of 336 completed anonymous questionnaires were submitted. Data were analyzed using IBM SPSS 24 and AMOS 24 (USA, SPSS Inc.). The initial inspection of the data confirmed that there were no floor and ceiling effects and that the database had less than 3% missing values. As the missing values were completely at random, they were replaced by personal mean scores, on the basis that there were few, and these supply minimally biased valuations in questionnaire research [##REF##20882358##37##]. Internal consistency of the questionnaire at this stage was assessed using interitem and item-total correlations. Items with very low interitem and item-total correlations (<italic>r</italic> &lt; 0.30) which would not be beneficial to a questionnaire were deleted [##REF##29942800##34##]. A subsample of the participants (<italic>n</italic> = 25) agreed to complete the RSQ again two weeks after they had submitted their questionnaires to provide a preliminary examination of the stability of the developing questionnaire. The test-retest reliability was assessed using two-way mixed intraclass correlations (ICC) with absolute agreement. An ICC of less than 0.40 is indicative of poor reliability, between 0.40 and 0.59 is considered fair, between 0.60 and 0.74 is good, and an ICC of 0.75 and above indicates excellent reliability [##UREF##23##38##]. These reliability analyses were undertaken before assessing construct validity to ensure all the items in each of the three dimensions had sufficient discriminatory power, as required for measuring the considered dimensions.</p>", "<p>Exploratory factor analysis (EFA; <italic>N</italic> = 200) and confirmatory factor analysis (CFA; <italic>N</italic> = 336) were performed to extract scale factors. EFA was performed using maximum likelihood with Promax rotation as this oblique rotation method allows factors to correlate with each other. In line with good practice, two statistical tests were applied to the data to ascertain factorability of the data [##UREF##24##39##]. The Kaiser-Meyer-Olkin (KMO) test was used to ensure that the ratio of the sample size to the number of items was sufficient. This would be confirmed by a KMO values ≥ 0.6 [##UREF##24##39##]. Bartlett's test of sphericity was used to assess the appropriateness of the correlations between variables in the factor model. A significant value (<italic>p</italic> ≤ 0.5) indicates factorability [##UREF##24##39##].</p>", "<p>The extent to which the initial questionnaire was compatible with the SCT was examined using confirmatory factor analysis (CFA) with maximum likelihood. This statistical method of determining the structural validity of the developed questionnaire was appropriate: CFA is a deductive test of hypothetical models, and this is not possible using other multivariate analyses [##REF##33170146##40##]. In this study, all variables were analyzed simultaneously to examine whether the model was consistent with the data. There are no definitive fit standards for CFA, but there are accepted test norms [##REF##29942800##34##, ##UREF##25##41##, ##UREF##26##42##] and for the indices used to test the CFA model fit in this study. These were the chi-square test (<italic>χ</italic><sup>2</sup>/df), the root mean square error of approximation (RMSEA), the goodness-of-fit index (GFI), the adjusted goodness-of-fit index (AGFI), and the comparative fit index (CFI). For a good model fit, the <italic>χ</italic><sup>2</sup>/df ratio should be low. There is no absolute standard, and sample size complicates the use of this measure beyond a description of goodness of fit; however, a <italic>χ</italic><sup>2</sup>/df ratio between 2 and 3 is generally regarded as an acceptable or good data-model fit. There is general agreement that RMSEA values ≤ 0.05 can be considered a good fit, and those between 0.05 and 0.08 are an adequate fit. Values of GFI and AGFI range from zero to one with values higher than 0.9 and 0.85 usually interpreted as an acceptable fit. CFI is an incremental relative fit index. Values range from zero to one and higher values are preferred. CFI ≥ 0.95 are indicative of a good fit [##UREF##23##38##, ##UREF##25##41##] although some sources suggest values ≥ 0.90 are acceptable [##UREF##25##41##].</p>", "<p>Following the CFA, analysis of reliability of the final Resilience Skills Questionnaire (RSQ) in terms of internal consistency was assessed using Cronbach's alpha and interitem and item-total correlations. Alpha coefficients ≥ 0.70 have been considered acceptable for most scales, although an alpha coefficient between 0.80 and 0.95 suggests that a scale has good psychometric quality [##UREF##25##41##].</p>" ]
[ "<title>3. Results</title>", "<title>3.1. Item Development</title>", "<p>The content validity assessments showed that 13 of the sample items did not meet the required CVR value (0.54), so these items were removed. These were four self-efficacy items (8, 9, 12, and 14), six self-regulation items (6, 8, 9, 12, 13, and 14), and three social support items (6, 9, and 10) (see ##TAB##0##Table 1##). The mean CVR score of the remaining 25 items was 0.92 (minimum = 0.69 to maximum = 1). The mean CVI for the 25 items was 0.93 (minimum = 0.84 to maximum = 1). These values indicate excellent content validity for all items. The impact score of each item was greater than 1.5, thus all items had face validity and were suitable for further analysis in the quantitative phase of the study.</p>", "<title>3.2. Quantitative Results</title>", "<p>A total of 336 health sector professionals completed the developed questionnaire. The mean age of the sample was 39.6 years (57.3% were female). The majority were married (74%). The samples were highly educated: 25.5% had a PhD, 38.8% had a master's degree, 32.5% had a bachelor's degree, and 3% had a high school diploma. We had confirmed the sufficiency of the sample size using G<sup>∗</sup>Power software, and even before the reduction of predictor items from 38 to 25, the sample size of 336 was sufficient to perform a CFA with the power which was set at the conventional 0.80. Construct validity of the three-dimensional model was examined by EFA followed by CFA. EFA of the data showed KMO = 0.89, and Bartlett's test of sphericity which was statistically significant indicated the factorability of correlation matrix. The findings of the initial model of the CFA indicated that the factor loadings of four items in self-efficacy construct (7, 10, 11, and 13), two items in the self-regulation construct (7, 10), and two items in the social support construct (1, 8) were lower than the cut-off point (<italic>β</italic> &lt; 0.50), and the fit indices of the model were inappropriate. Thus, these items were removed (see final scale at the appendix), and a desirable model was achieved, which explained 53.4% of the total variance (as shown in ##TAB##1##Table 2## and ##FIG##0##Figure 1##).</p>", "<p>Construct validity for dimensions of the developed 17-item Resilience Skills Questionnaire (RSQ) was measured based on the CFA. The standardized regression weights were significant for all items in the final RSQ (<italic>p</italic> &lt; 0.001). The loading factors of the RSQ items were significant (<italic>p</italic> &lt; 0.001). The factor loadings were 0.63 for the 6-item self-efficacy dimension, 0.74 for the 6-item self-regulation dimension, and 0.65 for the 5-item social support dimension—all of which indicate high and favorable correlation between items and factor. In addition, all items of the questionnaire had acceptable internal consistency, and the relationships of each item with total score of each dimension were appropriate. The Cronbach's alpha coefficient of the RSQ was 0.903. Cronbach's alpha coefficients of each dimension and mean score (range 1-5), corrected item-total correlation, and Cronbach's alpha if item deleted of each dimension are presented in ##TAB##2##Table 3##. The corrected item total correlations of the items in the three dimensions each had sufficient discriminatory power to measure the considered dimension. The table also presents an indication of excellent test-retest reliability (ICC) of the RSQ. Finally, Pearson's correlation analysis showed that RSQ scores were correlated with RAW scores which supported convergent validity of the RSQ (<italic>r</italic> = 0.588, <italic>p</italic> &lt; 0.01). Items in the Resilience Skills Questionnaire according to subscales: self-efficacy, self-regulation, social support can be found in ##TAB##3##Table 4##, which is presented as an appendix.</p>" ]
[ "<title>4. Discussion</title>", "<p>The aim of this study was to develop and validate an instrument for assessing resilience skills based on the SCT to fill an identified gap in the literature [##UREF##8##12##, ##UREF##9##13##]. The objective was to provide a reliable and valid Resilience Skills Questionnaire to support the development of educational intervention programs to decrease the prevalence of work-related stress and other common mental health problems. We used a robust mixed method study design, informed by and compliant with the ten general recommendations of the COSMIN study design checklist for new measurement instruments [##UREF##18##28##]. This was designed to evaluate the methodological quality of research examining measurement properties [##REF##27259179##43##]. Accordingly, the design and validation stages examined the content and face validity of the new instrument, including the metric properties of the considered items, the internal consistency of the SCT dimension items, and overall reliability and structural validity of the developed questionnaire. The final RSQ is comprised of 17 items and three theory-driven dimensions: self-efficacy (6 items), self-regulation (6 items), and social support (5 items).</p>", "<p>To strengthen the validity of the new tool in its construction, we took advantage of the views of experts in psychology, health promotion, and public health to include different perspectives of resilience [##UREF##17##27##, ##REF##29942800##34##]. The experts were involved in developing items and then in reducing the initial pool of potential items to include through assessing content validity. These steps contributed to ensuring that the final RSQ was parsimonious and made up of appropriate items [##REF##29942800##34##]. The results confirmed that all the questionnaire constructs were suitable in terms of relevance, necessity, and clarity. The Cronbach's alpha for the whole scale and its dimensions also indicate that the developed RSQ has excellent internal consistency. Our initial test-retest result was excellent, and further research will confirm the degree of stability of results over time.</p>", "<p>Previous studies have identified the potentials for resilience training to support employees and organizations to manage risks of work-related stress and improve performance [##UREF##8##12##, ##UREF##9##13##]. These studies also identified a gap in the availability of suitable robust scales to measure the skills that underpin resilience to support educational intervention programs. Pertinent to this study, the Hartman et al.'s review [##UREF##9##13##] included two scales developed to support further research in work-related performance. The 20-item Workplace Resilience Questionnaire [##UREF##9##13##] is a four-factor model that captures features of the resilient individual as a stable trait, and thus, this would be unsuitable as not in line with the assumption that resilience is a dynamic concept that can be improved. The RAW scale [##REF##24064782##35##] was developed in Australia on the basis that resilience is a capability that can be developed to preserve the physical health of employees as well as improve engagement with work. RAW is a 20-item scale concerned with seven dimensions of workplace resilience; however, as suggested by the authors and from other studies, the RAW scale may have suboptimal reliability for two or three components which impacts on its usefulness [##REF##24064782##35##, ##UREF##27##44##]. For example, a study to validate the scale for use in India [##UREF##27##44##] yielded only a partial replication, and the authors proceeded with a six-factor scale with 17 items. Also, another study in Australia reported a three-factor structure of the RAW [##UREF##28##45##]. Altogether, the RAW is insufficient for our overarching purpose of providing a tool to drive an intervention to improve employee's resilience skills to reduce the prevalence of work-related stress and other common mental health problems. This also supported our decision to take a theory-driven approach to developing a new scale based on SCT. Although the RAW scale has been criticized, it has been used as a valid measure of resilience, and hence, it was appropriate for examining convergent validity of the RSQ. This was shown.</p>", "<p>Alongside the observation that there are various resilience scales measuring the concept from different perspectives [##UREF##9##13##], it has also been argued that there is a need for more robust evidence to address the fundamental question of whether resilience can be taught [##UREF##7##11##, ##UREF##8##12##]. With the development of the psychometrically sound RSQ, based on SCT, this theory-driven study has successfully approached the previous gap in the literature in terms of being able to develop interventions. The SCT was used to develop the new tool because it was conceptually appropriate and because previous research had particularly identified three component parts of the SCT as determinants of resilience [##UREF##9##13##, ##UREF##11##15##]. Future educational intervention studies using the RSQ will be able to test whether the general assumption that resilience can be taught is true and to what extent and in what contexts. The use of the RSQ developed in this study in intervention studies will serve to address these questions as well as support workplace wellbeing.</p>", "<title>4.1. Strengths and Limitations</title>", "<p>A strength of this study is that it has successfully developed a suitable Resilience Skills Questionnaire that was needed as a precursor to providing intervention programs to help employees to thrive in potentially stressful situations in the workplace [##REF##22429170##22##]. It used a suitable sector to recruit participants in terms of the underpinning issues around managing work-related stress, particularly in 2021. There are many studies in the literature to confirm that the COVID-19 pandemic introduced an upward trend in the prevalence of mental health problems in the workforce, and a significant volume of these studies has referred to the health sector as being particularly as risk [##REF##33170146##46##]. This provided the impetus for setting this study in the health sector and provides confidence that an appropriate population provided the data that underpinned the findings we report. Nevertheless, the RSQ was designed to be used across the range of work, and it will be necessary to test the psychometric properties in other types of workers. It follows from the latter point that a limitation of the present study was that data was collected from only one sector and one city in Iran due to financial constraints; therefore, evaluating the effectiveness of the RSQ in other studies is necessary. Another limitation is that the data are self-reported, and there is potential for social desirability bias to impact on some responses. An attempt to mitigate this bias was made through the use of anonymous online data collection.</p>" ]
[ "<title>5. Conclusion</title>", "<p>This study developed and validated a questionnaire to measure determinants of resilience skills in health sector employees based on the SCT. The developed RSQ has appropriate psychometric properties, and it can be used as a standard and valid measure to evaluate the resilience skills of employees. This can, in turn, support longitudinal intervention programs to enhance employers' input into stress management and supporting the mental health of their workforce through the improvement of resilience skills. Future studies are required to confirm the generalizability of the RSQ in other areas of work.</p>" ]
[ "<p>Academic Editor: Dorota Formanowicz</p>", "<p>Good resilience skills support effective and timely adjustment to demanding situations in the workplace. Existing tools are insufficient to develop and evaluate workplace interventions to improve employee's resilience skills. The aim of this study was to develop and validate a Resilience Skills Questionnaire (RSQ) using the key constructs of social cognitive theory—self-efficacy, self-regulation, and social support—as a theoretical framework. Following DeVellis' guidelines for scale development, first an expert panel of thirteen professors was recruited to support the item development stages and determine content validity. At this stage, the initial pool of 38 items was reduced to 25 items and CVR and CVI were calculated as 0.92 and 0.93, respectively, indicating good content validity. A second panel of ten health professionals confirmed face validity. An online survey comprised of the 25 developed items was then completed by 336 health professionals working in urban healthcare centers in Shiraz, Iran, in November 2021. The data were used to assess the psychometrics of the questionnaire according to its hypothesized three-dimensional structure. Confirmatory factor analysis yielded a final model of seventeen items in three dimensions, self-efficacy (six items), social support (six items), and self-regulation (five items), with good psychometric properties (<italic>χ</italic><sup>2</sup>/df = 2.44 (<italic>p</italic> &lt; 0.001), RMSEA = 0.06, GFI = 0.92, AGFI = 0.90, IFI = 0.93, CFI = 0.93). All standardized factor loadings were significant (<italic>p</italic> &lt; 0.001). Internal consistency, as measured by Cronbach's alpha, was very good: RSQ (0.90), self-efficacy (0.86), social support (0.83), and self-regulation (0.86). Based on these results, the RSQ can be used as a standard and valid measure to develop and evaluate the effect of educational intervention programs to improve resilience skills and reduce job stress.</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>The authors would like to thank health center employees who participated in the study. This work was supported by Shiraz University of Medical Sciences for study design, data collection, interpretation of findings, and writing the manuscript. The university was not involved in the writing of this paper, nor did it read or approve it.</p>", "<title>Appendix</title>", "<title>The Resilience Skills Questionnaire</title>", "<p>Instructions. Please indicate your agreement with the following statements from your experiences in your workplace using the following responses: 1 = strongly disagree; 2 = agree; 3 = neutral; 4 = agree; 5 = strongly agree.</p>", "<title>Data Availability</title>", "<p>The data used to support the findings of this study are included within the article.</p>", "<title>Ethical Approval</title>", "<p>The study was approved by the Research Board Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1400.157). All methods were performed in accordance with the relevant guidelines and regulations of the Declaration of Helsinki.</p>", "<title>Consent</title>", "<p>First, the study purposes were explained to the participants. Then, written informed consent forms to fill questionnaire were obtained voluntarily before data collection.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' Contributions</title>", "<p>MA and MK contributed to the original design, the data collection, and the analysis and writing of the manuscript. HM contributed to the original design and editing the manuscript. RC, MJ, and CR approved and edited the final version of the manuscript. All authors read and approved the final manuscript.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Confirmatory factor analysis with standardized item loadings according to dimension for the final 17-item, 3-dimension Resilience Skills Questionnaire. efcy = self-efficacy; regu = self-regulation; supp = social support.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Sample items for assessing resilience according to SCT constructs.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">SCT construct</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Sample items</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Self-efficacy (efcy)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) When a problem upsets me a lot, I can evaluate my emotions, behavior, and beliefs about the problem.<break/>(2) If I hear my colleagues talking about me, I can still concentrate on my work.<break/>(3) If a colleague disrespects me, I can still consider the part I played in the incident.<break/>(4) When negative thoughts affect me, I can assess the pros and cons of those thoughts.<break/>(5) When I am very upset, I can reflect on the reason enough to approve or reject my negative thoughts.<break/>(6) When a problem makes me angry or distressed, I can think of ways to solve the problem.<break/>(7) Even when I am very busy, if unwanted thoughts come to me, I can dismiss them.<break/>(8) Even when I have a lot on my mind, I can listen to others when speaking with them.<break/>(9) While speaking with others, I wait for them to finish their point even if their conversation is too long or irrelevant.<break/>(10) Whenever I am talking with people, I can focus on their facial expressions.<break/>(11) If I am agitated by someone, I can plainly explain the reason to them.<break/>(12) I can comfort with my colleagues when a problem arises.<break/>(13) If I am asked to do something unusual, I can frankly decline to do it.<break/>(14) If I ever lose my temper with people, I can leave to room recognizing I need to calm down.</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Self-regulation (regu)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) I evaluate my thoughts regularly.<break/>(2) When I evaluate my thoughts, I know that I should modify my thoughts.<break/>(3) I evaluate my relationship with others regularly.<break/>(4) When I evaluate my relationship with others, I consider how I can improve my relationships.<break/>(5) I evaluate my emotions regularly.<break/>(6) When I evaluate my emotions, I consider how I can modify what I do.<break/>(7) I have undertaken some training to help me understand my thoughts.<break/>(8) I have undertaken training or studied books to develop my communication skills.<break/>(9) I have undertaken resilience skills training to improve control of my emotions.<break/>(10) I always ask myself whether I could clear away my negative thoughts.<break/>(11) I ask my colleagues for feedback on whether I have improved my communications with others.<break/>(12) I evaluate my deeds regularly to know whether if I have been successful in controlling my emotions.<break/>(13) When I write down about my negative thoughts, I encourage myself.<break/>(14) When I manage to control any negative emotions, I encourage and appreciate myself.</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Social support (supp)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) I have colleagues that help me with problems.<break/>(2) I have colleagues to motivate me when I am training to evaluate my thoughts.<break/>(3) My family help me to think about my communication skills.<break/>(4) I have at least one family member to guide me to know my emotions (e.g., anger, sadness, jealousy, anxiety, feeling blameworthy, and disappointment).<break/>(5) When I have negative emotions, I can talk about them with my family.<break/>(6) When I am upset or anxious, I can talk with my colleagues about it.<break/>(7) When I feel blameworthy for a mistake, I have friends to discuss them with me.<break/>(8) When I am under pressure at work, my family helps me with other issues.<break/>(9) When I am on leave, my colleagues help with my tasks.<break/>(10) If ever I need money, there are always some people who will help me.</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Fit indices of the confirmatory factor analysis of the RSQ.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Model fit index</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Modified model</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chi-square/degrees of freedom (<italic>χ</italic><sup>2</sup>/df)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">283.444/116 = 2.44<sup>∗∗</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Goodness-of-fit index</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.92</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adjusted goodness-of-fit index</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Incremental fit index</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.93</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Root mean square error of approximation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Mean (SD), corrected item-total correlations, Cronbach's alpha, <italic>R</italic><sup>2</sup>, and ICC of RSQ.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Dimension</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Item</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Mean (SD)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Corrected item-total correlation</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Cronbach's alpha if item deleted</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Cronbach's alpha</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>R</italic>\n<sup>2</sup>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">ICC</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"6\" colspan=\"1\">Self-efficacy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Efcy1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.37 (0.99)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.607</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.843</td><td align=\"center\" rowspan=\"6\" colspan=\"1\">0.859</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.384</td><td align=\"center\" rowspan=\"6\" colspan=\"1\">0.869</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Efcy2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.08 (1.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.626</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.841</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.464</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Efcy3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.54 (0.95)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.655</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.835</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.518</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Efcy4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.27 (0.97)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.737</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.820</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.621</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Efcy5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.30 (0.94)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.706</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.826</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.667</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Efcy6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.49 (0.99)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.581</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.848</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.367</td></tr><tr><td align=\"center\" colspan=\"8\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"6\" colspan=\"1\">Self-regulation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sr1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.31 (1.02)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.576</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.857</td><td align=\"center\" rowspan=\"6\" colspan=\"1\">0.863</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.385</td><td align=\"center\" rowspan=\"6\" colspan=\"1\">0.823</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sr2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.54 (0.85)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.715</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.831</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.542</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sr3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.56 (0.85)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">628</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.846</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.452</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sr4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.17 (0.96)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.741</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.825</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.625</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sr5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.51 (0.84)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.665</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.840</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.391</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sr11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.04 (0.97)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.643</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.843</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.498</td></tr><tr><td align=\"center\" colspan=\"8\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"5\" colspan=\"1\">Social support</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ss2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.41 (0.93)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.537</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.814</td><td align=\"center\" rowspan=\"5\" colspan=\"1\">0.825</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.359</td><td align=\"center\" rowspan=\"5\" colspan=\"1\">0.887</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ss3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.57 (0.95)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.689</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.769</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">596</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ss4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.64 (0.90)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.728</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.759</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.678</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ss5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.57 (0.94)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.607</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.794</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.447</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ss7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.55 (0.85)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.544</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.811</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.266</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>Items in the Resilience Skills Questionnaire according to subscales: self-efficacy, self-regulation, social support.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Statements</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) When a problem upsets me a lot, I can evaluate my emotions, behavior, and beliefs about the problem.<break/>(2) If I hear my colleagues talking about me, I can still concentrate on my work.<break/>(3) If a colleague disrespects me, I can still consider the part I played in the incident.<break/>(4) When negative thoughts affect me, I can assess the pros and cons of those thoughts.<break/>(5) When I am very upset, I can reflect on the reason enough to approve or reject my negative thoughts.<break/>(6) When a problem makes me angry or distressed, I can think of ways to solve the problem.</td></tr><tr><td align=\"center\" colspan=\"1\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) I evaluate my thoughts regularly.<break/>(2) When I evaluate my thoughts, I know that I should modify my thoughts.<break/>(3) I evaluate my relationship with others regularly.<break/>(4) When I evaluate my relationship with others, I consider how I can improve my relationships.<break/>(5) I evaluate my emotions regularly.<break/>(6) I ask my colleagues for feedback on whether I have improved my communications with others.</td></tr><tr><td align=\"center\" colspan=\"1\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(1) I have colleagues to motivate me when I am training to evaluate my thoughts.<break/>(2) My family help me to think about my communication skills.<break/>(3) I have at least one family member to guide me to know my emotions (e.g., anger, sadness, jealousy, anxiety, feeling blameworthy, and disappointment).<break/>(4) When I have negative emotions, I can talk about them with my family.<break/>(5) When I feel blameworthy for a mistake, I have friends to discuss it with me.</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[]
[ "<table-wrap-foot><fn><p>\n<sup>∗∗</sup>\n<italic>p</italic> &lt; 0.001.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>1 = strongly disagree; 2 = agree; 3 = neutral; 4 = agree; 5 = strongly agree, Response.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"BMRI2024-5660620.001\" position=\"float\"/>" ]
[]
[{"label": ["1"], "person-group": ["\n"], "surname": ["Mackay", "Cousins", "Kelly", "Lee", "McCaig"], "given-names": ["C. J.", "R.", "P. J.", "S.", "R. H."], "article-title": ["\u2018Management standards\u2019 and work-related stress in the UK: policy background and science"], "source": ["\n"], "italic": ["Work & Stress"], "year": ["2004"], "volume": ["18"], "issue": ["2"], "fpage": ["91"], "lpage": ["112"], "pub-id": ["10.1080/02678370410001727474", "2-s2.0-4444329397"]}, {"label": ["2"], "person-group": ["\n"], "surname": ["Cousins", "Mackay", "Clarke", "Kelly", "Kelly", "McCaig"], "given-names": ["R.", "C. J.", "S. D.", "C.", "P. J.", "R. H."], "article-title": ["\u2018Management standards\u2019 work-related stress in the UK: practical development"], "source": ["\n"], "italic": ["Work & Stress"], "year": ["2004"], "volume": ["18"], "issue": ["2"], "fpage": ["113"], "lpage": ["136"], "pub-id": ["10.1080/02678370410001734322", "2-s2.0-4444348581"]}, {"label": ["4"], "person-group": ["\n"], "surname": ["Margrove", "Smith"], "given-names": ["G.", "A. P."], "source": ["\n"], "italic": ["The demands-resources-individual effects (DRIVE) model"], "year": ["2022"], "publisher-name": ["IGI Global"], "pub-id": ["10.4018/978-1-6684-3937-1.ch002"]}, {"label": ["5"], "person-group": ["\n"], "surname": ["Cicchetti", "Garmezy"], "given-names": ["D.", "N."], "article-title": ["Prospects and promises in the study of resilience"], "source": ["\n"], "italic": ["Development and Psychopathology"], "year": ["1993"], "volume": ["5"], "issue": ["4"], "fpage": ["497"], "lpage": ["502"], "pub-id": ["10.1017/S0954579400006118", "2-s2.0-84971895294"]}, {"label": ["6"], "person-group": ["\n"], "surname": ["Masten", "Neemann", "Andenas"], "given-names": ["A. S.", "J.", "S."], "article-title": ["Life events and adjustment in adolescents: the significance of event independence, desirability, and chronicity"], "source": ["\n"], "italic": ["Journal of Research on Adolescence"], "year": ["1994"], "volume": ["4"], "issue": ["1"], "fpage": ["71"], "lpage": ["97"], "pub-id": ["10.1207/s15327795jra0401_5"]}, {"label": ["9"], "person-group": ["\n"], "surname": ["Eisenberg", "Lipson", "Posselt"], "given-names": ["D.", "S.", "J."], "article-title": ["Promoting resilience, retention, and mental health"], "source": ["\n"], "italic": ["New Directions for Student Services"], "year": ["2016"], "volume": ["156"], "fpage": ["87"], "lpage": ["95"], "pub-id": ["10.1002/ss.20194"]}, {"label": ["10"], "person-group": ["\n"], "surname": ["Yeager", "Dweck"], "given-names": ["D. S.", "C. S."], "article-title": ["Mindsets that promote resilience: when students believe that personal characteristics can be developed"], "source": ["\n"], "italic": ["Educational Psychologist"], "year": ["2012"], "volume": ["47"], "issue": ["4"], "fpage": ["302"], "lpage": ["314"], "pub-id": ["10.1080/00461520.2012.722805", "2-s2.0-84867758501"]}, {"label": ["11"], "person-group": ["\n"], "surname": ["Modesto-Lowe", "LaBell", "Meyers"], "given-names": ["V.", "J. C.", "N. Z."], "article-title": ["Can you teach resilience?"], "source": ["\n"], "italic": ["Journal of Clinical Psychiatry"], "year": ["2021"], "volume": ["82"], "issue": ["5, article 35481"], "pub-id": ["10.4088/JCP.21lr14035"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Robertson", "Cooper", "Sarkar", "Curran"], "given-names": ["I. T.", "C. L.", "M.", "T."], "article-title": ["Resilience training in the workplace from 2003 to 2014: a systematic review"], "source": ["\n"], "italic": ["Journal of Occupational and Organizational Psychology"], "year": ["2015"], "volume": ["88"], "issue": ["3"], "fpage": ["533"], "lpage": ["562"], "pub-id": ["10.1111/joop.12120", "2-s2.0-84937818048"]}, {"label": ["13"], "person-group": ["\n"], "surname": ["Hartmann", "Weiss", "Newman", "Hoegl"], "given-names": ["S.", "M.", "A.", "M. J."], "article-title": ["Resilience in the workplace: a multilevel review and synthesis"], "source": ["\n"], "italic": ["Applied Psychology"], "year": ["2020"], "volume": ["69"], "issue": ["3"], "fpage": ["913"], "lpage": ["959"], "pub-id": ["10.1111/apps.12191", "2-s2.0-85063961576"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Bandura"], "given-names": ["A."], "article-title": ["The self system in reciprocal determinism"], "source": ["\n"], "italic": ["American Psychologist"], "year": ["1978"], "volume": ["33"], "issue": ["4"], "fpage": ["344"], "lpage": ["358"], "pub-id": ["10.1037/0003-066X.33.4.344", "2-s2.0-0005402295"]}, {"label": ["15"], "person-group": ["\n"], "surname": ["Wood", "Bandura"], "given-names": ["R.", "A."], "article-title": ["Social cognitive theory of organizational management"], "source": ["\n"], "italic": ["Academy of Management Review"], "year": ["1989"], "volume": ["14"], "issue": ["3"], "fpage": ["361"], "lpage": ["384"], "pub-id": ["10.2307/258173"]}, {"label": ["19"], "person-group": ["\n"], "surname": ["Alonso-Tapia", "Garrido-Hernansaiz", "Rodr\u00edguez-Rey", "Ruiz", "Nieto"], "given-names": ["J.", "H.", "R.", "M.", "C."], "article-title": ["Personal factors underlying resilience: development and validation of the resiliency questionnaire for adults"], "source": ["\n"], "italic": ["International Journal of Mental Health Promotion"], "year": ["2017"], "volume": ["19"], "issue": ["2"], "fpage": ["104"], "lpage": ["117"], "pub-id": ["10.1080/14623730.2017.1297248", "2-s2.0-85014536159"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Cusack", "Smith", "Hegney"], "given-names": ["L.", "M.", "D."], "article-title": ["Exploring environmental factors in nursing workplaces that promote psychological resilience: constructing a unified theoretical model"], "source": ["\n"], "italic": ["Frontiers in Psychology"], "year": ["2016"], "volume": ["7"], "fpage": ["p. 600"], "pub-id": ["10.3389/fpsyg.2016.00600", "2-s2.0-84974715336"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Hayden"], "given-names": ["J."], "source": ["\n"], "italic": ["Introduction to health behavior theory"], "year": ["2022"], "publisher-name": ["Jones & Bartlett Learning"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Peccoralo", "Depierro", "Feingold"], "given-names": ["L.", "J.", "J. H."], "article-title": ["Building resilience and well-being in health care workers in the COVID-19 era: lessons learned and next steps\u2014reply to Modesto-Lowe et al"], "source": ["\n"], "italic": ["The Journal of Clinical Psychiatry"], "year": ["2021"], "volume": ["82"], "issue": ["5, article 35501"], "pub-id": ["10.4088/jcp.21lr14035b"]}, {"label": ["26"], "person-group": ["\n"], "surname": ["Gold"], "given-names": ["J. A."], "article-title": ["Covid-19: adverse mental health outcomes for healthcare workers"], "source": ["\n"], "italic": ["BMJ"], "year": ["2020"], "volume": ["369, article m1815"], "pub-id": ["10.1136/bmj.m1815"]}, {"label": ["27"], "person-group": ["\n"], "surname": ["DeVellis", "Thorpe"], "given-names": ["R. F.", "C. T."], "source": ["\n"], "italic": ["Scale Development: Theory and Applications"], "year": ["2021"], "publisher-name": ["Sage Publications"]}, {"label": ["28"], "person-group": ["\n"], "surname": ["Mokkink", "Prinsen", "Patrick"], "given-names": ["L. B.", "C. A.", "D. L."], "source": ["\n"], "italic": ["COSMIN Study Design Checklist for Patient-Reported Outcome Measurement Instruments"], "year": ["2019"], "publisher-loc": ["Amsterdam, The Netherlands"], "publisher-name": ["BMJ Publishing Group"]}, {"label": ["30"], "person-group": ["\n"], "surname": ["Kyriazos"], "given-names": ["T. A."], "article-title": ["Applied psychometrics: sample size and sample power considerations in factor analysis (EFA, CFA) and SEM in general"], "source": ["\n"], "italic": ["Psychology"], "year": ["2018"], "volume": ["9"], "issue": ["8"], "fpage": ["2207"], "lpage": ["2230"], "pub-id": ["10.4236/psych.2018.98126"]}, {"label": ["31"], "person-group": ["\n"], "surname": ["Memon", "Ting", "Cheah", "Thurasamy", "Chuah", "Cham"], "given-names": ["M. A.", "H.", "J. H.", "R.", "F.", "T. H."], "article-title": ["Sample size for survey research: review and recommendations"], "source": ["\n"], "italic": ["Journal of Applied Structural Equation Modeling"], "year": ["2020"], "volume": ["4"], "issue": ["2"], "fpage": ["1"], "lpage": ["20"]}, {"label": ["32"], "person-group": ["\n"], "surname": ["Taherdoost"], "given-names": ["H."], "article-title": ["Validity and reliability of the research instrument; how to test the validation of a questionnaire/survey in a research"], "source": ["\n"], "italic": ["International Journal of Academic Research in Management"], "year": ["2016"], "volume": ["5"], "fpage": ["28"], "lpage": ["36"], "pub-id": ["10.2139/ssrn.3205040"]}, {"label": ["36"], "person-group": ["\n"], "surname": ["Gregory"], "given-names": ["R. J."], "source": ["\n"], "italic": ["Psychological Testing: History, Principles, and Applications"], "year": ["2015"], "edition": ["7th"], "publisher-loc": ["England"], "publisher-name": ["Pearson Education Ltd"]}, {"label": ["38"], "person-group": ["\n"], "surname": ["Rosner"], "given-names": ["B."], "source": ["\n"], "italic": ["Fundamentals of Biostatistics"], "year": ["2015"], "edition": ["8th"], "publisher-loc": ["Boston, US"], "publisher-name": ["Cengage Learning"]}, {"label": ["39"], "person-group": ["\n"], "surname": ["Shrestha"], "given-names": ["N."], "article-title": ["Factor analysis as a tool for survey analysis"], "source": ["\n"], "italic": ["American Journal of Applied Mathematics and Statistics"], "year": ["2021"], "volume": ["9"], "issue": ["1"], "fpage": ["4"], "lpage": ["11"], "pub-id": ["10.12691/ajams-9-1-2"]}, {"label": ["41"], "person-group": ["\n"], "surname": ["Hsu", "Su", "Kao", "Shu", "Lin", "Tseng"], "given-names": ["I.-Y.", "T.-S.", "C.-S.", "Y.-L.", "P.-R.", "J.-M."], "article-title": ["Analysis of business safety performance by structural equation models"], "source": ["\n"], "italic": ["Safety Science"], "year": ["2012"], "volume": ["50"], "issue": ["1"], "fpage": ["1"], "lpage": ["11"], "pub-id": ["10.1016/j.ssci.2011.04.012", "2-s2.0-80053213279"]}, {"label": ["42"], "person-group": ["\n"], "surname": ["Schermelleh-Engel", "Moosbrugger", "M\u00fcller"], "given-names": ["K.", "H.", "H."], "article-title": ["Evaluating the fit of structural equation models: tests of significance and descriptive goodness of fit measures"], "source": ["\n"], "italic": ["Methods of Psychological Research Online"], "year": ["2003"], "volume": ["8"], "fpage": ["23"], "lpage": ["74"]}, {"label": ["44"], "person-group": ["\n"], "surname": ["Malik", "Garg"], "given-names": ["P.", "P."], "article-title": ["Psychometric testing of the resilience at work scale using Indian sample"], "source": ["\n"], "italic": ["Vikalpa"], "year": ["2018"], "volume": ["43"], "issue": ["2"], "fpage": ["77"], "lpage": ["91"], "pub-id": ["10.1177/0256090918773922", "2-s2.0-85048961241"]}, {"label": ["45"], "person-group": ["\n"], "surname": ["Holdsworth", "Turner", "Scott-Young"], "given-names": ["S.", "M.", "C. M."], "article-title": ["\u2026 not drowning, waving. Resilience and university: a student perspective"], "source": ["\n"], "italic": ["Studies in Higher Education"], "year": ["2018"], "volume": ["43"], "issue": ["11"], "fpage": ["1837"], "lpage": ["1853"], "pub-id": ["10.1080/03075079.2017.1284193", "2-s2.0-85012284380"]}]
{ "acronym": [ "RSQ:", "SCT:", "CVR:", "CVI:", "CFA:", "\nχ\n2/df:", "GFI:", "AGFI:", "CFI:", "RMSEA:", "COSMIN:", "RAW:", "ICC:", "efcy:", "regu:", "supp:" ], "definition": [ "Resilience Skills Questionnaire", "Social cognitive theory", "Content validity ratio", "Content validity index", "Confirmatory factor analysis", "Chi-square/degrees of freedom ratio", "Goodness-of-fit index", "Adjusted goodness-of-fit index", "Comparative fit index", "Root mean square error of approximation", "COnsensus Study for the Selection of health status Measurement INstruments", "Resilience at Work", "Intraclass correlation", "Self-efficacy", "Self-regulation", "Social support." ] }
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2024-01-14 23:43:50
Biomed Res Int. 2024 Jan 6; 2024:5660620
oa_package/4b/b8/PMC10787653.tar.gz
PMC10787654
0
[ "<title>1. Introduction</title>", "<p>Intussusception is commonly seen in children but rarely seen in adulthood and is often secondary to tumors, motility disorders, or inflammatory diseases [##REF##30059860##1##]. Intussusception in adults accounts for approximately 1% of all bowel obstructions [##REF##30059860##1##]. Colonic lipomas are usually asymptomatic and are found incidentally during autopsy, colonoscopy, or surgery, but larger ones may cause symptoms like bleeding, constipation, or pain [##REF##28224035##2##]. Larger lipomas may be difficult to differentiate from malignancy before surgery; hence, diagnosis can be made from histological evaluation [##REF##28224035##2##, ##REF##34768668##3##]. Herein, we present an uncommon case of colonic lipoma causing colo-colic intussusception in an adult.</p>" ]
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[ "<title>3. Discussion</title>", "<p>Colon lipoma is a rare benign adipose tumor of the gastrointestinal tract, first described by Bauer in 1757 [##REF##19927786##4##, ##REF##20966621##5##]. The incidence ranges between 0.2 and 4.4% with a female predominance, frequently affecting women aged between 40 and 70 years [##REF##8956963##6##–##REF##9548092##8##]. Colonic lipomas typically arise from the submucosal area in approximately 90% of cases as in the index case, but may extend into the muscularis propria and 10% are subserosal, with sizes varying between 2 mm and 30 cm [##REF##20966621##5##, ##REF##8956963##6##, ##REF##4026070##9##]. In an up-to-date systematic review in 2021 by Menegon Tasselli et al., colonic lipoma causing colo-colic intussusception was predominantly localized in the transverse colon, followed by the sigmoid colon, the cecum, the ascending colon, the descending colon, and least commonly found in the rectum [##REF##34768668##3##]. Our case was comparatively consistent with the literature with regard to the gender, age, and location of the lipoma.</p>", "<p>Generally, they are small and asymptomatic, although 6%-25% of patients with colonic lipoma have symptoms, with abdominal pain being the most common, followed by constipation, rectal bleeding, and diarrhea [##REF##16400357##10##, ##REF##12072607##11##]. Adult intussusception is rare, constituting less than 5% of all intussusception cases, and a majority of them have a malignant etiology. Uncommon causes of intussusception include adenomas, polyps, endometriosis, and lipomas [##REF##30059860##1##, ##REF##19152443##12##]. Lipomas larger than 4 cm are considered giant lipomas and 88% of them cause colo-colic intussusception [##REF##34768668##3##].</p>", "<p>Computed tomography having a sensitivity of 71–87% and a specificity of up to 100% remains the preferred radiological modality for the diagnosis of intussusception [##REF##8661763##13##, ##REF##24171703##14##]. It allows for the clear identification of the intussuscepting tissue as well as the lead point, presenting the typical appearance known as the “target sign” or “sign of donut” as seen in our case [##REF##19152443##12##, ##REF##24966269##15##]. Studies have shown the use of MRI being superior due to its effectiveness in highlighting adipose lesions because of the peculiar characteristics of the signal intensity of this tissue, especially T1-weighted and fat-suppressed images [##REF##19927786##4##, ##REF##21153727##16##]. Colonoscopy has been used for direct vision of the lesion, biopsies, and therapeutic resection of lipomas with pedicles and diameters of less than 2 cm. Pathognomonic signs for lipoma on a colonoscopy include the “naked fat sign” or “bare fat mark sign” which is seen as leakage of fat after biopsy, “tenting sign” portraying a tent-like appearance once the covering mucosa is lifted with forceps, and “pillow mark sign” or “cushion sign” representing a soft lesion with a cushion-like mucosal indentation when pressed with closed biopsy forceps [##REF##19927786##4##, ##REF##19152443##12##, ##REF##29510213##17##, ##UREF##0##18##]. In our case, intussusception was present and diagnosis of lipoma was established via a CT scan before the surgery; hence, an MRI or colonoscopy was not required.</p>", "<p>The majority of authors advocate surgery as the standard method of treatment for every colonic lipoma greater than 2 cm in size [##REF##30059860##1##, ##REF##34712536##19##], although surgical resection remains the treatment of choice and produces an excellent prognosis. Lipomas with a diameter smaller than 2 cm or pedunculated lipomas with a thin stalk can be resected endoscopically [##REF##24171703##14##, ##UREF##1##20##]. Surgical treatment includes resection, colotomy with local excision, limited colon resection, segmental resection, hemicolectomy, or subtotal colectomy; the choice of the surgical interventions mainly depends on the size, location of the lipomas, and the presence or absence of definite preoperative diagnosis or disease complications [##REF##17948945##21##]. Surgical excision is highly recommended in the event of intussusception and intestinal obstruction, especially in elderly patients, because of the high risk of underlying malignancy [##UREF##2##22##]. The prognosis depends on the complete removal of the tumor [##REF##32542410##23##].</p>" ]
[ "<title>4. Conclusion</title>", "<p>Colo-colic intussusception is a rare complication of a rarely occurring colon lipoma. CT imaging remains the method of choice for studying abdominal lipomas, particularly those rising into the layers of the colonic wall. Surgical resection remains the treatment of choice and produces excellent prognosis.</p>" ]
[ "<p>Academic Editor: George Rallis</p>", "<p>Intussusception is rarely seen in adulthood but is commonly seen in the pediatric age group. Causes of intussusception in adults are commonly due to tumors and inflammatory diseases. Intussusception in adults accounts for less than 5% of intestinal obstruction. Colonic lipomas are usually asymptomatic and are mostly managed surgically with promising outcomes as seen in our case.</p>" ]
[ "<title>2. Case Presentation</title>", "<p>A 57-year-old female presented to our outpatient surgical clinic with a one-month history of left lower abdominal pain, which was colicky in nature, nonradiating, and associated with nonprojectile bilious vomiting with episodes of bloodstained mucoid stools. There was no history of abdominal distension, abdominal surgery, or weight loss. She had visited several health centers with no relief of symptoms despite medical therapy. She denied a history of weight loss and cancers in her family.</p>", "<p>Upon arrival, she was not pale, mildly dehydrated, not jaundiced with vitals within normal range and saturating at 98% on room air. Her abdominal examination revealed a nondistended abdomen with symmetrical abdominal contours, no mass nor organomegaly with 4 bowel sounds heard per minute on auscultation. A digital rectal examination revealed an anal tag at the 6 o'clock position, a normal anal tone, and no mass palpated. The gloved finger was stained with a copious amount of bloody stained mucoid stool.</p>", "<p>Her lab results showed a leucocyte count of 11.39 × 10<sup>9</sup>/L, hemoglobin of 11.2 g/dL, platelet of 583 × 10<sup>9</sup>/L, creatinine of 52 <italic>μ</italic>mol/L, and normal liver enzymes. Her abdominal CT scan revealed a target sign at the descending colon suggestive of colo-colic intussusception measuring 11.6 × 5.2 × 5.3 cm with a lipoma measuring 3.7 × 4.3 × 3.5 cm as a lead point (##FIG##0##Figure 1##). She was admitted and was taken for an emergency laparotomy, whereby a colo-colic intussusception with a 6 × 5 cm lipomatous intraluminal mass in the distal transverse colon was found with an edematous colon and its mesentery. The intussusception was successfully reduced manually, then the mass with a 5 cm negative margin of the transverse colon was resected, and a transverse double-barrel colostomy was raised successfully as the colon was not prepared to have fecal content within.</p>", "<p>Her postoperative recovery was uneventful and was discharged on day three postoperatively with instructions on colostomy care and to return to the surgical outpatient clinic for follow-up and wound care. Histology of the resected mass revealed a submucosal mass completely obstructing the large bowel lumen with microscopic features of reactive colon mucosa, a tumor composed of lobules of bland mature fat cells suggestive of a lipoma with negative margins (##FIG##1##Figure 2##).</p>", "<p>She was then reviewed in the outpatient clinic and scheduled for colostomy closure after 3 months. Her preoperative blood works were within normal range. Her previous incision had healed with a functioning transverse colostomy (##FIG##2##Figure 3##). Colostomy closure was done after thorough bowel preparation, was discharged two days later, and had an uneventful follow-up visit.</p>" ]
[ "<title>Acknowledgments</title>", "<p>The authors would like to thank the patient for the permission to share her medical history for educational purposes and publication.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that there is no conflict of interest regarding the publication of this article.</p>", "<title>Authors' Contributions</title>", "<p>JS, MS, and JL conceptualized and drafted the manuscript, and AM and DM reviewed the medical records. AM reviewed and reported the histology films, and AS reported and prepared the radiology images. All authors have read and approved the final manuscript.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>CT scan showing intussusception due to colon lipoma. Contrasted abdomen and pelvis CT. Axial (a), coronal (b, c), and sagittal (d) views show intussuscepted transverse colon (intussusceptum) with the mesenteric fat and vessels telescoped into the lumen of the descending colon (intussuscipiens) as shown in yellow arrows. The lead point is a lipoma (blue arrows), which is clearly demonstrated distally. Free fluid in the abdomen.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>Photomicrographs showing colon lipoma histology. (a) Benign well-circumscribed colonic neoplasm composed of mature adipose tissue arising from submucosa with normal overlying mucosa. H&amp;E stained low power (20x original magnification). (b) Histopathology of colonic lipoma. Intermediate magnification (40x original magnification).</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>Pre- and postoperative clinical photographs. (a) Clinical photograph after first laparotomy showing double-barrel transverse colostomy. (b) Post colostomy closure (second surgery).</p></caption></fig>" ]
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[{"label": ["18"], "person-group": ["\n"], "surname": ["Dandin", "Balta", "Sucullu", "Yucel", "Yildirim"], "given-names": ["\u00d6.", "A. Z.", "I.", "E.", "S."], "article-title": ["Case report: a rare cause of abdominal pain and anemia: giant colonic lipoma"], "source": ["\n"], "italic": ["Turkish Journal of Colorectal Disease"], "year": ["2011"], "volume": ["21"], "issue": ["3"], "fpage": ["116"], "lpage": ["119"], "pub-id": ["10.5505/KRHD.2011.64935"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Atmatzidis", "Chatzimavroudis", "Patsas"], "given-names": ["S.", "G.", "A."], "article-title": ["Pedunculated cecal lipoma causing colo-colonic intussusception: a rare case report"], "source": ["\n"], "italic": ["Case Reports in Surgery"], "year": ["2012"], "volume": ["2012"], "fpage": ["3"], "pub-id": ["279213", "10.1155/2012/279213"]}, {"label": ["22"], "person-group": ["\n"], "surname": ["Croome", "Colquhoun"], "given-names": ["K. P.", "P. H."], "article-title": ["Intussusception in adults"], "source": ["\n"], "italic": ["Canadian Journal of Surgery"], "year": ["2007"], "volume": ["50"], "issue": ["6"], "fpage": ["E13"], "lpage": ["E14"]}]
{ "acronym": [], "definition": [] }
23
CC BY
no
2024-01-14 23:43:50
Case Rep Surg. 2024 Jan 6; 2024:7777258
oa_package/e9/bb/PMC10787654.tar.gz
PMC10787655
0
[ "<title>1. Introduction</title>", "<p>Point-of-care ultrasonography (POCUS) is defined as the acquisition, interpretation, and immediate clinical integration of ultrasonographic imaging performed by acute care clinicians at the patient's bedside rather than by a radiologist or cardiologist [##REF##34670045##1##, ##REF##21345104##2##]. With the increasing quality and availability of ultrasound equipment, POCUS is being more widely performed by a variety of specialists [##REF##30577046##3##]. Despite the growth in the importance and presumed benefits of POCUS, there is no comprehensive and generally accepted consensus on the required level of investigation at the time of writing this article [##REF##34670045##1##, ##REF##33467969##4##–##REF##31359188##20##]. The absence of a training programme and the introduction of this diagnostic method without clearly defined examination protocols and rules for its use led the Joint Commission on Accreditation of Healthcare Organizations and the Emergency Care Research Institute to identify the adoption of POCUS as a major health technology hazard in 2020 [##UREF##4##21##]. In the same year, a statement on the need for structured educational programmes for POCUS was published by the Ultrasound Working Group of the European Federation of Internal Medicine [##REF##31836177##22##]. Additionally, in 2020, the American Society of Echocardiography stated that to ensure high-quality care, cardiologists should be involved in the education and direct training of clinicians who perform cardiac ultrasonography [##REF##32122742##23##]. With reference to Dr. Kimura's original work, the different levels of cardiac ultrasound examination are described according to the extent of the examination [##REF##32122742##23##, ##REF##28259843##24##]. The definition of these terms is essential to define a universal POCUS training programme for use across different specialties (internal medicine, emergency medicine, intensive care, anaesthesia, etc.). Cardiac ultrasound categories are shown in ##TAB##0##Table 1##. The findings of cardiac POCUS should always be evaluated in the context of a comprehensive examination with the awareness of the potential for misinterpretation in view of the known limitations of this method (time constraints, limited patient examinability, examiner experience, etc.) [##REF##34382077##12##, ##REF##32034674##19##, ##REF##29529170##25##, ##UREF##5##26##]. In 2013, the European Association of Cardiovascular Imaging (EACVI) recommended following the ABCD approach when performing emergency echocardiography, which is shown in ##TAB##1##Table 2## [##REF##29529170##25##, ##REF##23239795##27##].</p>" ]
[]
[]
[ "<title>4. Discussion</title>", "<p>POCUS is not defined by the scope of the examination but by the conditions under which the examination is performed and the level of expertise of the examining physician. We believe that it is good practice to teach cardiac POCUS to physicians with a wider range of specialties to ensure safe and confident use of this examination method in clinical practice. The scope of the actual examination must always be adapted to the clinical condition of the patient; this is the essence of POCUS. Compared to the established multiday courses for POCUS, the limited echocardiography training programme at the University Hospital Hradec Králové is time-consuming and requires the accessibility of the echocardiography department facilities and the availability of specialist physicians to consult with the candidate regarding their ultrasound findings.</p>", "<title>4.1. Limitation of the Analysis</title>", "<p>The main limitation of this analysis is the fact that the analysed subgroup consisted only of hospitalised patients. This may have led to underestimating errors in patients who were discharged due to an unrecognised echocardiographic finding. This limitation will be addressed by the ENDEMIC study. Another limitation is the delay of the supervision examination (4.2 days), during which some findings could have spontaneously resolved (transient systolic dysfunction, etc.).</p>" ]
[ "<title>5. Conclusion</title>", "<p>Our experience confirms that the concept of sonography training enables physicians of different specialties to perform standardised ultrasound examinations that, according to our retrospective evaluation, are accurate and reproducible and meet the requirements for safe use according to the ABCD approach. However, high accuracy alone does not justify the cost of a training programme, and there is currently little to no evidence of the clinical benefit of POCUS echocardiography by noncardiologists. To overcome this lack of evidence in patients with chest pain, the prospective, randomised ENDEMIC trial (<ext-link xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT05306730\" ext-link-type=\"uri\">NCT05306730</ext-link>) was initiated. Publication of the study results is anticipated in mid-2024.</p>" ]
[ "<p>Academic Editor: Canan Akman</p>", "<p>Point-of-care ultrasound examinations performed by physicians of different specialties are a rapidly growing phenomenon, which has led to a worldwide effort to create a standardised approach to ultrasound examination training. The implementation of emergency echocardiography by noncardiologists is mainly aimed at the standardisation of the procedure, a structured training system, and an agreement on competencies. This article summarises the current training programmes for nonechocardiographers at the University Hospital in Hradec Králové. In cooperation with cardiologists specialised in cardiac ultrasound (ECHO), an extended acute echo protocol dedicated to emergency department physicians was developed and validated in daily practice. According to our retrospective evaluation, after one year of clinical practice, we can confirm that point-of-care ultrasound examinations performed using the standardised limited echo protocol are safe and accurate. The observed concordance with comprehensive ECHO was 78%. This trial is registered with <ext-link xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT05306730\" ext-link-type=\"uri\">NCT05306730</ext-link>.</p>" ]
[ "<title>2. Cardiac POCUS Training Programme in the University Hospital Hradec Králové</title>", "<p>In 2020, on the initiative of the physicians of the First Internal Cardiology Clinic and the physicians of the Emergency Medicine Department, a pilot training programme for limited echocardiography was launched for physicians without cardiology qualifications. The British Society of Echocardiography (BSE) Level 1 protocol was chosen as the echocardiography training scope, extended by apical two- and three-chamber views and measurement of the tricuspid valve gradient for pulmonary hypertension estimation and the ascending aortic dimension. Color Doppler of the aortic and mitral valves from parasternal long and short axis views and the mitral valve from apical views was also added. This protocol was chosen to cover major abnormalities of the right and left ventricles and aortic, mitral, and tricuspid valves to assess pulmonary hypertension and pericardial effusion. It may also raise suspicion of a dissection of the ascending aorta. The protocol also includes a lung ultrasound (see ##FIG##0##Figure 1##) [##UREF##6##28##] to assess the most significant pulmonary diseases. The ultrasound training programme consists of the following steps.</p>", "<title>2.1. Step One: Basic Echocardiography Introduction—Theoretical Course</title>", "<p>Physicians who had not previously received systematic training in any form of ultrasonography were offered participation in the training programme.</p>", "<p>A three-hour introductory seminar is provided to introduce students to the principles of focused ultrasound examination and the required echocardiography protocol. The theoretical training includes a presentation of possible pathological findings on lung ultrasound and their place in the overall examination protocol. For theoretical preparation, a basic textbook on echocardiography is recommended (e.g., Echo Made Easy or Point of Care Ultrasound [##UREF##7##29##, ##UREF##8##30##]).</p>", "<p>Following the introduction, trainees under the guidance of a cardiologist learn how to operate the ultrasound machine to optimise the projection display and archive individual recordings.</p>", "<title>2.2. Step Two: Training in Clinical Practice</title>", "<p>The actual clinical training takes place at the Department of Emergency Medicine and the Department of Noninvasive Cardiology of the First Department of Internal Medicine. Under the supervision of trainers, who are board-certified cardiologists with many years of experience in echocardiography, trainees perform an ultrasound examination of the heart according to a defined protocol. Individual digital images are archived in a central repository. A structured record of each examination is stored in the hospital information system.</p>", "<p>The duration of this phase can vary, and it is necessary for trainees to become familiar with the most common pathologies required for the logbook. The minimum period of clinical training is three months.</p>", "<title>2.3. Step Three: The Final Assessment</title>", "<p>The formal examination takes place in the presence of two cardiologists with extensive echocardiography experience, one holding EACVI accreditation in adult transthoracic echocardiography and the other being proficient in emergency medicine.</p>", "<p>The examination consists of an independently performed echocardiographic examination as per protocol under the supervision of examiners who assess the quality and completeness of the study. The candidate makes a report based on acquired images, which is scrutinised for accuracy and guidance for the next management of the patient. In the second part, the candidate is presented with a randomly selected echo study based on the protocol. Correct interpretation of the findings is marked.</p>", "<p>In the case of a positive evaluation by both examiners, the trainees receive a certificate on the successful completion of the course. Based on this, the candidates' personal work competencies are extended by independently performing and reporting on emergency cardiac ultrasound examinations.</p>", "<p>To maintain examination quality and competence, a minimum of fifty examinations per year must be documented, and once per year, a four-hour stay at the echo department under the supervision of the supervisor is required to formally verify the maintenance of examination quality. All examinations must be recorded in the hospital information system.</p>", "<title>3. Effectiveness of the Training Programme: Experience after One Year of Practice</title>", "<p>In 2022, a total of 560 patients were examined in the emergency department using the limited echocardiography protocol by a noncardiologist who had successfully completed the training programme. Of these, 111 were admitted to the hospital. Full echocardiography examination was recommended by the physician performing the basic echo study in all cases and performed in 69 (62%) of the patients. In this group, the results of both scans were compared with the focus on left and right ventricle dimensions, left ventricle global and regional function, ascending aorta dimension, pulmonary hypertension, significant valvular abnormality and assessment of pericardial space, and inferior vena cava dimension assessment. The study was approved by the local ethics committee. The main focus was the revelation of unrecognised aortic dilatation, unrecognised or misrecognised significant left ventricular dysfunction (left ventricle ejection fraction &lt;40% or focal akinesis), right ventricular dilatation, pulmonary hypertension, significant valvular regurgitation, incorrect assessment of the pericardium and inferior vena cava, or any other missed significant findings resulting in incorrect clinical management. The comparison was limited by the fact that the mean time difference between basic and comprehensive echo studies was 4.2 days (median 2 days), and the clinical status of some patients varied by hours. We can, however, conclude in retrospect that concordance was found for 78% of the patients examined. In 15 cases (the remaining 22%), a discrepancy was found (1 case of overestimated aorta dimension, 2 cases of overestimated LV function, 1 case of underestimated LV function, 1 case of undescribed PK dilatation, 3 cases of undescribed pulmonary hypertension, 1 case of overestimated and 1 case of underestimated valvular lesion, 1 case of undescribed tricuspid annuloplasty ring, 1 case of suspected LV thrombus that was not confirmed, and 1 case of an undescribed regional wall motion abnormality). Of note, none of these discrepancies would have changed or influenced the therapeutic management of the patients.</p>" ]
[ "<title>Acknowledgments</title>", "<p>This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Specific Research Project No: SV/FVZ202101) and the Ministry of Defence of the Czech Republic—DRO of the University of Defence, Faculty of Military Health Sciences Hradec Kralove, Czech Republic—Clinical Disciplines II (DZRO-FVZ22-KLINIKA II).</p>", "<title>Data Availability</title>", "<p>Dataset is available from the corresponding author upon reasonable request.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no conflicts of interest.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Limited ECHO protocol.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Cardiac ultrasound categories.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"center\" colspan=\"4\" rowspan=\"1\">Point-of-care ultrasound</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Consultative ultrasound</th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\">UAPE</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">FOCUS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CCE</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Limited ECHO</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Comprehensive ECHO</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diagnostic expectations</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">“US stethoscope” used to augment bedside examination</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Focused exams with specific imaging protocols based upon suspicion of specific disease</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Focused on a collection of specific views/findings pertinent to the care of the critically ill (e.g., cardiac output, fluid response)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Limited imaging protocol applied to answer a specific question</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Comprehensive, all findings, use advanced technics</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Quantification</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Usually absent</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Optional</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Typically</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Typically</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mandatory</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Documentation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Not recorded</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Images archived, formal report</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Images archived, formal report</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Images archived on PACS, formal report</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Images archived on PACS, formal report</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Teaching required</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Weeks</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Weeks to months</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Months</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Months to years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Years</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>ABCD approach.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(A) Awareness</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Avoiding routine</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(B) Be suspicious</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Verification of ultrasound findings in the context of clinical and other paraclinical investigations</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(C) Comprehensiveness</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Do as complete examination as suitable</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(D) Double R—record, review</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The study should be recorded and reviewed</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><fn><p>CCE, critical care echocardiography; POCUS, point-of-care ultrasound; UAPE, ultrasound-assisted physical examination; FOCUS, focused cardiac ultrasound. Source: Kimura BJ. Point-of-care cardiac ultrasound techniques in the physical examination: better at the bedside. Heart 2017; 103: 987–994.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Source: Neskovic AN, Hagendorff A, Lancellotti P, et al. Emergency echocardiography: the European Association of Cardiovascular Imaging recommendations. Eur Heart J Cardiovasc Imaging. 2013; 14 (1): 1–11.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"EMI2024-9974284.001\" position=\"float\"/>" ]
[]
[{"label": ["6"], "person-group": ["\n"], "surname": ["Kline", "Golinski", "Selai", "Horsch", "Hornbaker"], "given-names": ["J.", "M.", "B.", "J.", "K."], "article-title": ["The effectiveness of a blended POCUS curriculum on achieving basic focused bedside transthoracic echocardiography (TTE) proficiency. A formalized pilot study"], "source": ["\n"], "italic": ["Cardiovascular Ultrasound"], "year": ["2021"], "volume": ["19"], "issue": ["1"], "fpage": ["p. 39"], "pub-id": ["10.1186/s12947-021-00268-9"]}, {"label": ["8"], "person-group": ["\n"], "surname": ["Frederiksen", "Juhl-Olsen", "Andersen", "Sloth"], "given-names": ["C. A.", "P.", "N. H.", "E."], "article-title": ["Assessment of cardiac pathology by point-of-care ultrasonography performed by a novice examiner is comparable to the gold standard"], "source": ["\n"], "italic": ["Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine"], "year": ["2013"], "volume": ["21"], "issue": ["1"], "fpage": ["p. 87"], "pub-id": ["10.1186/1757-7241-21-87", "2-s2.0-84890691242"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Farsi", "Hajsadeghi", "Hajighanbari"], "given-names": ["D.", "S.", "M. J."], "article-title": ["Focused cardiac ultrasound (FOCUS) by emergency medicine residents in patients with suspected cardiovascular diseases"], "source": ["\n"], "italic": ["Journal of Ultrasound in Medicine"], "year": ["2017"], "volume": ["20"], "issue": ["2"], "fpage": ["133"], "lpage": ["138"], "pub-id": ["10.1007/s40477-017-0246-5", "2-s2.0-85019734370"]}, {"label": ["18"], "person-group": ["\n"], "surname": ["Tanael"], "given-names": ["M."], "article-title": ["Users\u2019 guide to point-of-care ultrasonography"], "source": ["\n"], "italic": ["Journal of the American College of Emergency Physicians Open"], "year": ["2020"], "volume": ["1"], "issue": ["6"], "fpage": ["p. 1777"], "pub-id": ["10.1002/emp2.12314"]}, {"label": ["21"], "collab": ["Ecri Institute"], "source": ["\n"], "italic": ["Top 10 Health Technology Hazards for 2020"], "year": ["2020"], "publisher-loc": ["Oakbrook Terrace, IL, USA"], "publisher-name": ["The Joint Commission, ECRI Institute"]}, {"label": ["26"], "person-group": ["\n"], "surname": ["Laursen", "Nielsen", "Riishede"], "given-names": ["C. B.", "K.", "M."], "article-title": ["A framework for implementation, education, research and clinical use of ultrasound in emergency departments by the Danish Society for Emergency Medicine"], "source": ["\n"], "italic": ["Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine"], "year": ["2014"], "volume": ["22"], "issue": ["1"], "fpage": ["p. 25"], "pub-id": ["10.1186/1757-7241-22-25", "2-s2.0-84899624295"]}, {"label": ["28"], "person-group": ["\n"], "surname": ["Grenar", "M\u011bd\u00edlek", "Nov\u00fd"], "given-names": ["P. M. K.", "K.", "J."], "article-title": ["Echocardiography in first-contact care"], "source": ["\n"], "italic": ["Military Medical Science Letters"], "year": ["2022"], "volume": ["91"], "issue": ["3"], "fpage": ["235"], "lpage": ["243"], "pub-id": ["10.31482/mmsl.2021.051"]}, {"label": ["29"], "person-group": ["\n"], "surname": ["Kaddoura"], "given-names": ["S."], "source": ["\n"], "italic": ["Echo Made Easy"], "year": ["2016"], "edition": ["3rd"], "publisher-loc": ["Amsterdam, Netherlands"], "publisher-name": ["Elsevier"]}, {"label": ["30"], "person-group": ["\n"], "surname": ["Nilam J Soni", "Kory"], "given-names": ["R. A.", "P."], "source": ["\n"], "italic": ["Point of Care Ultrasound"], "year": ["2019"], "edition": ["2nd"], "publisher-loc": ["Amsterdam, Netherlands"], "publisher-name": ["Elsevier"]}]
{ "acronym": [ "A2CH:", "A3CH:", "A4CH:", "A5CH:", "BSE:", "CCE:", "FOCUS:", "POCUS:", "PLAX:", "SAX:", "S4CH:", "UAPE:" ], "definition": [ "Apical two-chamber view", "Apical three-chamber view", "Apical four-chamber view", "Apical five-chamber view", "British Society of Echocardiography", "Critical care echocardiography", "Focused cardiac ultrasound", "Point-of-care ultrasound", "Parasternal long axis", "Parasternal short axis", "Subcostal four-chamber view", "Ultrasound-assisted physical examination." ] }
30
CC BY
no
2024-01-14 23:43:50
Emerg Med Int. 2024 Jan 6; 2024:9974284
oa_package/70/c8/PMC10787655.tar.gz
PMC10787656
0
[ "<title>1. Introduction</title>", "<p>Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is prevalent among the elderly population and is the leading cause of dementia [##REF##36918389##1##, ##UREF##0##2##]. The prevalence of AD is increasing steadily and is predicted to double in the next 20 years [##REF##29233480##3##]. Its pathogenesis encompasses the accumulation of beta-amyloid plaques, neurofibrillary tangles, and neuronal loss in the brain. It results in memory and cognitive function deterioration, which affects the daily activities of the patients [##REF##29653606##4##, ##REF##36952330##5##]. Despite the significant progress made in understanding the mechanism and therapeutic targets of AD, there is a lack of a definitive cure or effective treatment [##REF##36878848##6##]. Consequently, AD represents a growing societal challenge and an unmet medical need [##REF##36918389##1##].</p>", "<p>Several hypotheses have been proposed over the years to explain AD's pathogenesis, with the amyloid hypothesis being the prevailing paradigm [##UREF##1##7##, ##REF##15485483##8##]. However, recent studies have questioned the validity of this hypothesis and suggested alternative explanations, including the tau hypothesis, chronic inflammation, and gut microbiota theories [##REF##29387348##9##, ##REF##29524051##10##]. Inflammation hypothesis, in particular, postulates that proinflammatory cytokine, tumour necrosis factor-alpha (TNF<italic>α</italic>), plays a crucial role in AD pathogenesis. TNF<italic>α</italic> is upregulated in the brains of individuals with AD and impairs cognitive function [##REF##9774383##11##, ##REF##21728035##12##]. Additionally, studies have shown that the modulation of TNF<italic>α</italic> leads to variations in amyloid plaque deposition, neuronal death, and cognitive deficits, which are hallmarks of AD [##REF##20692646##13##, ##UREF##2##14##]. In general, there is compelling evidence to suggest that TNF<italic>α</italic> plays a significant role in the pathogenesis of AD [##UREF##3##15##]. However, TNF inhibitors such as infliximab and etanercept do not cross the blood-brain barrier (BBB), which is a physical barrier that separates the brain from the peripheral circulation, limiting their efficacy in treating brain inflammation [##REF##11592357##16##]. Emerging evidence suggests that the plant <italic>Rauwolfia vomitoria</italic> (RV) possesses compounds capable of preventing neuronal damage and reducing inflammation in the brain with minimal side effects [##UREF##4##17##–##UREF##5##20##]. This plant has exhibited promising therapeutic effects on cognitive deficit, among other plants that may have a beneficial effect on cognitive function [##UREF##5##20##–##REF##34500759##22##]. <italic>Rauwolfia vomitoria</italic> is an ethnomedicinal plant commonly used in traditional African medicine for various ailments, including inflammation [##UREF##6##23##–##UREF##8##25##].</p>", "<p>Plant-derived compounds have been a focal point in drug discovery for centuries, and recent advances in computational chemistry and molecular modelling have expedited the process of identifying promising drug candidates from natural sources [##UREF##9##26##, ##REF##23049178##27##]. <italic>In silico</italic> methods have been used to predict the biological activities of plant-derived compounds, thereby speeding up the process of identifying promising drug candidates at a reduced cost [##REF##35673392##28##–##REF##34577194##30##]. This is particularly important for developing countries, where plant diversity is high and access to modern drug discovery technologies is limited.</p>", "<p>Drug repurposing is the process of identifying new therapeutic uses for existing drugs. One approach to drug repurposing is based on the similarity of chemical structures between drugs.</p>", "<p>Furthermore, drug repurposing, particularly based on structural similarity, can potentially lead to the identification of new therapeutic uses for existing drugs. Based on the idea that if two molecules share similar structures, then they may have similar bioactivities [##REF##33430988##31##–##UREF##12##34##]. This approach is commonly used and aimed at identifying an analogue of an existing drug molecule that shares mechanisms of action with the original drug or compound [##REF##33430988##31##, ##REF##32525938##35##, ##REF##30547439##36##]. Therefore, in this study, we explored the potential of <italic>yohimbine</italic>, the most dominant compound in the stem bark of RV, as a TNF<italic>α</italic> binder and potential drug candidate. Additionally, we identified an existing drug metoserpate for TNF<italic>α</italic> inhibition based on structural similarities.</p>" ]
[ "<title>2. Methods</title>", "<title>2.1. Study Workflow</title>", "<p>The study started with a systematic evaluation of the pharmacokinetic properties of <italic>yohimbine</italic>, the primary compound found in RV stem bark. <italic>In silico</italic> analysis using SwissADME was employed to comprehensively understand <italic>yohimbine's</italic> absorption, distribution, metabolism, and excretion (ADME) profile. Subsequently, molecular docking of <italic>yohimbine</italic> and the TNF<italic>α</italic> receptor was performed using SeeSAR software to determine their binding affinity. A structure similarity search for <italic>yohimbine</italic> was conducted to identify compounds with a similarity of at least 75%. The pharmacokinetic profile of the selected compound was evaluated using SwissADME. The most promising FDA-approved drug was chosen based on its pharmacokinetic properties and its ability to bind to the TNF<italic>α</italic> receptor. The selected drug underwent docking and molecular dynamics simulations using GROMACS software to assess its stability and potential <italic>in vivo</italic> performance.</p>", "<title>2.2. Retrieval and Preparation of 3D Protein Structure</title>", "<p>The three-dimensional (3D) conformation of TNF<italic>α</italic> (PDB ID: 2AZ5; X-ray diffraction resolution: 2.10 Å), as previously reported by He et al. [##REF##16284179##37##], was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) [##REF##12037327##38##] (<ext-link xlink:href=\"https://www.rcsb.org\" ext-link-type=\"uri\">https://www.rcsb.org</ext-link>). The retrieved protein structure was subjected to preparation using the Biovia Discovery Studio Visualizer v2021 [##UREF##13##39##]. During protein preparation, all multiple chains were eliminated from the structure, resulting in the retention of chain “<bold>A</bold>” for subsequent molecular docking. Additionally, the water molecules and heteroatoms that were irrelevant to the investigation were removed during the protein preparation process.</p>", "<title>2.3. Retrieval and Preparation of 3D Conformer Compounds</title>", "<p>The dominant compound, specifically <italic>yohimbine</italic>, in the stem bark of RV, was analysed in terms of their 3D conformer structure, as obtained from the PubChem database [##REF##36305812##40##] (<ext-link xlink:href=\"https://pubchem.ncbi.nlm.nih.gov/\" ext-link-type=\"uri\">https://pubchem.ncbi.nlm.nih.gov/</ext-link>). The structure of <italic>yohimbine</italic> was processed using Avogadro v1.2.0 [##REF##22236646##41##] (<ext-link xlink:href=\"https://avogadro.cc\" ext-link-type=\"uri\">https://avogadro.cc</ext-link>) with the MMFF96 force field applied for the minimisation of the ligand after the addition of hydrogen atoms and the refinement of the geometry.</p>", "<title>2.4. Pharmacokinetic Assessment of <italic>Yohimbine</italic></title>", "<p>In this study, a comprehensive analysis of the ADME (absorption, distribution, metabolism, and excretion) profile of the compounds was performed. Compliance with the Lipinski rule of 5 [##UREF##14##42##], which includes the parameters of molecular weight (MW), lipophilicity (log <italic>P</italic>), hydrogen bond acceptor (HBA), and hydrogen bond donor (HBD), was evaluated. Additionally, the GI absorption and penetration of the BBB of the compounds were examined using various models, Ghose's rule [##REF##10746014##43##], Egan's rule [##REF##11052792##44##], Muegge's rule [##UREF##15##45##], and Veber's rule [##REF##12036371##46##]. The Ghose rule defines acceptable compounds as having a molecular weight between 160 and 480 g/mol, a log <italic>P</italic> value between -0.4 and 5.6, the number of hydrogen bond donors less than or equal to 5, and the number of hydrogen bond acceptors less than or equal to 10. Egan's rule considers molecular weight, log <italic>P</italic>, the number of hydrogen bond donors, the number of hydrogen bond acceptors, and the number of rotatable bonds, while Veber's rule takes into account the number of rotatable bonds, the number of hydrogen bond donors, the number of hydrogen bond acceptors, and molecular weight. Muegge's rule assesses the acceptability of compounds based on their molecular weight, the number of hydrogen bond donors, the number of hydrogen bond acceptors, and the topological polar surface area (TPSA). The SwissADME open-access online tool was employed to evaluate the ADME profile of the compounds assessed in this study [##REF##28256516##47##] (<ext-link xlink:href=\"http://www.swissadme.ch\" ext-link-type=\"uri\">http://www.swissadme.ch</ext-link>).</p>", "<title>2.5. Molecular Docking of TNF-Alpha, <italic>Yohimbine</italic>, and Metoserpate Using SeeSAR</title>", "<p>Molecular docking simulations were carried out using the SeeSAR module in BioSolveIT, following the default parameters. To generate the receptor grid, the AutoGrid tool in SeeSAR was used and placed at the active site of the receptor protein (Cys69, Lys98, Ser99, Pro100, Cys101, Gln102, Arg103, Glu104, Thr105, Trp114, Tyr115, Glu116, and Pro117). The ligand was then docked into the receptor utilizing SeeSAR's standard precision (SP) mode. Finally, the top-ranking poses were analysed using the Pose Viewer tool integrated within SeeSAR [##UREF##16##48##] (<ext-link xlink:href=\"https://www.biosolveit.de\" ext-link-type=\"uri\">https://www.biosolveit.de</ext-link>).</p>", "<title>2.6. Structural Similarity Search of DrugBank Compounds</title>", "<p>In this study, we used the DrugBank and SwissSimilarity tool [##REF##35054998##49##] to investigate drug structural similarity using <italic>yohimbine</italic> structure as a query. Specifically, we used SwissSimilarity, which is an open-access web-based tool that allows molecular structure comparisons of drugs based on their chemical properties. The similarity search was performed against the DrugBank database [##REF##16381955##50##], which provides comprehensive data on the chemical structure, pharmacology, and clinical applications of drugs. Notably, we opted for the 2D and 3D combined DrugBank option of the SwissSimilarity web platform for the search of structurally similar drugs, employing a similarity threshold of 75% and above. Equally, the chemical structure search feature was used for the <italic>yohimbine</italic>-centered approach to investigate drugs similar to <italic>yohimbine</italic>.</p>", "<title>2.7. Pharmacokinetic Assessment of Identified DrugBank Compounds</title>", "<p>Pharmacokinetic assessment was carried out for identified drugs with structural similarity equal to or greater than 75% by evaluating the gastrointestinal (GI) absorption and BBB penetration for the selected drugs using the SwissADME web tool [##REF##28256516##47##] (<ext-link xlink:href=\"http://www.swissadme.ch\" ext-link-type=\"uri\">http://www.swissadme.ch</ext-link>) with focus on two important aspects (GI and BBB) of drug distribution in the body.</p>", "<title>2.8. Molecular Dynamics Simulation of the TNF<italic>α</italic>-Ligand Complex</title>", "<p>Molecular dynamics (MD) simulations were undertaken using the GROMACS package [##UREF##17##51##, ##REF##16211538##52##] (<ext-link xlink:href=\"https://www.gromacs.org\" ext-link-type=\"uri\">https://www.gromacs.org</ext-link>) within the myPresto portal v5 software, using default force field settings (AMBER ff99SB, TIP3P, and GAFF ver2.1) [##REF##27924276##53##, ##REF##33240741##54##]. The entire MD process was carried out using the autodynamics options for 100 nanoseconds (100 ns (1000 ps)) [##REF##11031278##55##, ##REF##15093837##56##]. The MD simulation was performed on TNF<italic>α</italic>-metoserpate and TNF<italic>α</italic>-cocrystallized ligand (small molecule (C<sub>32</sub> H<sub>32</sub> F<sub>3</sub> N<sub>3</sub> O<sub>2</sub>)) complexes.</p>" ]
[ "<title>3. Results and Discussion</title>", "<title>3.1. Pharmacokinetics of <italic>Yohimbine</italic></title>", "<p>The study of the pharmacokinetics of potentially therapeutic compounds is of clinical importance in the drug development process. Elsewhere, about 40% of drug candidates do not pass the clinical trial stages [##REF##15334309##57##] due to undesired absorption, distribution, metabolism, and excretion (ADME) profiles of the drug candidates. For a compound to be considered a good candidate depends on its exposure to the molecular target, which is determined by absorption and metabolism and particularly for central nervous system (CNS) drugs, an ability to cross the BBB [##REF##12369891##58##]. From ##TAB##0##Table 1##, it can be inferred that <italic>yohimbine</italic> demonstrated high GI absorption and lipophilicity making it easier to cross the blood-brain barrier. A few pharmacokinetic principles pioneered by Lipinski, Ghose, Veber, Egan, and Muegge were applied to assess the drug-likeness of the plant compound <italic>yohimbine</italic>. <italic>Yohimbine</italic> was subjected to Lipinski's rule of 5, per the rule; orally active drugs should not violate any of these four criteria: molecular weight ≤ 500, log <italic>P</italic> (lipophilicity) ≤ 5, number of hydrogen bond donors ≤ 5, and number of hydrogen bond acceptors ≤ 10 [##UREF##14##42##]. Based on the physicochemical properties of <italic>yohimbine</italic>, none of the rules were violated (##TAB##0##Table 1##); this confers its use as an oral pharmaceutical drug. The total polar surface area (TPSA) for <italic>yohimbine</italic> was 65.56 Å<sup>2</sup> which is less than 140 Å<sup>2</sup> indicating good permeability in cellular lipid membranes according to Veber's rule [##REF##12036371##46##]. It is evident in literature that there is a strong correlation between high TPSA and low blood-brain penetration [##REF##26305616##59##–##REF##30502633##61##]. The Ghose filter was applied to evaluate the drug-likeness of <italic>yohimbine</italic>; again, no rule was violated. Egan and Muegge's filters were employed to assess the oral bioavailability based on the physicochemical properties; once more, <italic>yohimbine</italic> was compliant with all the rules [##REF##33320017##62##].</p>", "<title>3.2. Molecular Docking of TNF<italic>α</italic>-<italic>Yohimbine</italic></title>", "<p>The molecular docking result obtained between TNF<italic>α</italic> and <italic>yohimbine</italic> showed that there was a binding affinity Hyde score of -1.0 kJ/mol between the nitrogen atom at position 5 of the ligand and the amino acid residue Gln102 of TNF<italic>α</italic> (##FIG##0##Figure 1(a)## and ##TAB##1##Table 2##). Additionally, there was another bond interaction (Hyde: 0.2 kJ/mol) between the oxygen atom at position 3 of the ligand and the amino acid residue Gln102. The observation of a binding affinity Hyde score of -1.0 kJ/mol between the nitrogen atom at position 5 of <italic>yohimbine</italic> and the amino acid residue Gln102 of TNF<italic>α</italic> suggests that <italic>yohimbine</italic> may bind to TNF<italic>α</italic>'s active site and inhibit its proinflammatory effects. Additionally, the bond interaction between the oxygen atom at position 3 of <italic>yohimbine</italic> and Gln102 may contribute to the overall stability of the <italic>yohimbine</italic>-TNF<italic>α</italic> complex. Upon analysing the docking pose using Biovia Discovery Studio Visualizer, it was observed that the ligand formed two conventional hydrogen bond networks with the amino acid residue Gln102 of TNF<italic>α</italic> (##FIG##0##Figure 1(b)##). In addition to the conventional hydrogen bond networks, a nonconventional hydrogen bond network was also detected between the ligand and the amino acid residue Cys101 of TNF<italic>α</italic>. Furthermore, two pi-alkyl bond network interactions were observed between the ligand and the amino acid residue Arg103 of TNF<italic>α</italic> (##FIG##0##Figure 1(b)##). Hydrophobic interaction was also observed between <italic>yohimbine</italic> and the TNF<italic>α</italic> residues Arg103 and Gln102. These results suggest that <italic>yohimbine</italic> has potential to bind to TNF<italic>α</italic> at its binding site and inhibit its proinflammatory effects.</p>", "<p>From our study, <italic>yohimbine</italic>, an alkaloid with purported aphrodisiac properties and used for treating erectile dysfunction [##REF##36263866##63##, ##REF##11744068##64##], has been identified as a potential inhibitor of TNF<italic>α</italic>, a cytokine that mediates inflammation in the central nervous system (CNS) and causes oxidative stress, apoptosis, and synaptic dysfunction in neurons [##REF##18925972##65##]. Neuroinflammation and resultant neurodegeneration can be precipitated by activated microglia, the resident immune cells of the CNS [##REF##32488063##66##, ##REF##19302036##67##]. Therefore, identifying small molecules capable of inhibiting TNF<italic>α</italic> could be therapeutically beneficial in treating neurodegenerative disorders associated with chronic inflammation. Here, we utilized SeeSAR, a structure-based drug design software tool, to study the interaction between <italic>yohimbine</italic> and TNF<italic>α</italic> [##REF##36318405##68##, ##REF##28913743##69##]. Our analysis has demonstrated that <italic>yohimbine</italic> exhibits a stable interaction with TNF<italic>α</italic>, as indicated by a Hyde score of -1.0 kJ/mol, suggesting favourable binding. Further examination of the molecular interactions has revealed key findings. Notably, a pi-alkyl bond network is formed between <italic>yohimbine</italic> and the amino acid residue arginine at position 103 (Arg103) of TNF<italic>α</italic>. Additionally, a conventional hydrogen bond is observed between the hydrogen of the imine functional group of <italic>yohimbine</italic> and the amino acid residue glutamine at position 102 (Glu102) of TNF<italic>α</italic>. Furthermore, a nonconventional hydrogen bond network interaction between <italic>yohimbine</italic> and TNF<italic>α</italic> is observed at the amino acid residue position Cys101, which contributes significantly to the binding process. These molecular interactions, as illustrated in Figures ##FIG##0##1(a)## and ##FIG##0##1(b)##, play a prominent role in driving the binding between <italic>yohimbine</italic> and TNF<italic>α</italic>.</p>", "<p>These findings provide valuable insights into the specific mechanisms underlying the interaction between <italic>yohimbine</italic> and TNF<italic>α</italic>, shedding light on the potential efficacy of <italic>yohimbine</italic> in modulating TNF<italic>α</italic> and its implications for addressing the pathogenesis of AD. Therefore, we posit that <italic>yohimbine</italic> may act as an inhibitor of TNF<italic>α</italic> and reduce its proinflammatory and neurotoxic effects in the CNS, which could explain the benefit reported for cognitive impairment and motor dysfunction [##REF##3613864##70##, ##REF##22746337##71##].</p>", "<p>Compared to other TNF<italic>α</italic> inhibitors like etanercept and infliximab, which are large molecules and have difficulty penetrating the blood-brain barrier and pose systemic safety concerns [##REF##16204273##72##–##REF##19689163##74##], <italic>yohimbine</italic> is a small molecule that can easily cross the blood-brain barrier and has a relatively good safety profile when used at low doses [##REF##3412496##75##]. Therefore, we propose <italic>yohimbine</italic> as a model molecule for the repurposing of an old FDA-approved drug (that may have superior bioavailability and safety profiles) for a new drug target (TNF<italic>α</italic>) inhibition, based on structure similarity search.</p>", "<title>3.3. Identified Structurally Similar DrugBank Compounds</title>", "<p>The primary objective of this study is to explore structure-based drug design strategies in order to identify and repurpose known compounds, like <italic>yohimbine</italic>, for potential therapeutic use for the management of AD. To this end, we conducted a search of the DrugBank database for FDA-approved compounds that exhibited a high percentage structural similarity to <italic>yohimbine</italic>. From our analysis, a total of 10 compounds with a structural similarity of at least 75% to <italic>yohimbine</italic> were retrieved (##TAB##2##Table 3##). These compounds include metoserpate, deserpidine, 18-methoxycoronaridine, CP-320626, rescinnamine, reserpine, raubasine, methoserpidine, (7as,12ar,12bs)-1,2,3,4,7a,12,12a,12b-Octahydroindolo[2,3-a]Quinolizin-7(6h)-One, and vinburnine. After assessing the retrieved compounds for their current FDA approval status, four of the entries were found to have FDA approval.</p>", "<p>Metoserpate (DB11530) demonstrated the highest percentage structure similarity (0.992%) to <italic>yohimbine</italic>, ability to traverse the BBB, high GI absorption, and preexisting approval for clinical use, thus making it an ideal candidate for further investigation (Tables ##TAB##2##3## and ##TAB##3##4##). The observed high degree of similarity between metoserpate and <italic>yohimbine</italic> can be attributed to the presence of a pentacyclic yohimban skeleton, involving the formation of a carbocyclic ring from the C-17 to C-18 bond in a corynantheine precursor, as previously reported [##REF##36263866##63##].</p>", "<title>3.4. Pharmacokinetics of the Identified Structurally Similar DrugBank Compounds</title>", "<p>We assessed the GI absorption and the capacity to cross the BBB of the 10 compounds retrieved from the DrugBank database. Our findings showed that all 10 compounds had high GI absorption, indicating that they are likely to be well absorbed in the gastrointestinal tract (##TAB##3##Table 4##). However, only five of the compounds had the capacity to cross the BBB (##TAB##3##Table 4##), indicating that they may have potential therapeutic applications for the treatment of CNS disorders. These five compounds may be able to penetrate the BBB due to their physicochemical properties, such as their lipophilicity and molecular weight.</p>", "<p>Further analysis revealed that out of the five compounds that are able to cross the BBB, only one (metoserpate) had FDA approval. Thus, metoserpate (DB11530) was the ideal candidate not only because it is the only FDA-approved drug, but also it exhibited high gastrointestinal absorption and a propensity to cross or permeate the blood-brain barrier. Metoserpate has a total polar surface area (TPSA) of 73.02 Å<sup>2</sup> contributing to its ability to permeate cellular membranes. It is evident in literature that TPSA values less than 73.02 Å<sup>2</sup> are indicative of good permeability and satisfy Veber's rule [##REF##12036371##46##]. Metoserpate was thus selected for further analysis.</p>", "<title>3.5. Molecular Docking of TNF-Alpha and Metoserpate</title>", "<p>The binding affinity of TNF<italic>α</italic> and metoserpate was assessed using Hyde's score method. This method seeks to address weak or questionable hydrogen bonds as well as indifferent scaffolds not contributing to the free energy in the protein-ligand complex [##REF##2987660##76##, ##REF##23269578##77##]. From ##FIG##1##Figure 2(a)##, it can be observed that the Hyde score was -1.1 kg/mol which confers a favourable interaction [##REF##23269578##77##]. The docking analysis revealed one hydrogen bond between the nitrogen atom at position 8 of metoserpate and the amino acid residue Gln102 of TNF<italic>α</italic>. These results suggest that the interaction between metoserpate and TNF<italic>α</italic> at this site may have potential therapeutic implications for the treatment of TNF<italic>α</italic>-related diseases (##FIG##1##Figure 2(a)## and ##TAB##1##Table 2##).</p>", "<p>When the docking simulation result was visualized using Biovia Discovery Studio Visualizer, one pi-alkyl bond network between metoserpate and TNF<italic>α</italic> binding site amino acid (AA) residue Arg103 and two salt bridge interactions between metoserpate and the binding site AA residue Glu104 of TNF<italic>α</italic> were observed. In addition, one conventional hydrogen bond network was observed between metoserpate and TNF<italic>α</italic> binding site residue Gln102 (##FIG##1##Figure 2(b)##). It is documented that conventional hydrogen bonds aid in the stability of complexes, hence conferring a good binding affinity [##REF##34319314##78##, ##REF##20298519##79##]. Consequently, the glutamic acid (Glu104) of the protein participated in two cation-pi interactions between the imine functional group and the benzene ring of metoserpate is shown in yellow. Cation-pi interactions play an important role in determining protein structure as well as contributing significantly to the binding energy of the complex formation [##REF##15726638##80##]. Arginine (Arg103) of the protein residue participated in a pi-alkyl interaction with the benzene ring of our target drug metoserpate. According to literature, pi-alkyl interactions have a greater propensity for stability when compared to alkyls bound to nonaromatic moieties in a ligand [##UREF##18##81##–##REF##23020662##83##].</p>", "<title>3.6. Molecular Dynamics Simulations</title>", "<title>3.6.1. TNF<italic>α</italic>-Small Molecule and TNF<italic>α</italic>-Metoserpate</title>", "<p>Numerous significant pharmaceuticals and hundreds of natural products with promising bioactivities contain indole alkaloids or have structures that are like indole alkaloids. Despite not always adhering to Lipinski's rules, such compounds frequently exhibit favourable pharmacokinetic profiles with respect to cyclic molecules. The values of the root mean square deviation (RMSD) affirm whether a close-match docked pose was predicted between the crystal and the predicted structures. It is evident in literature that an RMSD value ≤ 0.2 nm is fairly good [##REF##28106755##84##–##REF##34641761##86##]. Figures ##FIG##2##3(a)## and ##FIG##2##3(b)## highlight the results of TNF<italic>α</italic> and the cocrystallized small molecule and TNF<italic>α</italic> and the target drug metoserpate both having their RMSD value ≤ 2 Å (0.2 nm) which confers a latent stable protein-ligand complex.</p>", "<p>The RMSD between the TNF<italic>α</italic>-small molecule complex and the TNF<italic>α</italic>-metoserpate complex remained consistent throughout a 100 ns simulation. However, when comparing the TNF<italic>α</italic>-small molecule complex (##FIG##2##Figure 3(a)##) to the TNF<italic>α</italic>-metoserpate complex (##FIG##2##Figure 3(b)##), a more stable trajectory was observed in the TNF<italic>α</italic>-metoserpate complex. In the case of the TNF<italic>α</italic>-small molecule complex, it displayed stability from 20 ns to approximately 30 ns, followed by a deviation. It then regained stability until around 55 ns but experienced another deviation until 60 ns. From this point, it became stable again until approximately 75 ns, with another observed deviation until around 82 ns. Finally, it regained stability and remained stable until the end of the simulation at 100 ns. On the other hand, the trajectory of the TNF<italic>α</italic>-metoserpate complex showed stability from around 15 ns to approximately 70 ns, with a slight deviation occurring until 80 ns. After this point, it regained stability and remained stable until the end of the simulation at 100 ns. Both complexes exhibited deviations within a range of 0.05 nm.</p>", "<p>The observation of stable RMSD values throughout a 100 ns simulation suggests that the overall conformation of the TNF<italic>α</italic>-small molecule complex and TNF<italic>α</italic>-metoserpate complex remained relatively consistent during the simulation period [##REF##31910338##87##]. This stability is an important characteristic as it indicates that the complexes maintained their structural integrity and did not undergo significant conformational changes. TNF<italic>α</italic>-metoserpate complex exhibited a more stable trajectory compared to the TNF<italic>α</italic>-small molecule complex suggesting that the binding of metoserpate, a small compound, may have induced more favourable interactions and a more stable complex formation. This could be attributed to specific molecular interactions, such as hydrogen bonding, electrostatic interactions, or hydrophobic interactions between metoserpate and TNF<italic>α</italic>. These interactions may contribute to a stronger binding affinity and a more stable conformation for the TNF<italic>α</italic>-metoserpate complex [##REF##20808434##88##].</p>", "<p>To describe the local conformational change in the TNF<italic>α</italic> and metoserpate and TNF<italic>α</italic>-small molecule complexes, the root mean square fluctuation (RMSF) was required. Figures ##FIG##3##4(a)## and ##FIG##3##4(b)## highlight the RMSF profile of the TNF<italic>α</italic>-small molecule and TNF<italic>α</italic>-metoserpate complexes, respectively. From the graph, stable fluctuations were observed with RMSF ≤ 0.2 nm in both instances [##REF##35385549##89##]. The TNF<italic>α</italic>-small molecule complex (##FIG##3##Figure 4(a)##) and TNF<italic>α</italic>-metoserpate complex (##FIG##3##Figure 4(b)##) both displayed reasonably low RMSF. However, the TNF<italic>α</italic>-metoserpate complex exhibited slightly higher fluctuations compared to the TNF<italic>α</italic>-small molecule complex. It is important to note that all the observed fluctuations in the TNF<italic>α</italic>-metoserpate complex were generally around 0.2 nm. On the other hand, in the TNF<italic>α</italic>-small molecule complex, fluctuations around atom positions 180 and 1520 were observed to be around 0.3 nm.</p>", "<p>These fluctuations, measured in nanometers, indicate the degree of movement or flexibility of specific atoms within the complexes. The relatively low RMSF values suggest that overall, the complexes remained relatively stable during the simulation [##UREF##19##90##]. However, the slightly higher fluctuations in the TNF<italic>α</italic>-metoserpate complex could imply that the binding of metoserpate induced some additional dynamics or flexibility in certain regions of the complex compared to the TNF<italic>α</italic>-small molecule complex [##UREF##19##90##]. The specific atom positions 180 and 1520 in the TNF<italic>α</italic>-small molecule complex experienced slightly higher fluctuations around 0.3 nm. These positions could correspond to specific residues or functional regions within the complex. The increased fluctuation at these positions may indicate potential conformational changes or greater flexibility in those regions, possibly influenced by the presence of the small compound or specific interactions between the compound and TNF<italic>α</italic> [##UREF##19##90##].</p>", "<p>The radius of gyration (Rg) monitors the compactness of the protein structure coupled with the binding patterns of the drug and protein in direct relation to the folding rate [##REF##16400647##91##]. A conformational change occurs when a ligand or lead molecule attaches to the protein, changing the radius of gyration [##REF##20401516##92##]. The TNF<italic>α</italic>-small molecule complex (##FIG##4##Figure 5(a)##) and TNF<italic>α</italic>-metoserpate complex (##FIG##4##Figure 5(b)##) exhibited similar total radius of gyration values, both measuring approximately 1.52 nm. A smaller radius of gyration indicates a more compact and tightly packed structure, while a larger radius of gyration suggests a more extended or flexible conformation [##UREF##19##90##]. The fact that both the TNF<italic>α</italic>-small molecule complex and TNF<italic>α</italic>-metoserpate complex demonstrated a total radius of gyration around 1.52 nm suggests that they possess comparable overall compactness, indicating a compact and stable conformation [##REF##35061725##93##]. This similarity in size could indicate that the binding of both the small molecule and metoserpate did not significantly alter the overall conformation or compactness of the TNF<italic>α</italic> complex.</p>", "<title>3.6.2. Bond Network Evaluation of Metoserpate and TNF-Alpha Complex following Molecular Dynamics Simulation</title>", "<p>The post-MD simulation analysis revealed significant changes in the metoserpate-TNF<italic>α</italic> complex compared to the pre-MD simulation complex. Our findings demonstrated that metoserpate established multiple bond network interactions with the AAs in the binding site of TNF<italic>α</italic>. Specifically, a conventional hydrogen bond (cH-bond) was formed between the oxygen of the carboxylic acid methyl ester of metoserpate and the amino acid residue Lys98 of TNF<italic>α</italic>. Conventional hydrogen bonds are known for their strength and contribute to strong binding affinity. Additionally, several nonconventional hydrogen bonds (ncH-bonds) were observed between metoserpate and the AAs Ser99, Glu104, Pro113, Tyr115, and Glu116. Metoserpate also engaged in a pi-alkyl interaction with Tyr115 and Pro117, as well as two cation-pi interactions with Glu104 and Glu116. These interactions played a crucial role in the stability and specificity of the complex (##FIG##5##Figure 6##). Hydrophobic contact area was also established between metoserpate and the binding site AA residues Lys98 and Tyr115.</p>", "<p>In contrast, the bond network analysis conducted prior to the MD simulation revealed specific interactions between metoserpate and TNF<italic>α</italic>, including a cH-bond network with Gln102, a pi-alkyl interaction with Arg103, and cation-pi interactions with Glu104. However, the subsequent MD simulation analysis yielded intriguing findings, indicating an enhanced binding affinity and selectivity of metoserpate towards TNF<italic>α</italic>. This improvement in binding was accompanied by the generation of more favourable and specific interactions.</p>", "<p>These results are further supported by the observed flexibility in the root mean square fluctuation (RMSF) output of the TNF<italic>α</italic> and metoserpate complex. The MD simulations have provided valuable insights into the intricate molecular interactions between the TNF<italic>α</italic> and metoserpate, unravelling the complexities of protein-ligand complexes.</p>", "<p>By elucidating the dynamic behaviour and uncovering the structural changes that occur during the simulation, the MD simulations offer a deeper understanding of the binding mechanism and contribute to the overall comprehension of the interactions between TNF<italic>α</italic> and metoserpate.</p>", "<title>3.6.3. Overall Bond Network Assessment</title>", "<p>The Hyde scoring method has proven to be a valuable computational tool in drug discovery for estimating the binding affinity between a protein and a ligand, utilizing their interaction energy [##REF##17124629##94##]. In the present study, we employed the Hyde score assessment method to evaluate the binding affinity of two ligands, metoserpate and <italic>yohimbine</italic>, with the protein TNF<italic>α</italic>, with a specific focus on the amino acid residues within the binding site (##TAB##1##Table 2##).</p>", "<p>The study's findings revealed that metoserpate exhibited a slightly lower Hyde score (-1.1 kJ/mol) in comparison to <italic>yohimbine</italic> (-1.0 kJ/mol and 0.2 kJ/mol) when interacting with Gln102 (##TAB##1##Table 2##). This indicates that metoserpate possesses a marginally better binding affinity with TNF<italic>α</italic> when compared to <italic>yohimbine</italic>, although the difference observed is relatively small. These results shed light on the relative strengths of the interactions between metoserpate and TNF<italic>α</italic>, providing insights into the binding affinity. This information contributes to the understanding of the potential efficacy of metoserpate as a potential therapeutic agent targeting TNF<italic>α</italic> in the context of AD management.</p>", "<p>The post-MD simulation analysis revealed the involvement of amino acid Lys98 in the conventional hydrogen bond formation, as well as the formation of a nonconventional hydrogen bond network with Pro113, which was originally not part of the binding site AA residues (##FIG##5##Figure 6##). This post-MD simulation analysis generated more bond diversity, and bond number compared to the TNF<italic>α</italic>-<italic>yohimbine</italic> and TNF<italic>α</italic>-metoserpate complexes. This demonstrates the importance of post-MD simulation analysis in providing a more comprehensive understanding of protein-ligand interactions beyond what can be predicted through initial scoring methods alone.</p>", "<p>The findings of this study also suggest that the binding affinity of a ligand with a protein may be influenced by amino acid residues outside of the initial binding site. This is consistent with previous studies that have shown the importance of protein flexibility and dynamics in ligand binding [##REF##31244000##95##]. It is possible that the nonconventional hydrogen bond network identified in the post-MD simulation analysis plays a critical role in the binding affinity between TNF<italic>α</italic> and metoserpate.</p>" ]
[ "<title>3. Results and Discussion</title>", "<title>3.1. Pharmacokinetics of <italic>Yohimbine</italic></title>", "<p>The study of the pharmacokinetics of potentially therapeutic compounds is of clinical importance in the drug development process. Elsewhere, about 40% of drug candidates do not pass the clinical trial stages [##REF##15334309##57##] due to undesired absorption, distribution, metabolism, and excretion (ADME) profiles of the drug candidates. For a compound to be considered a good candidate depends on its exposure to the molecular target, which is determined by absorption and metabolism and particularly for central nervous system (CNS) drugs, an ability to cross the BBB [##REF##12369891##58##]. From ##TAB##0##Table 1##, it can be inferred that <italic>yohimbine</italic> demonstrated high GI absorption and lipophilicity making it easier to cross the blood-brain barrier. A few pharmacokinetic principles pioneered by Lipinski, Ghose, Veber, Egan, and Muegge were applied to assess the drug-likeness of the plant compound <italic>yohimbine</italic>. <italic>Yohimbine</italic> was subjected to Lipinski's rule of 5, per the rule; orally active drugs should not violate any of these four criteria: molecular weight ≤ 500, log <italic>P</italic> (lipophilicity) ≤ 5, number of hydrogen bond donors ≤ 5, and number of hydrogen bond acceptors ≤ 10 [##UREF##14##42##]. Based on the physicochemical properties of <italic>yohimbine</italic>, none of the rules were violated (##TAB##0##Table 1##); this confers its use as an oral pharmaceutical drug. The total polar surface area (TPSA) for <italic>yohimbine</italic> was 65.56 Å<sup>2</sup> which is less than 140 Å<sup>2</sup> indicating good permeability in cellular lipid membranes according to Veber's rule [##REF##12036371##46##]. It is evident in literature that there is a strong correlation between high TPSA and low blood-brain penetration [##REF##26305616##59##–##REF##30502633##61##]. The Ghose filter was applied to evaluate the drug-likeness of <italic>yohimbine</italic>; again, no rule was violated. Egan and Muegge's filters were employed to assess the oral bioavailability based on the physicochemical properties; once more, <italic>yohimbine</italic> was compliant with all the rules [##REF##33320017##62##].</p>", "<title>3.2. Molecular Docking of TNF<italic>α</italic>-<italic>Yohimbine</italic></title>", "<p>The molecular docking result obtained between TNF<italic>α</italic> and <italic>yohimbine</italic> showed that there was a binding affinity Hyde score of -1.0 kJ/mol between the nitrogen atom at position 5 of the ligand and the amino acid residue Gln102 of TNF<italic>α</italic> (##FIG##0##Figure 1(a)## and ##TAB##1##Table 2##). Additionally, there was another bond interaction (Hyde: 0.2 kJ/mol) between the oxygen atom at position 3 of the ligand and the amino acid residue Gln102. The observation of a binding affinity Hyde score of -1.0 kJ/mol between the nitrogen atom at position 5 of <italic>yohimbine</italic> and the amino acid residue Gln102 of TNF<italic>α</italic> suggests that <italic>yohimbine</italic> may bind to TNF<italic>α</italic>'s active site and inhibit its proinflammatory effects. Additionally, the bond interaction between the oxygen atom at position 3 of <italic>yohimbine</italic> and Gln102 may contribute to the overall stability of the <italic>yohimbine</italic>-TNF<italic>α</italic> complex. Upon analysing the docking pose using Biovia Discovery Studio Visualizer, it was observed that the ligand formed two conventional hydrogen bond networks with the amino acid residue Gln102 of TNF<italic>α</italic> (##FIG##0##Figure 1(b)##). In addition to the conventional hydrogen bond networks, a nonconventional hydrogen bond network was also detected between the ligand and the amino acid residue Cys101 of TNF<italic>α</italic>. Furthermore, two pi-alkyl bond network interactions were observed between the ligand and the amino acid residue Arg103 of TNF<italic>α</italic> (##FIG##0##Figure 1(b)##). Hydrophobic interaction was also observed between <italic>yohimbine</italic> and the TNF<italic>α</italic> residues Arg103 and Gln102. These results suggest that <italic>yohimbine</italic> has potential to bind to TNF<italic>α</italic> at its binding site and inhibit its proinflammatory effects.</p>", "<p>From our study, <italic>yohimbine</italic>, an alkaloid with purported aphrodisiac properties and used for treating erectile dysfunction [##REF##36263866##63##, ##REF##11744068##64##], has been identified as a potential inhibitor of TNF<italic>α</italic>, a cytokine that mediates inflammation in the central nervous system (CNS) and causes oxidative stress, apoptosis, and synaptic dysfunction in neurons [##REF##18925972##65##]. Neuroinflammation and resultant neurodegeneration can be precipitated by activated microglia, the resident immune cells of the CNS [##REF##32488063##66##, ##REF##19302036##67##]. Therefore, identifying small molecules capable of inhibiting TNF<italic>α</italic> could be therapeutically beneficial in treating neurodegenerative disorders associated with chronic inflammation. Here, we utilized SeeSAR, a structure-based drug design software tool, to study the interaction between <italic>yohimbine</italic> and TNF<italic>α</italic> [##REF##36318405##68##, ##REF##28913743##69##]. Our analysis has demonstrated that <italic>yohimbine</italic> exhibits a stable interaction with TNF<italic>α</italic>, as indicated by a Hyde score of -1.0 kJ/mol, suggesting favourable binding. Further examination of the molecular interactions has revealed key findings. Notably, a pi-alkyl bond network is formed between <italic>yohimbine</italic> and the amino acid residue arginine at position 103 (Arg103) of TNF<italic>α</italic>. Additionally, a conventional hydrogen bond is observed between the hydrogen of the imine functional group of <italic>yohimbine</italic> and the amino acid residue glutamine at position 102 (Glu102) of TNF<italic>α</italic>. Furthermore, a nonconventional hydrogen bond network interaction between <italic>yohimbine</italic> and TNF<italic>α</italic> is observed at the amino acid residue position Cys101, which contributes significantly to the binding process. These molecular interactions, as illustrated in Figures ##FIG##0##1(a)## and ##FIG##0##1(b)##, play a prominent role in driving the binding between <italic>yohimbine</italic> and TNF<italic>α</italic>.</p>", "<p>These findings provide valuable insights into the specific mechanisms underlying the interaction between <italic>yohimbine</italic> and TNF<italic>α</italic>, shedding light on the potential efficacy of <italic>yohimbine</italic> in modulating TNF<italic>α</italic> and its implications for addressing the pathogenesis of AD. Therefore, we posit that <italic>yohimbine</italic> may act as an inhibitor of TNF<italic>α</italic> and reduce its proinflammatory and neurotoxic effects in the CNS, which could explain the benefit reported for cognitive impairment and motor dysfunction [##REF##3613864##70##, ##REF##22746337##71##].</p>", "<p>Compared to other TNF<italic>α</italic> inhibitors like etanercept and infliximab, which are large molecules and have difficulty penetrating the blood-brain barrier and pose systemic safety concerns [##REF##16204273##72##–##REF##19689163##74##], <italic>yohimbine</italic> is a small molecule that can easily cross the blood-brain barrier and has a relatively good safety profile when used at low doses [##REF##3412496##75##]. Therefore, we propose <italic>yohimbine</italic> as a model molecule for the repurposing of an old FDA-approved drug (that may have superior bioavailability and safety profiles) for a new drug target (TNF<italic>α</italic>) inhibition, based on structure similarity search.</p>", "<title>3.3. Identified Structurally Similar DrugBank Compounds</title>", "<p>The primary objective of this study is to explore structure-based drug design strategies in order to identify and repurpose known compounds, like <italic>yohimbine</italic>, for potential therapeutic use for the management of AD. To this end, we conducted a search of the DrugBank database for FDA-approved compounds that exhibited a high percentage structural similarity to <italic>yohimbine</italic>. From our analysis, a total of 10 compounds with a structural similarity of at least 75% to <italic>yohimbine</italic> were retrieved (##TAB##2##Table 3##). These compounds include metoserpate, deserpidine, 18-methoxycoronaridine, CP-320626, rescinnamine, reserpine, raubasine, methoserpidine, (7as,12ar,12bs)-1,2,3,4,7a,12,12a,12b-Octahydroindolo[2,3-a]Quinolizin-7(6h)-One, and vinburnine. After assessing the retrieved compounds for their current FDA approval status, four of the entries were found to have FDA approval.</p>", "<p>Metoserpate (DB11530) demonstrated the highest percentage structure similarity (0.992%) to <italic>yohimbine</italic>, ability to traverse the BBB, high GI absorption, and preexisting approval for clinical use, thus making it an ideal candidate for further investigation (Tables ##TAB##2##3## and ##TAB##3##4##). The observed high degree of similarity between metoserpate and <italic>yohimbine</italic> can be attributed to the presence of a pentacyclic yohimban skeleton, involving the formation of a carbocyclic ring from the C-17 to C-18 bond in a corynantheine precursor, as previously reported [##REF##36263866##63##].</p>", "<title>3.4. Pharmacokinetics of the Identified Structurally Similar DrugBank Compounds</title>", "<p>We assessed the GI absorption and the capacity to cross the BBB of the 10 compounds retrieved from the DrugBank database. Our findings showed that all 10 compounds had high GI absorption, indicating that they are likely to be well absorbed in the gastrointestinal tract (##TAB##3##Table 4##). However, only five of the compounds had the capacity to cross the BBB (##TAB##3##Table 4##), indicating that they may have potential therapeutic applications for the treatment of CNS disorders. These five compounds may be able to penetrate the BBB due to their physicochemical properties, such as their lipophilicity and molecular weight.</p>", "<p>Further analysis revealed that out of the five compounds that are able to cross the BBB, only one (metoserpate) had FDA approval. Thus, metoserpate (DB11530) was the ideal candidate not only because it is the only FDA-approved drug, but also it exhibited high gastrointestinal absorption and a propensity to cross or permeate the blood-brain barrier. Metoserpate has a total polar surface area (TPSA) of 73.02 Å<sup>2</sup> contributing to its ability to permeate cellular membranes. It is evident in literature that TPSA values less than 73.02 Å<sup>2</sup> are indicative of good permeability and satisfy Veber's rule [##REF##12036371##46##]. Metoserpate was thus selected for further analysis.</p>", "<title>3.5. Molecular Docking of TNF-Alpha and Metoserpate</title>", "<p>The binding affinity of TNF<italic>α</italic> and metoserpate was assessed using Hyde's score method. This method seeks to address weak or questionable hydrogen bonds as well as indifferent scaffolds not contributing to the free energy in the protein-ligand complex [##REF##2987660##76##, ##REF##23269578##77##]. From ##FIG##1##Figure 2(a)##, it can be observed that the Hyde score was -1.1 kg/mol which confers a favourable interaction [##REF##23269578##77##]. The docking analysis revealed one hydrogen bond between the nitrogen atom at position 8 of metoserpate and the amino acid residue Gln102 of TNF<italic>α</italic>. These results suggest that the interaction between metoserpate and TNF<italic>α</italic> at this site may have potential therapeutic implications for the treatment of TNF<italic>α</italic>-related diseases (##FIG##1##Figure 2(a)## and ##TAB##1##Table 2##).</p>", "<p>When the docking simulation result was visualized using Biovia Discovery Studio Visualizer, one pi-alkyl bond network between metoserpate and TNF<italic>α</italic> binding site amino acid (AA) residue Arg103 and two salt bridge interactions between metoserpate and the binding site AA residue Glu104 of TNF<italic>α</italic> were observed. In addition, one conventional hydrogen bond network was observed between metoserpate and TNF<italic>α</italic> binding site residue Gln102 (##FIG##1##Figure 2(b)##). It is documented that conventional hydrogen bonds aid in the stability of complexes, hence conferring a good binding affinity [##REF##34319314##78##, ##REF##20298519##79##]. Consequently, the glutamic acid (Glu104) of the protein participated in two cation-pi interactions between the imine functional group and the benzene ring of metoserpate is shown in yellow. Cation-pi interactions play an important role in determining protein structure as well as contributing significantly to the binding energy of the complex formation [##REF##15726638##80##]. Arginine (Arg103) of the protein residue participated in a pi-alkyl interaction with the benzene ring of our target drug metoserpate. According to literature, pi-alkyl interactions have a greater propensity for stability when compared to alkyls bound to nonaromatic moieties in a ligand [##UREF##18##81##–##REF##23020662##83##].</p>", "<title>3.6. Molecular Dynamics Simulations</title>", "<title>3.6.1. TNF<italic>α</italic>-Small Molecule and TNF<italic>α</italic>-Metoserpate</title>", "<p>Numerous significant pharmaceuticals and hundreds of natural products with promising bioactivities contain indole alkaloids or have structures that are like indole alkaloids. Despite not always adhering to Lipinski's rules, such compounds frequently exhibit favourable pharmacokinetic profiles with respect to cyclic molecules. The values of the root mean square deviation (RMSD) affirm whether a close-match docked pose was predicted between the crystal and the predicted structures. It is evident in literature that an RMSD value ≤ 0.2 nm is fairly good [##REF##28106755##84##–##REF##34641761##86##]. Figures ##FIG##2##3(a)## and ##FIG##2##3(b)## highlight the results of TNF<italic>α</italic> and the cocrystallized small molecule and TNF<italic>α</italic> and the target drug metoserpate both having their RMSD value ≤ 2 Å (0.2 nm) which confers a latent stable protein-ligand complex.</p>", "<p>The RMSD between the TNF<italic>α</italic>-small molecule complex and the TNF<italic>α</italic>-metoserpate complex remained consistent throughout a 100 ns simulation. However, when comparing the TNF<italic>α</italic>-small molecule complex (##FIG##2##Figure 3(a)##) to the TNF<italic>α</italic>-metoserpate complex (##FIG##2##Figure 3(b)##), a more stable trajectory was observed in the TNF<italic>α</italic>-metoserpate complex. In the case of the TNF<italic>α</italic>-small molecule complex, it displayed stability from 20 ns to approximately 30 ns, followed by a deviation. It then regained stability until around 55 ns but experienced another deviation until 60 ns. From this point, it became stable again until approximately 75 ns, with another observed deviation until around 82 ns. Finally, it regained stability and remained stable until the end of the simulation at 100 ns. On the other hand, the trajectory of the TNF<italic>α</italic>-metoserpate complex showed stability from around 15 ns to approximately 70 ns, with a slight deviation occurring until 80 ns. After this point, it regained stability and remained stable until the end of the simulation at 100 ns. Both complexes exhibited deviations within a range of 0.05 nm.</p>", "<p>The observation of stable RMSD values throughout a 100 ns simulation suggests that the overall conformation of the TNF<italic>α</italic>-small molecule complex and TNF<italic>α</italic>-metoserpate complex remained relatively consistent during the simulation period [##REF##31910338##87##]. This stability is an important characteristic as it indicates that the complexes maintained their structural integrity and did not undergo significant conformational changes. TNF<italic>α</italic>-metoserpate complex exhibited a more stable trajectory compared to the TNF<italic>α</italic>-small molecule complex suggesting that the binding of metoserpate, a small compound, may have induced more favourable interactions and a more stable complex formation. This could be attributed to specific molecular interactions, such as hydrogen bonding, electrostatic interactions, or hydrophobic interactions between metoserpate and TNF<italic>α</italic>. These interactions may contribute to a stronger binding affinity and a more stable conformation for the TNF<italic>α</italic>-metoserpate complex [##REF##20808434##88##].</p>", "<p>To describe the local conformational change in the TNF<italic>α</italic> and metoserpate and TNF<italic>α</italic>-small molecule complexes, the root mean square fluctuation (RMSF) was required. Figures ##FIG##3##4(a)## and ##FIG##3##4(b)## highlight the RMSF profile of the TNF<italic>α</italic>-small molecule and TNF<italic>α</italic>-metoserpate complexes, respectively. From the graph, stable fluctuations were observed with RMSF ≤ 0.2 nm in both instances [##REF##35385549##89##]. The TNF<italic>α</italic>-small molecule complex (##FIG##3##Figure 4(a)##) and TNF<italic>α</italic>-metoserpate complex (##FIG##3##Figure 4(b)##) both displayed reasonably low RMSF. However, the TNF<italic>α</italic>-metoserpate complex exhibited slightly higher fluctuations compared to the TNF<italic>α</italic>-small molecule complex. It is important to note that all the observed fluctuations in the TNF<italic>α</italic>-metoserpate complex were generally around 0.2 nm. On the other hand, in the TNF<italic>α</italic>-small molecule complex, fluctuations around atom positions 180 and 1520 were observed to be around 0.3 nm.</p>", "<p>These fluctuations, measured in nanometers, indicate the degree of movement or flexibility of specific atoms within the complexes. The relatively low RMSF values suggest that overall, the complexes remained relatively stable during the simulation [##UREF##19##90##]. However, the slightly higher fluctuations in the TNF<italic>α</italic>-metoserpate complex could imply that the binding of metoserpate induced some additional dynamics or flexibility in certain regions of the complex compared to the TNF<italic>α</italic>-small molecule complex [##UREF##19##90##]. The specific atom positions 180 and 1520 in the TNF<italic>α</italic>-small molecule complex experienced slightly higher fluctuations around 0.3 nm. These positions could correspond to specific residues or functional regions within the complex. The increased fluctuation at these positions may indicate potential conformational changes or greater flexibility in those regions, possibly influenced by the presence of the small compound or specific interactions between the compound and TNF<italic>α</italic> [##UREF##19##90##].</p>", "<p>The radius of gyration (Rg) monitors the compactness of the protein structure coupled with the binding patterns of the drug and protein in direct relation to the folding rate [##REF##16400647##91##]. A conformational change occurs when a ligand or lead molecule attaches to the protein, changing the radius of gyration [##REF##20401516##92##]. The TNF<italic>α</italic>-small molecule complex (##FIG##4##Figure 5(a)##) and TNF<italic>α</italic>-metoserpate complex (##FIG##4##Figure 5(b)##) exhibited similar total radius of gyration values, both measuring approximately 1.52 nm. A smaller radius of gyration indicates a more compact and tightly packed structure, while a larger radius of gyration suggests a more extended or flexible conformation [##UREF##19##90##]. The fact that both the TNF<italic>α</italic>-small molecule complex and TNF<italic>α</italic>-metoserpate complex demonstrated a total radius of gyration around 1.52 nm suggests that they possess comparable overall compactness, indicating a compact and stable conformation [##REF##35061725##93##]. This similarity in size could indicate that the binding of both the small molecule and metoserpate did not significantly alter the overall conformation or compactness of the TNF<italic>α</italic> complex.</p>", "<title>3.6.2. Bond Network Evaluation of Metoserpate and TNF-Alpha Complex following Molecular Dynamics Simulation</title>", "<p>The post-MD simulation analysis revealed significant changes in the metoserpate-TNF<italic>α</italic> complex compared to the pre-MD simulation complex. Our findings demonstrated that metoserpate established multiple bond network interactions with the AAs in the binding site of TNF<italic>α</italic>. Specifically, a conventional hydrogen bond (cH-bond) was formed between the oxygen of the carboxylic acid methyl ester of metoserpate and the amino acid residue Lys98 of TNF<italic>α</italic>. Conventional hydrogen bonds are known for their strength and contribute to strong binding affinity. Additionally, several nonconventional hydrogen bonds (ncH-bonds) were observed between metoserpate and the AAs Ser99, Glu104, Pro113, Tyr115, and Glu116. Metoserpate also engaged in a pi-alkyl interaction with Tyr115 and Pro117, as well as two cation-pi interactions with Glu104 and Glu116. These interactions played a crucial role in the stability and specificity of the complex (##FIG##5##Figure 6##). Hydrophobic contact area was also established between metoserpate and the binding site AA residues Lys98 and Tyr115.</p>", "<p>In contrast, the bond network analysis conducted prior to the MD simulation revealed specific interactions between metoserpate and TNF<italic>α</italic>, including a cH-bond network with Gln102, a pi-alkyl interaction with Arg103, and cation-pi interactions with Glu104. However, the subsequent MD simulation analysis yielded intriguing findings, indicating an enhanced binding affinity and selectivity of metoserpate towards TNF<italic>α</italic>. This improvement in binding was accompanied by the generation of more favourable and specific interactions.</p>", "<p>These results are further supported by the observed flexibility in the root mean square fluctuation (RMSF) output of the TNF<italic>α</italic> and metoserpate complex. The MD simulations have provided valuable insights into the intricate molecular interactions between the TNF<italic>α</italic> and metoserpate, unravelling the complexities of protein-ligand complexes.</p>", "<p>By elucidating the dynamic behaviour and uncovering the structural changes that occur during the simulation, the MD simulations offer a deeper understanding of the binding mechanism and contribute to the overall comprehension of the interactions between TNF<italic>α</italic> and metoserpate.</p>", "<title>3.6.3. Overall Bond Network Assessment</title>", "<p>The Hyde scoring method has proven to be a valuable computational tool in drug discovery for estimating the binding affinity between a protein and a ligand, utilizing their interaction energy [##REF##17124629##94##]. In the present study, we employed the Hyde score assessment method to evaluate the binding affinity of two ligands, metoserpate and <italic>yohimbine</italic>, with the protein TNF<italic>α</italic>, with a specific focus on the amino acid residues within the binding site (##TAB##1##Table 2##).</p>", "<p>The study's findings revealed that metoserpate exhibited a slightly lower Hyde score (-1.1 kJ/mol) in comparison to <italic>yohimbine</italic> (-1.0 kJ/mol and 0.2 kJ/mol) when interacting with Gln102 (##TAB##1##Table 2##). This indicates that metoserpate possesses a marginally better binding affinity with TNF<italic>α</italic> when compared to <italic>yohimbine</italic>, although the difference observed is relatively small. These results shed light on the relative strengths of the interactions between metoserpate and TNF<italic>α</italic>, providing insights into the binding affinity. This information contributes to the understanding of the potential efficacy of metoserpate as a potential therapeutic agent targeting TNF<italic>α</italic> in the context of AD management.</p>", "<p>The post-MD simulation analysis revealed the involvement of amino acid Lys98 in the conventional hydrogen bond formation, as well as the formation of a nonconventional hydrogen bond network with Pro113, which was originally not part of the binding site AA residues (##FIG##5##Figure 6##). This post-MD simulation analysis generated more bond diversity, and bond number compared to the TNF<italic>α</italic>-<italic>yohimbine</italic> and TNF<italic>α</italic>-metoserpate complexes. This demonstrates the importance of post-MD simulation analysis in providing a more comprehensive understanding of protein-ligand interactions beyond what can be predicted through initial scoring methods alone.</p>", "<p>The findings of this study also suggest that the binding affinity of a ligand with a protein may be influenced by amino acid residues outside of the initial binding site. This is consistent with previous studies that have shown the importance of protein flexibility and dynamics in ligand binding [##REF##31244000##95##]. It is possible that the nonconventional hydrogen bond network identified in the post-MD simulation analysis plays a critical role in the binding affinity between TNF<italic>α</italic> and metoserpate.</p>" ]
[ "<title>4. Conclusion</title>", "<p>In summary, our study employed the Hyde score assessment method to evaluate the binding affinity of metoserpate and <italic>yohimbine</italic> with TNF<italic>α</italic>, with a specific focus on the binding site amino acid residues. While metoserpate generated a lower Hyde score than <italic>yohimbine</italic> with the key binding site amino acid Gln102, further investigation using postmolecular dynamics (MD) simulation analysis demonstrated the involvement of additional amino acid residues in the binding affinity. The results indicated that metoserpate has the potential to inhibit TNF<italic>α</italic> and thus presents as a promising candidate for further study as a therapeutic agent for TNF<italic>α</italic>-related diseases. Additionally, our work showcases the utility of <italic>yohimbine</italic> as a query compound to identify structurally similar drugs from the DrugBank database in the context of drug repurposing. Specifically, our study identified metoserpate as a potential inhibitor of TNF<italic>α</italic> using a computational approach that combined molecular docking and MD simulation. This approach allowed for a more comprehensive and nuanced understanding of the binding affinity of metoserpate with TNF<italic>α</italic> and provided insights into the potential mechanisms of inhibition. Furthermore, our use of <italic>yohimbine</italic> as a query compound helped identify metoserpate as a structurally similar compound with potential therapeutic properties. Overall, these findings represent a significant step forward in the development of metoserpate as a potential therapeutic agent for TNF<italic>α</italic>-related diseases. However, further research is needed to validate these findings through <italic>in vitro</italic> and <italic>in vivo</italic> (in a physiologically relevant cell line, fly models, and/or animal models) studies and to optimize the efficacy of metoserpate as a drug candidate.</p>" ]
[ "<p>Academic Editor: Min Hui Li</p>", "<title>Introduction</title>", "<p> Alzheimer's disease (AD) is a neurodegenerative disorder with no conclusive remedy. <italic>Yohimbine</italic>, found in <italic>Rauwolfia vomitoria</italic>, may reduce brain inflammation by targeting tumour necrosis factor-alpha (TNF<italic>α</italic>), implicated in AD pathogenesis. Metoserpate, a synthetic compound, may inhibit TNF<italic>α</italic>. The study is aimed at assessing the potential utility of repurposing metoserpate for TNF<italic>α</italic> inhibition to reduce neuronal damage and inflammation in AD. The development of safe and effective treatments for AD is crucial to address the growing burden of the disease, which is projected to double over the next two decades. </p>", "<title>Methods</title>", "<p> Our study repurposed an FDA-approved drug as TNF<italic>α</italic> inhibitor for AD management using structural similarity studies, molecular docking, and molecular dynamics simulations. <italic>Yohimbine</italic> was used as a reference compound. Molecular docking used SeeSAR, and molecular dynamics simulation used GROMACS. </p>", "<title>Results</title>", "<p> Metoserpate was selected from 10 compounds similar to <italic>yohimbine</italic> based on pharmacokinetic properties and FDA approval status. Molecular docking and simulation studies showed a stable interaction between metoserpate and TNF<italic>α</italic> over 100 ns (100000 ps). This suggests a reliable and robust interaction between the protein and ligand, supporting the potential utility of repurposing metoserpate for TNF<italic>α</italic> inhibition in AD treatment. </p>", "<title>Conclusion</title>", "<p> Our study has identified metoserpate, a previously FDA-approved antihypertensive agent, as a promising candidate for inhibiting TNF<italic>α</italic> in the management of AD.</p>" ]
[]
[ "<title>Data Availability</title>", "<p>The PDB file was obtained from the RCSB Protein Data Bank (<ext-link xlink:href=\"http://www.rcsb.org/\" ext-link-type=\"uri\">http://www.rcsb.org/</ext-link>). The 3D conformer structure of <italic>yohimbine</italic> was obtained from the PubChem database (<ext-link xlink:href=\"https://pubchem.ncbi.nlm.nih.gov/\" ext-link-type=\"uri\">https://pubchem.ncbi.nlm.nih.gov/</ext-link>). The data generated in this research, including the utilized compounds, molecular docking outcomes, and molecular dynamics simulation data, are accessible upon request to the corresponding author.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' Contributions</title>", "<p>TEJ was involved in the conceptualization, study design, molecular docking, molecular dynamics simulation, and drafting of the manuscript. OA was involved in the study design and revision of the initial draft manuscript. KDNO, BKS, SDL, MM, JTD, and O-AMY were involved in the study design. All authors read and approved the final manuscript.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>(a) A 3D representation of the complex formed between TNF<italic>α</italic> and <italic>yohimbine</italic> using SeeSAR software. The Hyde score indicates the binding affinity of the ligand to the protein. The ligand's N5 and O3 atoms form hydrogen bonds with the protein's Gln102 residue, contributing to the stability of the complex. (b) Molecular docking of <italic>yohimbine</italic> with TNF<italic>α</italic> protein. 3D structure of the protein-ligand complex visualized using Biovia Discovery Studio. The ligand (in grey, red, and purple) forms a hydrogen bond network with Gln102 (in green dashed lines) through its N5 and O3 atoms and a nonconventional hydrogen bond network between the O3 atom of <italic>yohimbine</italic> and Cys101 (in light green dashed lines). The ligand also interacts with Arg103 through a pi-alkyl interaction (mauve dashed line). The ligand is surrounded by a hydrophobic contact area (in light blue shade) involving Gln102 and Arg103.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>(a) A 3D representation of the complex formed between TNF<italic>α</italic> and metoserpate using SeeSAR software. The Hyde score indicates the binding affinity of the ligand to the protein. The ligand's N8 atom form hydrogen bonds with the protein's Gln102 residue, contributing to the stability of the complex. (b) Molecular interactions of TNF<italic>α</italic>-metoserpate complex. 3D visualization of the complex using Biovia Discovery Studio. The ligand (in grey, red, and purple) forms a hydrogen bond network with Gln102 through its N8 atom (in green dashed lines). The ligand also interacts with Arg103 through a pi-alkyl interaction (in mauve dashed line). A hydrophobic contact area (in light blue shade) is observed between the ligand and residues Gln102 and Glu104 (in light blue shade). Cation-pi interactions (in golden yellow) are formed between metoserpate and Glu104.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>Trajectories of the overall RMSD: (a) TNF<italic>α</italic>-small molecule complex; (b) TNF<italic>α</italic>-metoserpate complex. RMSD of the various complexes with respect to the starting structure over 100 ns MD simulation. The <italic>x</italic>-axis represents the simulation time in nanoseconds. The <italic>y</italic>-axis represents RMSD in nanometers.</p></caption></fig>", "<fig position=\"float\" id=\"fig4\"><label>Figure 4</label><caption><p>Residue-wise RMSF profiles of the TNF<italic>α</italic> and various ligand complexes: (a) TNF<italic>α</italic>-small molecule complex; (b) TNF<italic>α</italic>-metoserpate complex. The <italic>x</italic>-axis represents the atom number. The <italic>y</italic>-axis represents RMSF in nanometers.</p></caption></fig>", "<fig position=\"float\" id=\"fig5\"><label>Figure 5</label><caption><p>Radius of gyration profiles of the TNF<italic>α</italic> and various ligand complexes: (a) TNF<italic>α</italic>-small molecule complex; (b) TNF<italic>α</italic>-metoserpate complex. The <italic>x</italic>-axis represents the time in picoseconds. The <italic>y</italic>-axis represents Rg in nanometers.</p></caption></fig>", "<fig position=\"float\" id=\"fig6\"><label>Figure 6</label><caption><p>Molecular interactions between TNF<italic>α</italic> and metoserpate after molecular dynamics simulation. The protein residues involved in binding are represented in disc shape with different colours. The ligand is shown in ball-and-stick representation with different colours for different atom types. The molecular interactions are depicted by dashed lines with different colours indicating different types of interactions: conventional hydrogen bonds (deep green), nonconventional hydrogen bonds (light green), pi-alkyl interactions (mauve), and cation-pi interactions (golden yellow).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Pharmacokinetic properties of <italic>yohimbine.</italic></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">GI absorption</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">BBB permeant</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Lipinski</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Ghose</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Veber</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Egan</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Muegge</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">High</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Permeant</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Hyde's score estimates and TNF<italic>α</italic> binding site amino acid residues.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Compound</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Hyde score (kJ/mol)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Bond interaction (ligand ➔ protein)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Binding site amino acid residues</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Yohimbine</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-1.0<break/>0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N5 ➔ Gln102<break/>O3 ➔ Gln102</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">Cys69, Lys98, Ser99, Pro100, Cys101, Gln102, Arg103, Glu104, Thr105, Trp114, Tyr115, Glu116, and Pro117</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Metoserpate</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-1.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N8 ➔ Gln102</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Drugs that are 75% or more structurally similar to <italic>yohimbine.</italic></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Drug (ID)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Status</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">% similarity</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Chemical formula</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Metoserpate (DB11530)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vet approved</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.992</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>24</sub>H<sub>32</sub>N<sub>2</sub>O<sub>5</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Deserpidine (DB01089)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Approved</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.960</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>32</sub>H<sub>38</sub>N<sub>2</sub>O<sub>8</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">18-Methoxycoronaridine (DB15096)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Investigational</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.942</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>22</sub>H<sub>28</sub>N<sub>2</sub>O<sub>3</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CP-320626 (DB03383)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Experimental</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.764</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>23</sub>H<sub>23</sub>C<sub>l</sub>FN<sub>3</sub>O<sub>3</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rescinnamine (DB01180)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Approved</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.823</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>35</sub>H<sub>42</sub>N<sub>2</sub>O<sub>9</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Reserpine (DB00206)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Approved, investigational, withdrawn</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.809</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>33</sub>H<sub>40</sub>N<sub>2</sub>O<sub>9</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Raubasine (DB15949)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Experimental</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.873</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>21</sub>H<sub>24</sub>N<sub>2</sub>O<sub>3</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Methoserpidine (DB13631)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Experimental</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.812</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>33</sub>H<sub>40</sub>N<sub>2</sub>O<sub>9</sub></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(7as,12ar,12bs)-1,2,3,4,7a,12,12a,12b-Octahydroindolo[2,3-a]Quinolizin-7(6h)-One (DB02191)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Experimental</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.767</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>15</sub>H<sub>16</sub>N<sub>2</sub>O</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vinburnine (DB13793)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Experimental</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.751</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C<sub>19</sub>H<sub>22</sub>N<sub>2</sub>O</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>Pharmacokinetic properties of the 10 DrugBank compounds.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Drug</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">BBB permeant</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">GI absorption</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Metoserpate (DB11530)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Deserpidine (DB01089)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">18-Methoxycoronaridine (DB15096)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CP-320626 (DB03383)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rescinnamine (DB01180)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Reserpine (DB00206)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Raubasine (DB15949)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Methoserpidine (DB13631)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(7as,12ar,12bs)-1,2,3,4,7a,12,12a,12b-Octahydroindolo[2,3-a]Quinolizin-7(6h)-One (DB02191)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vinburnine (DB13793)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr></tbody></table></table-wrap>" ]
[]
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[ "<table-wrap-foot><fn><p>Permeant = blood-brain barrier permeant; Yes = no violation; BBB = blood-brain barrier.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Gln102 = the key amino acid residue involved in the hydrogen bond formation.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Status = FDA approval status; Drug ID = DrugBank ID.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>BBB = blood brain-barrier; GI = gastrointestinal.</p></fn></table-wrap-foot>" ]
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[{"label": ["2"], "person-group": ["\n"], "surname": ["Green", "Fujita", "Yang"], "given-names": ["G. S.", "M.", "H. S."], "source": ["\n"], "italic": ["Cellular dynamics across aged human brains uncover a multicellular cascade leading to Alzheimer\u2019s disease"], "year": ["2023"], "publisher-name": ["bioRxiv"], "pub-id": ["10.1101/2023.03.07.531493"]}, {"label": ["7"], "person-group": ["\n"], "surname": ["Kepp"], "given-names": ["K. P."], "article-title": ["Alzheimer\u2019s disease: how metal ions define "], "italic": ["\u03b2", "Coordination Chemistry Reviews"], "source": ["\n"], "year": ["2017"], "volume": ["351"], "fpage": ["127"], "lpage": ["159"], "pub-id": ["10.1016/j.ccr.2017.05.007", "2-s2.0-85019753866"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Tobinick", "Gross", "Weinberger", "Cohen"], "given-names": ["E.", "H.", "A.", "H."], "article-title": ["TNF-alpha modulation for treatment of Alzheimer's disease: a 6-month pilot study"], "source": ["\n"], "italic": ["Medscape General Medicine"], "year": ["2006"], "volume": ["8"], "issue": ["2"], "fpage": ["p. 25"]}, {"label": ["15"], "person-group": ["\n"], "surname": ["Rogers", "Cooper", "Webster"], "given-names": ["J.", "N. R.", "S."], "article-title": ["Complement activation by beta-amyloid in Alzheimer disease"], "source": ["\n"], "italic": ["Proceedings of the National Academy of Sciences"], "year": ["1992"], "volume": ["89"], "issue": ["21"], "fpage": ["10016"], "lpage": ["10020"], "pub-id": ["10.1073/pnas.89.21.10016", "2-s2.0-0026447529"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Ajao", "Imam", "Adana"], "given-names": ["M.", "A.", "M."], "article-title": ["Central cholinergic markers and spatial memory performance in rats following administration of rauwolfia vomitoria and chlorpromazine"], "source": ["\n"], "italic": ["Centre Point Journals"], "year": ["2015"], "volume": ["21"], "fpage": ["75"], "lpage": ["93"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Tettevi", "Maina", "Simpong", "Osei-Atweneboana", "Ocloo"], "given-names": ["E. J.", "M.", "D. L.", "M. Y.", "A."], "article-title": ["A Review of African Medicinal Plants and Functional Foods for the Management of Alzheimer's Disease-related Phenotypes, Treatment of HSV-1 Infection and/or Improvement of Gut Microbiota. Journal of Evidence-Based"], "source": ["\n"], "italic": ["Integrative Medicine"], "year": ["2022"], "volume": ["27, article 2515690X221114657"], "pub-id": ["10.1177/2515690X221114657"]}, {"label": ["23"], "person-group": ["\n"], "surname": ["Ajayi"], "given-names": ["O. A."], "article-title": ["Phytochemical and GC-MS analysis of bioactive components in ethanolic extract of Rauvolfia vomitoria leaves"], "source": ["\n"], "italic": ["Journal of Chemical Society of Nigeria"], "year": ["2021"], "volume": ["46"], "issue": ["4"], "pub-id": ["10.46602/jcsn.v46i4.642"]}, {"label": ["24"], "person-group": ["\n"], "surname": ["Bonheur", "D\u00e9sir\u00e9", "Ernestine"], "given-names": ["Y. D. D.", "D. D. P.", "N. Z."], "article-title": ["Anti-inflammatory and antioxidant effects of the stem bark aqueous extract of Rauwolfia vomitoria (Apocynaceae) in female Wistar rats"], "source": ["\n"], "italic": ["European Journal of Pharmaceutical and Medical Research"], "year": ["2015"], "volume": ["2"], "fpage": ["64"], "lpage": ["73"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Chinonye", "Chijioke", "Iwuji"], "given-names": ["I. I.", "C.", "C. S."], "article-title": ["Chemical and medicinal properties of Rauwolfia vomitoria (AFZEL) harvested from the South Eastern Nigeria"], "source": ["\n"], "italic": ["Asian Journal of Chemical Sciences"], "year": ["2021"], "volume": ["10"], "issue": ["4"], "fpage": ["56"], "lpage": ["71"], "pub-id": ["10.9734/ajocs/2021/v10i419103"]}, {"label": ["26"], "person-group": ["\n"], "surname": ["Jachak", "Saklani"], "given-names": ["S. M.", "A."], "article-title": ["Challenges and opportunities in drug discovery from plants"], "source": ["\n"], "italic": ["Current Science"], "year": ["2007"], "volume": ["92"], "issue": ["9"], "fpage": ["1251"], "lpage": ["1257"], "comment": ["\n"], "ext-link": ["https://www.jstor.org/stable/24097892"]}, {"label": ["32"], "person-group": ["\n"], "surname": ["Lo", "Torres"], "given-names": ["Y. C.", "J. Z."], "article-title": ["Chemical Similarity Networks for Drug Discovery"], "source": ["\n"], "italic": ["Special Topics in Drug Discovery"], "year": ["2016"], "volume": ["1"], "fpage": ["53"], "lpage": ["70"], "pub-id": ["10.5772/65106"]}, {"label": ["33"], "person-group": ["\n"], "surname": ["Shao", "Jiang", "Meng", "Xu"], "given-names": ["M.", "L.", "Z.", "J."], "article-title": ["Computational drug repurposing based on a recommendation system and drug\u2013drug functional pathway similarity"], "source": ["\n"], "italic": ["Molecules"], "year": ["2022"], "volume": ["27"], "issue": ["4"], "fpage": ["p. 1404"], "pub-id": ["10.3390/molecules27041404"]}, {"label": ["34"], "person-group": ["\n"], "surname": ["Wang", "Aldahdooh", "Hu"], "given-names": ["Y.", "J.", "Y."], "article-title": ["DrugRepo: a novel approach to repurposing drugs based on chemical and genomic features"], "source": ["\n"], "italic": ["Scientific Reports"], "year": ["2022"], "volume": ["12"], "issue": ["1, article 21116"], "pub-id": ["10.1038/s41598-022-24980-2"]}, {"label": ["39"], "person-group": ["\n"], "surname": ["BioviaDS"], "given-names": ["D. S."], "source": ["\n"], "italic": ["BIOVIA discovery studio visualizer"], "year": ["2021"], "volume": ["20"], "publisher-name": ["Softw. version"]}, {"label": ["42"], "person-group": ["\n"], "surname": ["Lipinski", "Lombardo", "Dominy", "Feeney"], "given-names": ["C. A.", "F.", "B. W.", "P. J."], "article-title": ["Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings"], "source": ["\n"], "italic": ["Advanced Drug Delivery Reviews"], "year": ["2012"], "volume": ["64"], "fpage": ["4"], "lpage": ["17"], "pub-id": ["10.1016/j.addr.2012.09.019", "2-s2.0-84870249445"]}, {"label": ["45"], "person-group": ["\n"], "surname": ["Muegge", "Oloff"], "given-names": ["I.", "S."], "article-title": ["Advances in virtual screening"], "source": ["\n"], "italic": ["Drug Discovery Today: Technologies"], "year": ["2006"], "volume": ["3"], "issue": ["4"], "fpage": ["405"], "lpage": ["411"], "pub-id": ["10.1016/j.ddtec.2006.12.002", "2-s2.0-33846487854"]}, {"label": ["48"], "person-group": ["\n"], "surname": ["See"], "given-names": ["S. A. R."], "source": ["\n"], "italic": ["Version 12.1"], "year": ["2022"], "publisher-loc": ["Sankt Augustin, Germany"], "publisher-name": ["BioSolveIT GmbH"], "comment": ["\n"], "ext-link": ["https://www.biosolveit.de"]}, {"label": ["51"], "person-group": ["\n"], "surname": ["Abraham", "Murtola", "Schulz"], "given-names": ["M. J.", "T.", "R."], "article-title": ["GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers"], "source": ["\n"], "italic": ["SoftwareX"], "year": ["2015"], "volume": ["1-2"], "fpage": ["19"], "lpage": ["25"], "pub-id": ["10.1016/j.softx.2015.06.001", "2-s2.0-84946416234"]}, {"label": ["81"], "person-group": ["\n"], "surname": ["Arthur", "Akoji", "Sahnoun"], "given-names": ["D. E.", "J. N.", "R."], "article-title": ["A theoretical insight in interactions of some chemical compounds as mTOR inhibitors"], "source": ["\n"], "italic": ["Bulletin of the National Research Centre"], "year": ["2021"], "volume": ["45"], "issue": ["1"], "fpage": ["1"], "lpage": ["12"], "pub-id": ["10.1186/s42269-021-00525-x"]}, {"label": ["90"], "person-group": ["\n"], "surname": ["V\u00e1zquez", "Hermosilla", "Guallar", "Estrada", "Vinacua"], "given-names": ["P.", "P.", "V.", "J.", "\u00c0."], "article-title": ["Visual analysis of protein\u2010ligand interactions"], "source": ["\n"], "italic": ["Computer Graphics Forum"], "year": ["2018"], "volume": ["37"], "issue": ["3"], "fpage": ["391"], "lpage": ["402"], "pub-id": ["10.1111/cgf.13428", "2-s2.0-85050298915"]}]
{ "acronym": [], "definition": [] }
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2024-01-14 23:43:50
Biomed Res Int. 2024 Jan 6; 2024:9985719
oa_package/76/bf/PMC10787656.tar.gz
PMC10787657
0
[ "<title>1. Introduction</title>", "<p>Aquaculture is one of the fastest growing food production industries globally, and finfish farming has accounted for the largest share of world aquaculture for decades. In 2020, farmed finfish reached 57.5 million tons and in particular, farmed Atlantic salmon has been one of the major contributors to growth in the global aquaculture production and global trade [##UREF##0##1##]. However, there are increasing challenges including environmental impact and cost of aquafeeds ranged 50%–60% of the operational costs. Currently, fishmeal is the primary ingredient used as a quality protein source in feeds for aquatic animals, particularly those used for salmon and other carnivorous fish species. Nevertheless, the utilization of fishmeal as the primary source of protein in aquafeeds is becoming unfeasible, both practically and economically, due to its limited supply and rapidly increasing price. Therefore, developing new alternative ingredients, including those derived from insects, to replace fishmeal is a priority for the aquaculture industry and scientists.</p>", "<p>Insect meals are among the most promising novel protein sources in aquafeeds due to their nutritional compositions such as protein content and amino acid profile as well as their potential for commercial-scale production to meet the demand for alternative protein sources in aquafeeds [##UREF##1##2##–##UREF##6##7##]. Mealworms are insects rich in proteins (47%–60% on a dry weight basis; DW) and lipids (31%–43% DW) and with favorable amino acid and fatty acid profiles for animal feeds [##UREF##7##8##]. There are increasing reports of partial to complete replacement of fishmeal by insect meals, including mealworm meals in the diets of carnivorous finfish such as rainbow trout, <italic>Oncorhynchus mykiss</italic> [##UREF##8##9##–##UREF##11##12##], European sea bass, <italic>Dicentrarchus labrax</italic> [##UREF##12##13##, ##REF##29229441##14##], gilthead seabream, <italic>Sparus aurata</italic> [##UREF##13##15##], and other finfish species such as African catfish, <italic>Clarias gariepinus</italic> [##UREF##14##16##, ##UREF##15##17##], common catfish, <italic>Ameiurus melas</italic> [##UREF##16##18##], yellow catfish, <italic>Pelteobagrus fulvidraco</italic> [##REF##28807649##19##], and Tilapia, <italic>Oreochromis nilotica</italic> [##UREF##17##20##]. Moreover, insects, including mealworms, contain varying amounts of chitin [##REF##19360565##21##] which may have immunomodulatory benefits in aquatic animals [##REF##29229441##14##, ##UREF##18##22##]. Few studies have reported the effects of mealworm meals on the immune system of fish, which mainly focus to European sea bass [##REF##29229441##14##], Jian carp, <italic>Cyprinus carpio</italic> var. Jian [##UREF##19##23##], and yellow catfish [##REF##28807649##19##].</p>", "<p>In fish nutrition studies, blood biochemistry and hepatic antioxidants are key parameters for evaluating fish health as it relates to nutritional manipulations [##REF##29229441##14##, ##UREF##20##24##–##UREF##27##32##]. The adaptive immune system of teleosts includes B cells, which produce three classes of immunoglobulins (Igs) such as IgM, IgD, and IgT (or IgZ in some species), with IgM+ being the predominant surface Ig isotype [##UREF##28##33##, ##UREF##29##34##]. The relative expression of target genes related to immune/health responses as a result of dietary substitutions is important because they can provide insights that show further specific mechanisms behind fish health [##REF##28807649##19##, ##UREF##19##23##, ##REF##26631806##35##, ##UREF##30##36##]. The gut microbiome includes both allochthonous (transient) members that are more associated with the dietary components found in the digesta in the lumen of the gut which is eventually excreted in the feces as well as autochthonous (residential) members which are associated with the host's gut epithelial surface or microvilli [##UREF##31##37##]. The gut microbiome changes due to diet, exposure to toxicants, stress, and other environmental factors such as salinity and temperature [##UREF##32##38##]. These changes in gut microbiome can affect the nutritional metabolism, immune regulation, fish development, and disease resistance of the fish host [##UREF##33##39##]. Studies have shown that the use of mealworm meal has had variable effects on the gut microbiome of rainbow trout, gilthead sea bream, and European sea bass [##UREF##34##40##, ##REF##33397465##41##].</p>", "<p>Furthermore, recent growth in land-based recirculating aquaculture systems (RASs) has created the need to develop diets suitable for use in closed systems. In RAS, feeds that result in physically stable feces are desired to minimize the disintegration of these waste products into suspended solids which can degrade water quality and may compromise fish health. In general, larger particle sizes (&gt;150 microns) of suspended solids are desired as more efficiently removed by mechanical filtration [##UREF##35##42##]. Feces stability is believed to be affected by the feed ingredients used because they may contain natural surfactants or other compounds that relate to particle binding or breakdown. This study hypothesis that diets produced with mealworm meals are suitable for use in RAS, particularly in terms of the resultant feces' stability and larger particle sizes of suspended solids, more efficiently removable by filters.</p>", "<p>This study was conducted to determine if the substitution of fishmeal with two mealworm meals in the diet of Atlantic salmon effected the growth performance, feed utilization, feces stability, condition indices, body composition, health parameters, digesta microbiome, and the relative expression of target genes related to fish growth, protein synthesis, and health.</p>" ]
[ "<title>2. Materials and Methods</title>", "<title>2.1. Experimental Diets</title>", "<p>\n##TAB##0##Table 1## shows the nutritional values of the test ingredients used in this study which included defatted mealworm meal, <italic>Tenebrio molitor</italic>, and whole mealworm meal, <italic>Alphitobius diaperinus</italic> (Ÿnsect, France). The formulations of the control diet (100% FM) produced with a high-quality fishmeal (FM) with 75% crude protein (SeaProTM 75, BioOregon Protein Inc., Warrenton, OR, USA) as a primary ingredient and three test diets (partial and full substitution of FM by defatted yellow mealworm meal (50% DMM and 100% DMM, respectively) and partial replacement of FM with whole mealworm meal (50% WMM)) are presented in ##TAB##1##Table 2##. Complete replacement of fishmeal with whole mealworm meal could not be achieved because the high lipid concentrations of WMM could not be balanced using practical feed formulations. All ingredient replacements were conducted on a crude protein basis. The four diets were formulated to be isonitrogenous, isolipidic, and isoenergetic while considering similar levels of total fiber and meeting the requirements of Atlantic salmon for essential amino acids and phosphorus. Guar gum (0.3% w/w on a dry weight basis) was added as a binding agent known to stabilize salmonid feces [##UREF##36##43##]. Diets were produced at the USDA-ARS facility in Bozeman, MT, USA, using a twin-screw extruder (DNDL-44, Buhler AG, Uzwil, Switzerland). Pellets were then dried, cooled, and vacuum coated with oil blends. Analyses of experimental diets for proximate, amino acid, and fatty acid compositions (Tables ##TAB##2##3## and ##TAB##3##4##) were carried out by the Experiment Station Chemical Laboratories (ESCL), University of Missouri, Columbia, MO, USA.</p>", "<title>2.2. Experimental System and Approach</title>", "<p>A 12-week growth trial was conducted to evaluate the four diets described above. Each diet was evaluated in four replicates using a randomized design. The use of experimental fish was under scientific research protocols of the University of Maine Institutional Animal Care and Use Committee (Protocol number A2021-09-04) and complied with all relevant international animal welfare laws, guidelines, and policies. Twenty feed-trained Atlantic salmon parr (38.5 ± 0.1 g initial weight) were stocked into each of the 16 experimental tanks of the RAS system, with tank serving as the experimental replicate. Diets were randomly assigned to tanks using a random number generator (random.org). Following stocking and acclimation, experimental diets were fed to Atlantic salmon three times daily to apparent satiation/ ad-libitum (09:00, 13:00, and 16:00 hr) and two times daily (09:00 and 16:00 hr) during weekends for 12 weeks. Feed given per tank was recorded daily by weighing feed before and after feeding time. Fish were weighed three times: at the beginning, middle, and end of the growth trial. Water quality parameters were maintained within suitable ranges for Atlantic salmon, including temperature (12–14°C), pH (7–8), dissolved oxygen (80%–120%), total ammonia nitrogen (0–0.8 mg/L), and nitrite nitrogen (0–0.6 mg/L). Photoperiod was 12-hr daylight and 12-hr darkness (artificial lighting controlled by a timer).</p>", "<title>2.3. Sample Collection</title>", "<p>At the beginning of the growth trial, samples of diets and 10 fish (∼40 g each) from the source population were collected and stored at −80°C for proximate, amino acid, and fatty acid composition analyses. At the end of the 12-week growth trial, fish in each tank were sampled after a 24-hr fasting period. All fish from each tank were bulk weighed and counted. Five fish were returned to their respective tanks and cultured for an additional 1 week at which point they were sampled for digesta microbiome analysis and evaluation of feces stability. Three fish from each experimental tank were bled following anesthesia with buffered tricaine methanesulfonate (MS-222; buffered with 200 mg/L of sodium bicarbonate) at approximately 75 mg/L. Blood samples were placed into 2 mL tubes, with lithium heparin (Greiner Bio-One North America Inc., Monroe, NC, USA) as anticoagulant. Plasma was extracted immediately by microcentrifugation at 3,000x <italic>g</italic> at 4°C for 10 min. Three additional fish were sampled and humanely euthanized with 250 mg/L buffered MS-222. The external surface of each euthanized fish was wiped with 70% ethanol to avoid contamination from external microbes, and liver was removed and placed into a 2 mL microcentrifuge tube and stored at −80°C for analysis of hepatic peroxide and antioxidant enzymes. Next, part of the midgut of the same three fish per tank was removed and placed in a separate group of sterile 2 mL tubes and stored at −80°C for conducting growth and immune-related gene expression assays. Eleven euthanized fish from each tank were individually weighed and measured to calculate the Fulton condition factor (K-factor); and then five fish were dissected, and their viscera were used to calculate viscerosomatic index (VSI). Six euthanized fish from each tank were stored at −80°C for subsequent whole-body proximate, amino acid, and fatty acid composition analyses.</p>", "<p>The remaining five fish per tank were fed with their respective experimental diets for one additional week and then euthanized, as described above. The digestive tracts of these fish were aseptically dissected and the digesta was removed by massaging the GI until the digesta pellet emerged and frozen at −80°C for microbiome analysis (two fish per tank and eight fish per treatment) and evaluation of feces stability (three fish per tank and 12 fish per treatment).</p>", "<title>2.4. Feces Stability Assay</title>", "<p>Fecal strands were thawed 2–3 hr prior to analysis. Approximately 50 mg (wet weight) of undisturbed fecal material was added to a laser diffraction particle size analyzer (Mastersizer 3000, Malvern Panalytical, Malvern, UK) equipped with a Hydro MV automated dispersion unit (Malvern Panalytical, Malvern, UK). Feces breakdown was achieved by stirring (800 rpm) each sample in distilled water, using the Hydro MV stirrer, for 20 s prior to the first measurement. The particle size distribution (percent by total particle volume) of each sample was measured five times and then averaged. The mean particle size at the 10<sup>th</sup>, 50<sup>th</sup>, and 90<sup>th</sup> percentiles of the resultant particle size distributions was used in statistical comparisons.</p>", "<title>2.5. Chemical Composition Analysis</title>", "<p>Test ingredients, feed, and whole-body samples were analyzed for total crude protein, energy, total (ether extracted) crude lipid, moisture, amino acid, and fatty acid compositions. The proximate composition of diets and whole-body samples was analyzed using standard procedures [##UREF##37##44##]. The amino acids and fatty acids analyses were conducted by the Experiment Station Chemical Laboratories (ESCL), University of Missouri, Columbia, MO, USA, using methods approved by the Association of Official Analytical Chemists (AOAC) and American Oil Chemists' Society (AOCS).</p>", "<title>2.6. Plasma Biochemical, Hepatic Peroxide, and Antioxidants Analyses</title>", "<p>Plasma biochemistry (alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), immunoglobulin M (IgM), and total iron-binding capacity (TIBC)) and hepatic peroxide (malondialdehyde (MDA)) content, antioxidants enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) activity, were analyzed spectrophotometrically using commercial kits (BioVision, Milpitas, CA, USA), as described in previous studies [##UREF##26##31##, ##UREF##27##32##, ##REF##26631806##35##, ##REF##25463293##45##].</p>", "<title>2.7. Extraction, Sequencing, and Sequence Analysis for Digesta Microbiome</title>", "<p>Triplicate samples of each of the feeds (<italic>n</italic> = 12) as well as duplicate digesta samples (<italic>n</italic> = 32) from each tank were sent to RTL Genomics (Lubbock, TX, USA) for 16S metabarcoding. Once received by RTL Genomics, the digesta and feed samples underwent DNA extraction with the Zymo ZR-96 Magbead kit (Irvine, CA, USA) on the Thermo Scientific KingFisher FLEX instrument (Waltham, MA, USA) using a modified version of the manufacturer's instructions which included a mechanical lysis step with a Qiagen TissueLyser (Hilden, Germany). Samples were amplified using the 28F (GAGTTTGATCNTGGCTCAG)-519R (GTNTTACNGCGGCKGCTG) PCR primers to amplify the V1–V3 region of the 16S gene [##UREF##38##46##]. The amplicons were sequenced on an Illumina MiSeq (San Diego, CA, USA).</p>", "<p>Demultiplexed raw sequences were analyzed with two Snakemake protocols: one focused on quality control and trimming using Trim Galore and FastX, respectively, and another based around QIIME2 (v2022.2) [##UREF##39##47##–##UREF##42##51##]. The second Snakemake used Dada2 to denoise the sequences and Vsearch to annotate the resulting amplicon sequence variants (ASVs) with an RESCRIPT-built version of the 138.1 SILVA database focusing on the V1–V3 region of 16S [##REF##17947321##52##–##UREF##45##57##]. ASVs associated with mitochondria, chloroplast, eukaryotes, and unassigned annotations were removed. See <italic>Supplementary <xref rid=\"supplementary-material-1\" ref-type=\"sec\">1</xref></italic> for further information regarding changes in number of sequences per sample through analysis.</p>", "<title>2.8. RNA Isolation, Reverse Transcription, and Real-Time qPCR Assays</title>", "<p>RNA isolation, reverse transcription, and real-time qPCR assays for target genes were conducted, according to previous studies [##UREF##26##31##, ##REF##26631806##35##, ##REF##26202077##58##]. Total RNA was isolated from the intestine of Atlantic salmon using TRIzol® reagent (Life Technologies, Carlsbad, CA, USA). A NanoDrop™ spectrophotometer (Thermo Scientific™, USA) was used to assess quality and quantity of the isolated RNA. The cDNA of samples was synthesized using PrimeScript™ RT reagent kit (Takara Bio, San Jose, CA, USA) following the manufacturer's instructions. Specific primers for target genes related to growth (insulin-like growth factor-I (IGF-I)), protein synthesis (target of rapamycin signaling pathway regulator-like (TIPRL)), and immunoglobulins (IgM, IgD, and IgT) were designed using online resources according to the partial cDNA sequences of the target genes using Atlantic salmon transcriptome analysis based on published sequences (##TAB##4##Table 5##). All primers of the target and housekeeping genes were synthesized by the Integrated DNA Technologies (IDT, Morrisville, NC, USA).</p>", "<p>Real-time qPCR was used to determine mRNA levels for the target genes and was performed according to standard protocols provided by the manufacturer. The real-time qPCR was carried out in a RT-qPCR System (QuantStudio3, Applied Biosystems). The thermocycling conditions for the target genes were initiated with the denaturation step at 95°C for 30 s followed by 40 cycles at 95°C for 5 s, 60°C for 34 s, and 95°C for 30 s, 95°C for 3 s, 60°C for 30 s, respectively. Melting curve analysis was performed to verify that a single PCR product was produced. Threshold cycle number (<italic>C</italic><sub>T</sub>) for each sample was determined using the software provided with the qPCR system, which was related to the concentration of the target genes. A housekeeping gene of Atlantic salmon (<italic>β</italic>-actin) was used to normalize the expression levels of the target genes. The amplification efficiencies of the target and housekeeping genes were quantified according to the specific gene standard curves generated from 10-fold serial dilutions. After verifying that the primers were amplified with 100% efficiency, the relative expression results were analyzed using the 2<sup>−<italic>ΔΔ</italic>Ct</sup> method [##REF##11846609##60##].</p>", "<title>2.9. Calculation</title>", "<p>The following performance parameters were used to assess the response of the experimental fish to the various dietary treatments:</p>", "<title>2.10. Statistical Analysis</title>", "<p>Growth performance, feces stability, body composition, physiobiochemical, and nutrigenomics data were validated for normality and homogeneity of variances by Shapiro–Wilk and Levene's tests, respectively. Thereafter, these data were analyzed using SPSS version 27 (SPSS, Chicago, IL, USA) and subjected to one-way analysis of variances (ANOVA) followed by Tukey's HSD test. All results were considered significantly different at the level of <italic>P</italic> &lt; 0.05.</p>", "<p>The final microbiome abundance table was imported into RStudio (version 2022.07.2, R version 4.2.0) with the following libraries loaded into the environment: phyloseq (1.42.0), vegan (2.6-4), ggplot2 (3.4.0), plyr (1.8.8), tidyverse (1.3.2), dplyr (1.0.10), reshape (0.8.9), DESeq2 (1.38.2), rstatix (0.7.1), and ANCOMBC (2.0.1) [##UREF##47##61##–##UREF##52##66##]. Sequences were filtered of low abundance ASVs (&lt;11 sequences) to reduce the possible noise they could introduce to patten detection at a community scale [##REF##23647974##67##]. The untransformed ASV counts were used to calculate the alpha diversity which was represented in this study by Shannon diversity. Shannon diversity was statistically correlated with observed ASVs and Simpson diversity (Spearman Rank Correlation Benjamini Hochberg adjusted <italic>P</italic>-values &lt; 0.05). Since the Shannon diversity was not normal (Shapiro–Wilk test <italic>P</italic>-value = 2.10E−7) and heteroscedastic (Bartlett test = 0.00069), the Welch's analysis of variance (ANOVA) and Games–Howell tests (with <italic>P</italic>-value adjusted using Tukey's method) were used to determine statistical significance between diets. To assess the beta diversity, abundances were variance-stabilizing transformed with DESeq2 before using Bray–Curtis to create a dissimilarity matrix which was imported to PRIMER7/PERMANOVA+ to calculate overall and pairwise permutational analysis of variance (PERMANOVA) between diets with 9,999 permutations, the unrestricted method, Type III Sum of Squares, and Monte Carlo adjusted <italic>P</italic>-values [##UREF##53##68##]. Differences between samples were visualized with a principal coordinate of analysis (PCoA). Differential abundance between the control diet (100% FM) and each of the three mealworm meal diets (50% DMM, 100% DMM, and 50% WMM) were determined with ANCOMBC2 (Analysis of Composition of Microbiomes with Bias Correction 2) with <italic>P</italic>-values adjusted with the default Holm method. The Snakemake, Conda environment, R scripts, and R session files can be found on GitHub (<ext-link xlink:href=\"https://github.com/djbradshaw2/Atlantic_salmon_mealworm_meal\" ext-link-type=\"uri\">https://github.com/djbradshaw2/Atlantic_salmon_mealworm_meal</ext-link>). Sequences were uploaded into the NCBI database (PRNJ accession number: PRJNA916945).</p>" ]
[ "<title>3. Results</title>", "<title>3.1. Growth Performance, Feed Utilization, and Feces Stability</title>", "<p>Growth performance, survival, feed utilization, and condition indices of Atlantic salmon fed the experimental diets for 12 weeks are presented in ##TAB##5##Table 6##. Survival of Atlantic salmon at 12 weeks of feeding trial was 100% in all treatments with the exception of the 100% DMM treatment which lost one fish and had a survival of 98.8% (ANOVA, <italic>P</italic>=0.426). Feeding rates were not significantly different among treatments (ANOVA, <italic>P</italic>=0.133). No significant differences were observed in the weight gain, feeding efficiency (FE) ratio, protein efficiency ratio, Fulton's condition factor (K-factor), and VSI. Fish fed with 100% DMM and 50% WMM displayed significantly lower hepatosomatic index than fish fed the control diet (100% FM; <italic>P</italic> &lt; 0.05). In addition, the substitution of FM with DMM and WMM did not significantly affect the final weight of Atlantic salmon at the middle and final weighing periods (##FIG##0##Figure 1##).</p>", "<p>Feces stability: particle size distributions as indicated by the mean particle size at the 10<sup>th</sup>, 50<sup>th</sup>, and 90<sup>th</sup> percentiles were not significantly different between treatments (ANOVA, <italic>P</italic>=0.096, 0.124, and 0.167, respectively). The resultant histograms are shown in ##FIG##1##Figure 2##.</p>", "<title>3.2. Whole-Body Proximate, Amino Acids and Fatty Acids Composition</title>", "<p>\n##TAB##6##Table 7## presents the whole-body proximate and amino acid compositions of Atlantic salmon fed the experimental diets for 12 weeks. There were no significant differences in the proximate and amino acid compositions between the treatments (ANOVA, <italic>P</italic> &gt; 0.05 for all indices). Whole-body fatty acid compositions of Atlantic salmon fed the experimental diets for 12 weeks are reported in ##TAB##7##Table 8##. The results showed that fatty acids (including EPA, linolenic, clupanodonic, homo-<italic>α</italic>-linolenic) were significantly influenced by the dietary substitutions (ANOVA, <italic>P</italic>=0.025, 0.001, 0.031, and 0.010, respectively) with lower values in fish fed 100% DMM. However, DHA (one of the omega-3 fatty acids) content in the whole body of Atlantic salmon was not significantly different between treatments (ANOVA, <italic>P</italic>=0.051). In addition, the ratios of fish to diet (FD ratio) for ARA, EPA, total PUFA, and total omega-3 fatty acids were similar in all treatments and were ≤1.0, while the FD ratio for DHA was &gt;1.0 in all groups (##FIG##2##Figure 3##).</p>", "<title>3.3. Plasma and Liver Biochemical Parameters</title>", "<p>\n##TAB##8##Table 9## shows plasma health parameters, hepatic peroxides (MDA), and antioxidants of Atlantic salmon fed the experimental diets for 12 weeks. Plasma ALT, ALP, TP, and TIBC were not significantly different among treatments (ANOVA, <italic>P</italic>=0.063, 0.557, 0.760, and 0.521, respectively). Plasma IgM was significantly different among treatments, with its higher concentrations measured in fish fed 50% DMM and 100% DMM compared to those fed the control diet (100% FM; <italic>P</italic> &lt; 0.05). In addition, neither MDA content nor the activities of SOD and GPx were significantly different between the dietary treatments (ANOVA, <italic>P</italic>=0.986, 0.383, and 0.322, respectively).</p>", "<title>3.4. Digesta Microbiome</title>", "<p>All 12 feed samples had less than 1,000 sequences after filtering and quality control, thus they were not analyzed further. The loss of sequences was mainly due to the filtering of chloroplast sequences resulting in an average loss of 19,615 sequences or a reduction of 97% of sequences on average (<italic>Supplementary <xref rid=\"supplementary-material-1\" ref-type=\"sec\">1</xref></italic>). Digesta samples lost on average 408 sequences or a 6.5% reduction of sequences. After digesta samples (<italic>n</italic> = 32) were filtered of ASVs with less than 10 occurrences, there were 302,661 sequences distributed across 846 ASVs (see <italic>Supplementary <xref rid=\"supplementary-material-1\" ref-type=\"sec\">1</xref></italic>) for a taxonomic rank breakdown).</p>", "<p>The Shannon diversity in digesta samples was statistically similar between all treatments based upon Welch's analysis of variance (Welch's ANOVA, <italic>F</italic> = 1.2974, <italic>P</italic>=0.3182) and pairwise using the Games–Howell test (adjusted <italic>P</italic>-values &gt; 0.05) (##FIG##3##Figure 4##). On average, the Shannon diversity was 3.15 ± 1.0 across all samples. In terms of beta diversity, PERMANOVA results indicated a statistically significant difference across all diets (PERMANOVA, Monte Carlo <italic>P</italic>=0.001). Pairwise PERMANOVAs revealed that there was significant difference between 50% WMM and 100% FM as well as 100% DMM (PERMANOVA, Monte Carlo <italic>P</italic>=0.025 and 0.049, respectively; <italic>Supplementary <xref rid=\"supplementary-material-1\" ref-type=\"sec\">1</xref></italic>). This is reflected in the PCoA which shows all 50% WMM samples clustering away from the other three diets (##FIG##4##Figure 5##).</p>", "<p>The most common phylum associated with the samples shifted from Firmicutes in 100% FM, 50% DMM, and 100% DMM to Proteobacteria in 50% WMM (<italic>Supplementary <xref rid=\"supplementary-material-1\" ref-type=\"sec\">2</xref></italic>). This diet also had the highest percentage of Actinobacteriota. The most common genus in all diets besides 100% FM was <italic>Pseudomonas</italic> followed by <italic>Clostridium</italic>; this order was reversed in 100% FM (##FIG##5##Figure 6##). ANCOMBC2 analysis revealed that <italic>Brevibacterium</italic> (log fold change = 3.48 ± 1.05 standard error; ANCOMBC2, Holm <italic>Q</italic> = 0.021) and <italic>Brachybacterium</italic> (log fold change = 3.40 ± 1.04 standard error; ANCOMBC2, Holm <italic>Q</italic> = 0.024) were significantly more abundant in 50% WMM than 100% FM. <italic>Tepidimicrobium</italic> was significantly lower in 100% DMM than in 100% FM (log fold change = −3.60 ± 0.91 standard error; ANCOMBC2, Holm <italic>Q</italic> = 0.002).</p>", "<p>Besides <italic>Pseudomonas</italic>, genera that have potentially pathogenic members for Atlantic salmon were not detected in this dataset including <italic>Francisella</italic> (associated disease = Francisellosis), <italic>Aeromonas</italic> (Furunculosis), <italic>Renibacterium</italic> (bacterial kidney disease), <italic>Tenacibaculum</italic> (tenacibaculosis), and <italic>Vibrio</italic> (vibriosis) [##UREF##54##69##–##UREF##57##73##]. None of the genera associated with epitheliocytis (<italic>Piscichlamydia, Branchiomonas, Sygnamidia</italic>, and <italic>Clavochlamydia</italic>) were detected in this study either [##UREF##58##74##]. <italic>Flavobacterium</italic> was detected in three samples, but the species was unknown [##UREF##58##74##]. Known lactic acid bacteria, a class of bacteria which have been shown to have positive benefits on gut health in general, that were not detected included: <italic>Lactobacillus, Pediococcus, Leuconostoc, Aerococcus, Enterococcus, Vagococcus</italic>, and <italic>Carnobacterium</italic> [##UREF##59##75##]. <italic>Lactococcus</italic> was detected in two subsamples (2-1FE and 2-2FE; both 50% WMM), and <italic>Streptococcus</italic> was detected in one subsample (12-1FE; 100% FM).</p>", "<title>3.5. Relative Expression of Growth and Immunity-Related Genes in Intestine</title>", "<p>Relative expressions of growth and immune-related genes which included insulin-like growth factor-I (IGF-I), target of rapamycin signaling pathway regulator-like (TIPRL), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin T (IgT) were analyzed in the intestine of Atlantic salmon and are presented in Figures ##FIG##6##7## and ##FIG##7##8##. The relative expressions of IGF-I and TIPRL genes showed upregulation trends with the dietary substitutions and with higher levels in fish fed 100% DMM and 50% WMM but these increases were not statistically significant (ANOVA, <italic>P</italic>=0.473 and 0.853, respectively; Figures ##FIG##6##7(a)## and ##FIG##6##7(b)##). The relative gene expression of IgM was significantly different among treatments (<italic>P</italic> &lt; 0.01) with higher levels observed in the groups fed 50% and 100% DMM diets compared to those fed the control (100% FM) diet (<italic>P</italic> &lt; 0.05, ##FIG##7##Figure 8(a)##). IgD gene expression of fish fed 100% DMM was significantly higher than those fed the control diet (<italic>P</italic> &lt; 0.05, ##FIG##7##Figure 8(b)##). The relative gene expression of IgT significantly increased in treatment with 100% DMM in contrast to the control and 50% WMM (<italic>P</italic> &lt; 0.05, ##FIG##7##Figure 8(c)##).</p>" ]
[ "<title>4. Discussion</title>", "<p>Mealworms are rich in protein and lipids and appear to have amino acid and fatty acid compositions that should be nutritional adequate for many cultured finfish species [##UREF##7##8##]. As a result, several studies have been reported in the diets of finfish including rainbow trout [##UREF##8##9##, ##UREF##9##10##], gilthead seabream [##UREF##13##15##], European sea bass [##UREF##12##13##, ##REF##29229441##14##], African catfish [##UREF##14##16##], common catfish, <italic>Ameiurus melas</italic> [##UREF##16##18##], yellow catfish [##REF##28807649##19##], and Tilapia [##UREF##17##20##]. This study reports the substitution of fishmeal by mealworm meals both defatted and whole products with high-protein quality (both growth and health benefits) in the diet of Atlantic salmon using growth performance, feed utilization, feces stability, body composition, health parameters, digesta microbiome, and the relative expression of target genes.</p>", "<p>The results of this study showed that fish fed diets produced with mealworm meals had similar growth metrics (final weight and weight gain), feed utilization, and protein efficiency when compared to those fed a control diet produced with a high-quality fishmeal. These results are in accordance with the previous reports of complete substitution of fishmeal by insect meals even in the diets of carnivorous finfish [##UREF##4##5##, ##UREF##60##76##–##UREF##63##79##]. In addition, the present study showed that whole-body proximate composition (crude protein, crude fat, fiber), whole-body amino acids, essential amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, and tryptophan) and taurine (conditional essential amino acid) were similar among treatments. Taken as a whole, these results suggest that the mealworm meals tested in this study provided adequate levels of nutrients needed to support protein metabolism (i.e., synthesis of proteins and amino acids) and growth of Atlantic salmon.</p>", "<p>Feces stability is believed to be affected by the feed ingredients used because they may contain natural surfactants or other compounds that relate to particle binding or breakdown [##UREF##35##42##]. In this study, the particle size distributions of feces breakdown products were not affected by dietary treatments. However, it should be noted that the diets used in this study contained 0.3% gum guar as a binding agent [##UREF##36##43##] and the results should be viewed in this context. These results suggest that neither of the mealworm meals used in this study facilitated breakdown of the feces when mechanically agitated. From this perspective, mealworm meals appear to be suitable for use in RASs when included in stabilized feeds.</p>", "<p>In this study, the whole-body essential fatty acids (EPAs) were influenced by the substitution of fishmeal with 100% mealworm meal. The results showed that EPA, linolenic, clupanodonic, homo-<italic>α</italic>-linolenic were significantly influenced by the dietary substitutions, with lower values in fish fed 100% DMM. However, DHA content in the whole body of Atlantic salmon was not significantly different between the treatments. In general, the differences observed can be attributed to the fact that the diets were formulated to be isolipidic but the fatty acid content was allowed to vary so long as similar levels of EPAs (DHA, EPA, and ARA) were obtained in the experimental diets. To obtain these concentrations, the high levels of lipid provided by the whole mealworm meal (50% WMM) were offset by reducing the amount of poultry oil added to this diet. Similarly, less fish oil was added to the fishmeal-based diet (100% FM) since fishmeal contains notable levels of EPAs (including DHA and EPA). Likewise, the observed differences should be viewed as oil blends that impacted the whole-body fatty acid concentrations measured in the salmon. As reviewed by Tran et al. [##UREF##3##4##], the concentrations of unsaturated fatty acids are high in mealworm and linoleic acid (18:2<italic>n</italic>-6) concentrations are much greater than that of <italic>α</italic>-linolenic acid (18:3<italic>n</italic>-3). In contrast to fish oil, terrestrial insects contain greater quantities of <italic>n</italic>-6 polyunsaturated fatty acids and negligible amounts of EPA (20:5<italic>n</italic>-3) and DHA (22:6<italic>n</italic>-3) [##UREF##3##4##]. It is also reported that salmonids can synthetize limited quantities of EPA and DHA from APA so dietary inclusion is more efficient [##UREF##2##3##]. Furthermore, one method to evaluate the adequacy of EFA provided in a diet is to evaluate the ratio of specific EFA in fish tissues with respect to the EFA provided by the diet, i.e., the fish to diet (FD) ratio [##UREF##64##80##]. This FD ratio could help to understand which EFA might be conserved, deficient, or selectively retained in terms of EFA in fish juveniles [##UREF##27##32##, ##UREF##64##80##]. In the present study, the FD ratios for the total omega-3 fatty acids and PUFA for all treatments were around 1 (≤1), suggesting that the requirements for EFAs were met when Atlantic salmon parr fed the mealworm diets as fishmeal does.</p>", "<p>In fish nutrition studies, blood parameters including total protein, ALP, ALT, IgM, and TIBC are important indicators for health status in response to dietary manipulations of European sea bass [##REF##29229441##14##], Indian major carps, <italic>Labeo rohita</italic>, <italic>Catla catla</italic>, and <italic>Cirrhinus mrigala</italic> [##REF##19063976##26##], red seabream [##UREF##22##27##], blunt snout bream, <italic>Megalobrama amblycephala</italic> [##REF##26631806##35##, ##REF##25463293##45##, ##UREF##65##81##], largemouth bass, <italic>Micropterus salmoides</italic> [##UREF##24##29##, ##UREF##26##31##, ##UREF##66##82##], and Florida pompano, <italic>Trachinotus carolinus</italic> [##UREF##27##32##]. The total protein in plasma is related to the enhancement of digested protein [##UREF##65##81##, ##UREF##67##83##, ##UREF##68##84##]. The presence of ALP activity in plasma is directly related to the release of ALP enzymes from cells to the extracellular fluids [##UREF##69##85##, ##REF##18670985##86##] and elevated activity of ALP may occur when there is cell growth, tissue necrosis, or leakage of ALP [##REF##18670985##86##]. High plasma activity of ALT indicates a damage or weakening of normal liver function [##UREF##27##32##, ##UREF##65##81##, ##UREF##70##87##]. In the present study, plasma total protein, ALP, ALT, and TIBC levels were statistically similar among the dietary treatments. Immunoglobulin production is a specific immune response after being stimulated by antigen and the IgM class is the predominant immunoglobulin in fish [##REF##19063976##26##]. In addition, IgM is not only the major antibody of primary response but also a key part of the adaptive immune response of fish [##UREF##71##88##]. In this study, plasma IgM levels were significantly higher in fish fed 50% DMM and 100% DMM when compared to those fed the control (100% FM diet). These results suggest that the substitution of fishmeal with mealworm meal in the diet of Atlantic salmon may result in health benefits through enhancing the adaptive immune system of Atlantic salmon. On the other hand, the elevated levels of IgM may indicate a specific immune response to the dietary ingredient. In either case, the results are in agreement with a previous study that reported plasma IgM levels increased significantly with increasing dietary mealworm contents in yellow catfish, <italic>Pelteobagrus fulvidraco</italic> [##REF##28807649##19##]. More research is needed to evaluate the specific role and mechanisms of this response.</p>", "<p>Fish liver typically has high concentrations of unsaturated fatty acids with a risk of oxidative damages that can result in the imbalance of reactive oxygen species (ROS) [##UREF##72##89##]. MDA is well-known as an oxidative stress marker [##UREF##30##36##, ##REF##15765761##90##] that can be used to compare nutritional stress in fish. To overcome an oxidative stressor, fish are equipped with an antioxidant defense system to maintain endogenous ROS at a low level and attenuate oxidative damage resulting from high ROS reactivity [##UREF##21##25##, ##UREF##30##36##]. In this study, substitution of fishmeal with dietary mealworm meals did not influence the MDA content of Atlantic salmon suggesting that there was relatively low lipid peroxidation and, presumably, no oxidative damage. SOD, GPx, catalase, and glutathione are the key proteins in the antioxidants defense system [##UREF##73##91##] and their enzymatic activities can be correlated with fish nutritional factors [##UREF##23##28##, ##UREF##26##31##, ##UREF##27##32##, ##REF##26631806##35##, ##REF##25463293##45##, ##UREF##66##82##, ##REF##25148964##92##]. In fish, SOD can catalyze dismutation of superoxide radicals to hydrogen peroxide that can be removed by GPx [##UREF##30##36##, ##UREF##74##93##, ##UREF##75##94##]. In the present study, SOD and GPx activities were not statistically affected by the substitution of mealworms meals indicating that antioxidative defense mechanisms were not activated which could be due to low levels of lipid peroxides as indicated by the measured MDA concentrations.</p>", "<p>This study acknowledges that major differences exist between the transient and residential gut microbiomes, but some members of the community overlap, making comparisons possible [##UREF##76##95##]. Additionally, they are necessary in this case due to the limited examples of studies replacing fishmeal with mealworm conducting microbiome analysis. Other studies have also shown a dominance of Proteobacteria and Firmicutes at the phyla level as well as <italic>Pseudomonas</italic> and <italic>Clostridium</italic> at the general level as part of the normal microbiome in Atlantic salmon and other freshwater fish [##REF##28714057##96##–##UREF##79##99##]. <italic>Pseudomonas</italic> members may be involved in protein utilization and cellular homeostasis allowing it to play a prominent role in ingestion performance [##UREF##80##100##]. On the other hand, some members of Pseudomonas such as <italic>Pseudomonas anguilliseptica</italic> are potential pathogens in Atlantic salmon [##UREF##81##101##]. Some <italic>Clostridium</italic> species such as <italic>Clostridium butyricum</italic> are considered mutualistic symbionts and/or probiotics due to their ability to supply fatty acids and vitamins to the host or stimulate the immune response, disease resistance, and enhance growth, respectively [##UREF##77##97##, ##REF##16490324##102##–##UREF##83##104##].</p>", "<p>There are conflicting reports in other studies into the replacement of fishmeal with <italic>Tenebrio molitor</italic> meal as to whether alpha diversity is statistically affected, which can be explained by differing fish physiology [##REF##24847735##105##]. Shannon diversity was not statistically different in gilthead sea bream (<italic>Sparus aurata</italic>) or European sea bass (<italic>Dicentrarchus labrax</italic>) but was lower in rainbow trout (<italic>Oncorhynchus mykiss</italic>) when 50% of the fishmeal was replaced with <italic>Tenebrio molitor</italic> [##UREF##34##40##]. Furthermore, 25%, 50%, and 75% replacement of fishmeal with <italic>Tenebrio molitor</italic> did not change the Shannon diversity in European perch (<italic>Perca fluviatilis</italic>) [##UREF##84##106##]. In contrast to the above study, 100% replacement of fishmeal with <italic>Tenebrio molitor</italic> in the diet of rainbow trout (<italic>O. mykiss</italic>) led to a higher Shannon diversity in the intestinal mucosa microbiome [##REF##33397465##41##]. Changes in richness can be associated with negative consequences such as dysbiosis or loss of functional redundancy. It could be considered a positive result that this study shows no significant differences between the diets in terms of Shannon diversity.</p>", "<p>When considering the beta diversity, a significant shift in the microbiome was found between fish fed 50% WMM and the control (100% FM) diets and between 50% WMM and 50% DMM. Significant differences in beta diversity or in specific genera between insect meal diets and the control have been noted in other studies [##UREF##34##40##, ##REF##33397465##41##, ##UREF##84##106##]. The addition of defatted mealworm (<italic>Tenebrio molitor</italic>) to the diet of European perch (<italic>Perca fluviatilis</italic>) led to significantly reduced relative percentages of <italic>Lactobacillus</italic> and <italic>Streptococcus</italic> [##UREF##84##106##]. Although no differentially abundant genera were noted in rainbow trout fed insect meal diets [##REF##33397465##41##]. Both <italic>Lactobacillus</italic> and <italic>Streptococcus</italic> were not detected in most samples in this study, although other genera were shown to be differentially abundant between the control (100% FM) and mealworm meal diets. These included <italic>Brevibacterium, Brachybacterium</italic>, and <italic>Tepidimicrobium</italic>. <italic>Brevibacterium</italic> has been found to be enriched in a black soldier fly prepupae meal diet in comparison to a control diet in rainbow trout [##UREF##85##107##]. <italic>Brevibacterium</italic> members are Gram-positive, obligate aerobes that can survive carbohydrate starvation and reduce nitrates to nitrites [##UREF##86##108##]. It has been identified as a potential probiotic due to its potential contribution to nutritional processes in Artic charr (<italic>Salvelinus alpinus</italic>) [##UREF##87##109##]. This is a positive change in the microbiome that is statistically higher in the 50% WMM diet in comparison to the 100% FM diet in this study. Brachybacterium members are Gram-positive, aerobes that can grow slowly in anaerobic environments and have been isolated from the gut of several fish including Atlantic salmon [##UREF##88##110##, ##REF##17349083##111##]. Although the positive effects of any members of these genera have not been explored as a probiotic, a study showed that an exopolysaccharide isolated from a strain of Brachybacterium isolated from Asian seabass exhibited antibacterial activity and suggested further exploration as a source of marine drugs [##UREF##89##112##]. No conclusions could be made regarding whether its increased presence in fish fed 50% WMM could be considered a positive or negative. <italic>Tepidimicrobium</italic> species are mostly strict or facultative anaerobes with broad fermentative capabilities that are capable of Fe(III) reduction [##REF##16449442##113##]. Since it has been identified as a potential commensal species in Atlantic salmon due to its broad fermentative capabilities, its decrease in the 100% DMM samples may be considered a negative if other potential commensals do not overcome its absence [##REF##33613868##114##].</p>", "<p>The organ growth of fish is controlled by the endocrine system, particularly through the growth hormone—insulin-like growth factor (IGF) axis [##UREF##90##115##]. Target of rapamycin pathway (TOR pathway) regulates protein synthesis genes [##UREF##26##31##, ##REF##26631806##35##, ##UREF##30##36##]. In this study, no statistical differences were observed in the relative expressions of IGF-I and TIPRL genes among treatments, consistent with the similarity in growth metrics among treatments. Immune response (activation of the immune system) comes either from the imbalance between supply and utilization of nutrients or nutritional and environmental stressors [##UREF##30##36##]. In teleosts, three classes of immunoglobulins (Igs) have been identified, such as IgM, IgD, and IgT, with IgM+ being the predominant surface Ig isotype [##UREF##28##33##, ##UREF##29##34##]. IgM and IgD isotypes are evolutionary conserved and present in all teleost species, IgT is only found in some teleosts, including Atlantic salmon [##UREF##91##116##, ##UREF##92##117##]. In this study, the relative expression of IgM gene was significantly upregulated in the groups fed 50% and 100% DMM diets compared to those fed the control diet. IgD gene expression of fish fed 100% DMM was significantly higher than the control. The relative gene expression of IgT significantly increased in treatment with 100% DMM in contrast to the control and 50% WMM. These immunoglobulin genes support the plasma IgM results in this study. Su et al. [##REF##28807649##19##] also reported significant upregulation of IgM-related genes in yellow catfish fed with mealworm diets. The immunomodulatory functions could be due to chitin availability in insects including mealworms [##REF##29229441##14##, ##REF##19360565##21##, ##UREF##19##23##]. Those signaling molecules help in understanding the specific mechanisms behind fish growth and health benefits of mealworm meal that guide future research directions of Atlantic salmon (finfish), as well as its responses to dietary substitutions.</p>" ]
[ "<title>5. Conclusions</title>", "<p>Across all treatments, Atlantic salmon in this study showed high survival, and no significant differences were observed in growth performance, feces stability, body composition (except unsaturated fatty acids), health parameters (except IgM), the relative expression of growth-related genes, and alpha diversity of digesta microbiome. The most common genus in all treatments was <italic>Pseudomonas</italic>, which has been previously reported to have both commensal and pathogenic members. Plasma IgM content and intestinal immune genes (IgM, IgD, and IgT) expression were significantly upregulated by the dietary substitutions of defatted mealworm meal, suggesting that mealworm substitution in the diet of Atlantic salmon could increase health benefits through enhancing the adaptive immune system. Overall, the mealworm meals performed well indicating similar growth, survival, and health benefits to fishmeal. The findings of this study provide valuable information and insights for studies on fish nutrition, particularly those geared toward the optimization of nutritionally balanced (healthy), cost-effective, and environment-friendly commercial feeds for Atlantic salmon and other farmed species.</p>" ]
[ "<p>Academic Editor: Yanjiao Zhang</p>", "<p>A 12-week growth trial was conducted to assess the effects of mealworm meals, as a substitution for fishmeal, on the growth, physiobiochemical responses, digesta microbiome, and immune-related genes expression of Atlantic salmon (<italic>Salmo salar</italic>). Twenty Atlantic salmon parr (38.5 ± 0.1 g, initial weight) were stocked into each of 16 tanks in a recirculating aquaculture system. A fishmeal-based diet (100% FM) was used as the control treatment and was compared with three test diets where: (1) fishmeal was partially (50%) replaced with defatted mealworm meal, <italic>Tenebrio molitor</italic> (50% DMM), (2) fishmeal was fully replaced with defatted mealworm meal (100% DMM), and (3) fishmeal was partially replaced with whole lesser mealworm meal, <italic>Alphitobius diaperinus</italic> (50% WMM). All substitutions were done on a crude protein basis. Each of the four experimental diets was evaluated in quadruplicate tanks as part of randomized design. The results indicated that Atlantic salmon showed high survival (greater or equal to 98.8%), and no significant difference in final growth, feed efficiency, feces stability and condition indices. Hepatosomatic index was lower in fish fed 100% DMM and 50% WMM when compared to fish fed the control diet (100% FM). Whole-body proximate and amino acid compositions were not statistically different between treatments, while essential fatty acids, including linolenic, eicosapentaenoic acid, and homo-a-linolenic, were lower in fish fed 100% DMM. Plasma parameters (total protein, alanine aminotransferase, alkaline phosphatase, and total iron-binding capacity), hepatic peroxide, and antioxidant enzymes were not significantly affected by dietary substitutions, whereas plasma immunoglobulin M showed significantly higher levels in fish fed 50% DMM and 100% DMM when compared to fish fed the control diet (100% FM). The inclusion of mealworm meals significantly impacted the overall microbiome composition but not the richness and evenness of the salmon digesta microbiomes compared to control. The most common genus in all treatments was <italic>Pseudomonas</italic>, which has been previously shown to have both commensal and pathogenic members. The relative expressions of growth (IGF-I) and protein synthesis (TIPRL) were not significantly different between the treatments, whereas immunoglobulin genes (IgM, IgD, and IgT) were significantly upregulated in fish fed the DMM diets when compared to fish fed the control diet. Overall, this study suggests that the mealworm meals tested could be suitable alternatives to fishmeal in the diet of Atlantic salmon.</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>This work was supported by the Ÿnsect France (Project #: Ynsect: 2021-0445). The salaries of H.-M.H.-T., M.H., and K.-M.M. were supported by the U.S. Department of Agriculture, Agricultural Research Service by NACA Agreement Number 58-8030-0-004 with the University of Maine's Aquaculture Research Institute. The authors are very grateful to the University of Maine—Aquaculture Research Institute (ARI) team, for their help during the growth trial (fish stocking, fish husbandry, and sample collection). The authors would like to extend a special thanks to Alex Sullivan for his help in fish husbandry and data recording. The authors are also very grateful to Dr. Gibson Gaylord (USFWS, Bozeman, MT, USA) for his help during the experimental diets production.</p>", "<title>Data Availability</title>", "<p>The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.</p>", "<title>Ethical Approval</title>", "<p>The University of Maine holds the Office of Laboratory Animal Welfare (OLAW) of the National Institutes of Health assurance for vertebrate animals used in research, teaching, and outreach (Assurance #: A3754-01). The use of experimental fish was under scientific research protocols of the University of Maine, Institutional Animal Care and Use Committee (IACUC Protocol #: A2021-09-04) and complied with all relevant international animal welfare laws, guidelines, and policies.</p>", "<title>Disclosure</title>", "<p>Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA). USDA is an equal opportunity provider and employer.</p>", "<title>Conflicts of Interest</title>", "<p>All authors declare that there are no conflicts of interest.</p>", "<title>Supplementary Materials</title>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Effects of FM substitution by mealworm meals (at 50% DMM, 100% DMM, and 50% WMM) on the final weight at different weighing periods (initial, middle, and final) of Atlantic salmon fed the experimental diets for 12 weeks. Values are means with standard errors represented by vertical bars (<italic>n</italic> = 4). FM, fishmeal; DMM, defatted mealworm meal; WMM, whole mealworm meal; IW, initial weight; MW, middle weight; and FW, final weight.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>Histogram showing particle size distribution (log scale; % volume) of feces breakdown products after physical agitation for 20 s. Treatments are represented by different colors: blue = 50% DMM, red =  50% WMM, green = 100% DMM, and purple = 100% FM.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>Ratio (fish : diet) of fatty acids in Atlantic salmon (<italic>Salmo salar</italic>) parr after a 12-week feeding trial with a control diet (100% FM), and three test diets such as 50% DMM, 100% DMM, and 50% WMM. The dashed line (ratio = 1) indicates equal amounts of fatty acids in fish and the diet. DMM, defatted mealworm meal; WMM, whole mealworm meal; ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; total PUFA, total polyunsaturated fatty acids; and total <italic>ω</italic>-3 FA, total omega-3 fatty acids.</p></caption></fig>", "<fig position=\"float\" id=\"fig4\"><label>Figure 4</label><caption><p>Boxplots showing digesta Shannon diversity by experimental diet. Bars denote largest and smallest values within 1.5 times the interquartile range while dots represent values outside that range. The middle line is the medium and ends of boxes are the first and third quartiles.</p></caption></fig>", "<fig position=\"float\" id=\"fig5\"><label>Figure 5</label><caption><p>Principal coordinates of analysis of Bray–Curtis distances between samples. Shape and color represent the experimental diet. FM stands for fishmeal, DMM stands for defatted mealworm meal, and WMM stands for whole mealworm meal.</p></caption></fig>", "<fig position=\"float\" id=\"fig6\"><label>Figure 6</label><caption><p>Stacked bar graphs showing the genera, which are represented by color, that had a mean relative abundance greater than 1% across all samples summarized by experimental treatment.</p></caption></fig>", "<fig position=\"float\" id=\"fig7\"><label>Figure 7</label><caption><p>Effects of FM substitution by mealworm meals (at 50% DMM, 100% DMM, and 50% WMM) on the relative gene expression of insulin-like growth factor I (IGF-I) (a) and target of rapamycin signaling pathway regulator-like (TIPRL) (b) in Atlantic salmon fed the experimental diets for 12 weeks. Values are means with standard errors represented by vertical bars (<italic>n</italic> = 12). FM, fishmeal; DMM, defatted mealworm meal; and WMM, whole mealworm meal.</p></caption></fig>", "<fig position=\"float\" id=\"fig8\"><label>Figure 8</label><caption><p>Effects of FM substitution by mealworm meals (at 50% DMM, 100% DMM, and 50% WMM) on the relative gene expression of immunoglobulin M (IgM) (a), immunoglobulin D (IgD) (b), and immunoglobulin T (IgT) (c) in Atlantic salmon fed the experimental diets for 12 weeks. Values are means with standard errors represented by vertical bars (<italic>n</italic> = 8). FM, fishmeal; DMM, defatted mealworm meal; and WMM, whole mealworm meal. Mean values with different letters are significantly different (<italic>P</italic> &lt; 0.05).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Nutrient composition of test ingredients (mealworm meals) <sup><italic>∗</italic></sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Nutritional value</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">WMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">DMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Energy (kcal/kJ)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">510/2142</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">554/2317</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude protein (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">59.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">70.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude fat (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fibers (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ashes (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Moisture</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.0</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Total amino acids (g/100 g)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Alanine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Arginine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Aspartic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Cysteine + cystine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Glutamic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Glycine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Histidine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Isoleucine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Leucine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lysine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Methionine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Phenylalanine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Proline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Serine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Threonine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tryptophan</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tyrosine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Valine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.2</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Fatty acids (percentage of total fats)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C12:0 (lauric acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C14:0 (myristic acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C16:0 (palmitic acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C18:0 (stearic acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C18:1-<italic>n</italic>9c (oleic acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C18:2-<italic>n</italic>6c (linoleic acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> C18:3-<italic>n</italic>3 (alpha-linolenic acid)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Saturated fatty acids</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Sum monosaturated fatty acids (MUFA)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Sum polyunsaturated fatty acids (PUFA)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Total trans fatty acids</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Fatty acids sum of omega 3 calc.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Fatty acids sum of omega 6 calc.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.9</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">Minerals (mg/kg)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Calcium (Ca)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">520</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">633.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Phosphorus (P)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7,633.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9,125.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Potassium (K)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9,600.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10,335.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Magnesium (Mg)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1,150</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2,912.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Iron (Fe)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">51.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">75.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Copper (Cu)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Manganese (Mn)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Zinc (Zn)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">135.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Formulation and composition of the experimental diets<sup>1</sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Ingredients</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">Control diet (100% FM)</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">Test diets (g/100 g diet) <sup><italic>∗</italic></sup></th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fishmeal (SeaPro<sup>TM</sup> 75) <sup><italic>∗∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Defatted mealworm meal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Whole mealworm meal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poultry meal, pet food grade</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Soybean meal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Soy protein concentrate 1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Corn protein concentrate (75% CP)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Wheat gluten meal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Wheat flour 70% starch 2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fish oil</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poultry oil</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vitamin premix ARS 702</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Stay C 35%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Choline chloride</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Trace mineral premix ARS 1440</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lysine HCl</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">DL-methionine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Taurine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Astaxanthin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gum guar</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>α</italic>-Cellulose</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Diet composition (calculated, in dry matter)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Crude protein (CP, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lipid (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Digestible energy (kcal/100 g)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">431.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">430.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">427.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">433.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Fiber (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lysine (Lys, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Methionine (Met, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Total sulfur amino acids (TSAA, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Threonine (Thr, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Arginine (Arg, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Taurine (Tau, %)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Phosphorus (P)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.2</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Analyzed proximate and amino acids composition of the experimental diets.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Proximate composition (%, as is)</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Diets (%)</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control (100% FM)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude protein <sup><italic>∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44.80</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45.30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46.30</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Moisture</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude fat</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.89</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.38</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude fiber</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ash</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.58</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.86</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Essential amino acids (W/W%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Threonine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.63</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Valine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.39</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.28</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Methionine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.36</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.35</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Isoleucine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Leucine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.61</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.68</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Phenylalanine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lysine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.69</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Histidine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.99</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Arginine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.52</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.47</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.52</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tryptophan</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Conditional essential amino acid (W/W%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Taurine<sup>§</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.70</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.67</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.69</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Nonessential amino acids (W/W%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hydroxyproline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Aspartic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Serine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.67</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Glutamic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.70</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.91</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Proline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.80</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.89</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lanthionine<sup>§</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Glycine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.37</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Alanine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.58</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Cysteine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tyrosine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.73</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hydroxylysine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ornithine<sup>§</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total AAs (W/W%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44.41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44.82</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45.58</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>Analyzed fatty acids composition of the experimental diets.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Fatty acids profile</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Diets (%)</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control (100% FM)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C14:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.66</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myristoleic (9c-14:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C15:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C15:1<italic>n</italic>5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitic (16:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.97</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.85</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.71</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitoleic (9c-16:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.79</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.48</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Margaric (17:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10c-17:1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Stearic (18:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.70</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.87</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Elaidic (9t-18:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Oleic (9c-18:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.80</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.76</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccenic (11c-18:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.87</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.44</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linoelaidic (18:2t)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linoleic (18:2<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.88</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.55</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linolenic (18:3<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.22</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.25</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.10</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.08</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">g-Linolenic (C18:3<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Stearidonic (18:4<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.00</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.63</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.00</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.74</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Arachidic (20:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gonodic (20:1<italic>n</italic>9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.67</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.83</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C20:2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Homo-g-linolenic (C20:3<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Homo-a-linolenic (20:3<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.11</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.09</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.10</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.11</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ARA (20:4<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.81</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EPA (20:5<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>8.24</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>7.74</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>7.26</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>9.03</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C21:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Behenoic (22:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Erucic (22:1<italic>n</italic>9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C22:2<italic>n</italic>6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adrenic (C22:4<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C22:5<italic>n</italic>6 <sup><italic>∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clupanodonic (22:5<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.27</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.23</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.18</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.41</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">DHA (22:6<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>5.36</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>4.90</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>4.33</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>5.54</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C23:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lignoceric (24:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nervonic (24:1<italic>n</italic>9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab5\"><label>Table 5</label><caption><p>Primers sequences for real-time qPCR.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Target gene</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Primer sequences (5′–3′)</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">Accession number</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">Product size (bp)</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">Source</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Forward</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Reverse</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">IGF-I</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TGGGGATGTCTAGCGGTCAT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">AGTGAGAGGGTGTGGGTACA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">XM_014208346.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">94</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">New design</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TIPRL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CTGCACGACCACGGAGTATC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CTCCATCCACTCGCAGGAAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">XM_014136761.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">97</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">New design</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">IgM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">AGGCGGAAATTCCCTGACTG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CACGGAGTTGACTGACTCCC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y12457.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">83</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">New design</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">IgD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CGTCTACTCCATCGCTCCAC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TTTGGCGTCATACGCAGAGT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">AF141607.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">104</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">New design</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">IgT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CAAAGGGCAACCTGAACAGC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GAACGACCGGTGTGTCTTCA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GQ907004.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">117</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">New design</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>β</italic>-actin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CCAAAGCCAACAGGGAGAA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">AGGGACAACACTGCCTGGAT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">BG933897</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">102</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##46##59##]</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab6\"><label>Table 6</label><caption><p>Growth performance, survival, feed utilization, and condition indices of Atlantic salmon fed the experimental diets for 12 weeks.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Parameters</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Diets</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control (100% FM)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Initial weight (g)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.5 ± 0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.7 ± 0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.4 ± 0.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.4 ± 0.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Survival (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 0.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">98.8 ± 2.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 0.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Final weight (g)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">182.8 ± 5.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">179.0 ± 2.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">191.4 ± 5.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">191.5 ± 3.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Weight gain (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">374.7 ± 14.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">363.4 ± 6.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">392.7 ± 9.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">398.8 ± 8.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Feeding rate (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.53 ± 0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.50 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.50 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.60 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Feed efficiency ratio</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.23 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.20 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.25 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.23 ± 0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PER (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.64 ± 0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.54 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.69 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.57 ± 0.04</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">K-factor (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.14 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.19 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.18 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.25 ± 0.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">VSI (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.00 ± 0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.83 ± 0.28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.61 ± 0.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.89 ± 0.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HSI (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.48 ± 0.05<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.43 ± 0.05<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.28 ± 0.04<sup>bc</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.24 ± 0.05<sup>c</sup></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab7\"><label>Table 7</label><caption><p>Whole-body proximate and amino acids composition of Atlantic salmon fed the experimental diets for 12 weeks.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Proximate composition (as is)</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">Initial</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Diets</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control (100% FM)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude protein <sup><italic>∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54.84</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">51.34 ± 0.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52.40 ± 0.37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">51.35 ± 0.44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">51.91 ± 1.44</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Moisture</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.27 ± 0.56</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.48 ± 0.56</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.89 ± 0.31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.98 ± 0.54</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crude fat</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39.27 ± 0.66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37.66 ± 0.67</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39.12 ± 0.87</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37.22 ± 0.78</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fiber</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 ± 0.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ash</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.84 ± 0.18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.11 ± 0.26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.26 ± 0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.60 ± 0.44</td></tr><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">Essential amino acids (W/W%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Threonine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.27 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.31 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.28 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.32 ± 0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Valine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.70 ± 0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.76 ± 0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.69 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.75 ± 0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Methionine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.56</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.53 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.55 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.56 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.56 ± 0.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Isoleucine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.35 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.42 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.33 ± 0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.41 ± 0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Leucine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.77 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.84 ± 0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.72 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.83 ± 0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Phenylalanine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.04 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.09 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.06 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.09 ± 0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lysine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.47 ± 0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.56 ± 0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.40 ± 0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.55 ± 0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Histidine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.30 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.33 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.31 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.33 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Arginine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.96 ± 0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.07 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.23 ± 0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.09 ± 0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tryptophan</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.56 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.56 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59 ± 0.02</td></tr><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">Conditional essential amino acid (W/W%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Taurine<sup>§</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.01 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.01 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 ± 0.13</td></tr><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">Nonessential amino acids (W/W%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hydroxyproline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.31 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.56 ± 0.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.41 ± 0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Aspartic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.85</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.74 ± 0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.83 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.82 ± 0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.86 ± 0.07</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Serine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.81 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.82 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.86 ± 0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.83 ± 0.04</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Glutamic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.31 ± 0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.27 ± 0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.27 ± 0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.34 ± 0.12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Proline</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.78 ± 0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.83 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.13 ± 0.31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.87 ± 0.04</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Lanthionine<sup>§</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.27 ± 0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Glycine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.79 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.87 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.57 ± 0.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.99 ± 0.09</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Alanine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.95 ± 0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.01 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.16 ± 0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.04 ± 0.05</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Cysteine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.58 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.58 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tyrosine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.94</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.10 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.18 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.12 ± 0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.18 ± 0.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hydroxylysine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ornithine<sup>§</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total AAs (W/W%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">47.76 ± 0.86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.70 ± 0.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49.67 ± 1.87</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49.18 ± 0.80</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab8\"><label>Table 8</label><caption><p>Whole-body fatty acids composition of Atlantic salmon fed the experimental diets for 12 weeks.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Fatty acid composition</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">Initial</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Diets</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control (100% FM)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C14:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.84 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.93 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.09 ± 0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.04 ± 0.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myristoleic (9c-14:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.00<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.00<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 ± 0.00<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.00<sup>b</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C15:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitic (16:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.39 ± 0.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.37 ± 0.24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.21 ± 0.59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.13 ± 0.46</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitoleic (9c-16:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.79</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.60 ± 0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.54 ± 0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.92 ± 0.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.52 ± 0.12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Margaric (17:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18 ± 0.00<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19 ± 0.00<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.01<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.21 ± 0.01<sup>a</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10c-17:1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 ± 0.00<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 ± 0.00<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.26 ± 0.01<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.27 ± 0.00<sup>a</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Stearic (18:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.85</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.36 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.31 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.45 ± 0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.52 ± 0.09</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Elaidic (9t-18:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Oleic (9c-18:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.51</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.68 ± 0.33<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.86 ± 0.12<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.29 ± 0.57<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.87 ± 0.26<sup>ab</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccenic (11c-18:1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.31 ± 0.10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.49 ± 0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.51 ± 0.19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.64 ± 0.12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linoelaidic (18:2t)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linoleic (18:2<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.17 ± 0.09<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.16 ± 0.11<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.83 ± 0.17<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.59 ± 0.03<sup>c</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Linolenic (18:3<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.85</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.06 ± 0.01</bold>\n<sup>a</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.09 ± 0.01</bold>\n<sup>a</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.94 ± 0.03</bold>\n<sup>b</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.97 ± 0.01</bold>\n<sup>b</sup>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">g-Linolenic (C18:3<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Stearidonic (18:4<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.00</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.29 ± 0.17</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.60 ± 0.01</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.54 ± 0.03</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.49 ± 0.16</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Arachidic (20:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.01<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 ± 0.01<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.00<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.01<sup>ab</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gonodic (20:1<italic>n</italic>9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.25 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.29 ± 0.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.26 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.29 ± 0.03</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C20:2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.84 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.81 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.53 ± 0.17</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Homo-g-linolenic (C20:3<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.21 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Homo-a-linolenic (20:3<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.12</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.13 ± 0.00</bold>\n<sup>a</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.13 ± 0.00</bold>\n<sup>a</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.11 ± 0.01</bold>\n<sup>b</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.13 ± 0.00</bold>\n<sup>a</sup>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Arachidonic (20:4<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.70 ± 0.01<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.70 ± 0.01<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54 ± 0.04<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59 ± 0.03<sup>b</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EPA (20:5<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>3.73</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>4.62 ± 0.09</bold>\n<sup>ab</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>4.64 ± 0.13</bold>\n<sup>ab</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>3.76 ± 0.38</bold>\n<sup>b</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>4.99 ± 0.27</bold>\n<sup>a</sup>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C21:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.023 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.023 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Behenoic (22:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Erucic (22:1<italic>n</italic>9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23 ± 0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23 ± 0.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C22:2<italic>n</italic>6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adrenic (C22:4<italic>n</italic>6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.00<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 ± 0.00<sup>ab</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 ± 0.01<sup>bc</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 ± 0.00<sup>c</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C22:5<italic>n</italic>6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.00<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16 ± 0.01<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.01<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 ± 0.01<sup>ab</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clupanodonic (22:5<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.33</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>2.10 ± 0.04</bold>\n<sup>ab</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>2.03 ± 0.08</bold>\n<sup>ab</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>1.63 ± 0.15</bold>\n<sup>b</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>2.11 ± 0.15</bold>\n<sup>a</sup>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">DHA (22:6<italic>n</italic>3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>5.53</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>7.91 ± 0.14</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>7.75 ± 0.27</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>5.87 ± 0.72</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>7.56 ± 0.63</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">C23:0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.01</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lignoceric (24:0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nervonic (24:1<italic>n</italic>9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.28 ± 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.26 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 ± 0.02</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.26 ± 0.03</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab9\"><label>Table 9</label><caption><p>Plasma health parameters, and hepatic peroxide and antioxidants of Atlantic salmon fed the experimental diets for 12 weeks.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Parameters</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Diets</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Control (100% FM)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">100% DMM</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">50% WMM</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Plasma health parameters</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> ALT (mU/ml)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.93 ± 1.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.16 ± 1.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.75 ± 1.23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.07 ± 0.85</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> ALP (U/l)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">253.29 ± 35.97</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">246.32 ± 28.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">268.81 ± 24.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">299.91 ± 26.44</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> TP (g/l)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.48 ± 3.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30.42 ± 4.68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32.72 ± 4.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.03 ± 4.54</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> IgM (<italic>µ</italic>g/ml)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">547.07 ± 17.66<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">777.46 ± 82.72<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">718.90 ± 57.60<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">669.61 ± 40.87<sup>ab</sup></td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> TIBC (<italic>µ</italic>mol/l)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78.55 ± 7.48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">86.55 ± 17.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.76 ± 7.63</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">80.58 ± 13.70</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Hepatic peroxide and antioxidants</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> MDA (nmol/mg)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.56 ± 5.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.04 ± 3.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.75 ± 6.49</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.99 ± 6.32</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> SOD (U/mg)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.70 ± 0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.72 ± 0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.33 ± 0.22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.47 ± 0.17</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> GPx (U/ml)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.89 ± 0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.62 ± 0.12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90 ± 0.19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.06 ± 0.18</td></tr></tbody></table></table-wrap>" ]
[ "<disp-formula id=\"EEq1\"><label>(1)</label><mml:math id=\"M1\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Survival </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">%</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Final population</mml:mtext><mml:mo>/</mml:mo><mml:mtext>initial population</mml:mtext></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq2\"><label>(2)</label><mml:math id=\"M2\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Weight gain </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">%</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Final body weight</mml:mtext><mml:mo>−</mml:mo><mml:mtext>initial body weight</mml:mtext></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>initial body weight</mml:mtext></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq3\"><label>(3)</label><mml:math id=\"M3\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Feeding rate</mml:mtext><mml:mo>,</mml:mo><mml:mtext> percentage of body weight per day </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>BW </mml:mtext><mml:msup><mml:mtext>day</mml:mtext><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Dry feed intake </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:msup><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>initial body weight</mml:mtext><mml:mo>×</mml:mo><mml:mtext>final body weight </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mrow><mml:mn>1</mml:mn><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup><mml:mo>/</mml:mo><mml:mtext>days on feed</mml:mtext></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq4\"><label>(4)</label><mml:math id=\"M4\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Feed efficiency </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>FE</mml:mtext></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mtext>Weight gain </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mtext>dry feed consumed </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq5\"><label>(5)</label><mml:math id=\"M5\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Protein efficiency ratio </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>PER</mml:mtext><mml:mo>,</mml:mo><mml:mtext> </mml:mtext><mml:mi mathvariant=\"normal\">%</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Weight gain </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi><mml:mo>,</mml:mo><mml:mtext> wet weight</mml:mtext></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mtext>protein intake </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi><mml:mo>,</mml:mo><mml:mtext> dry weight</mml:mtext></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq6\"><label>(6)</label><mml:math id=\"M6\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Fulton condition factor</mml:mtext><mml:mo>,</mml:mo><mml:mtext> </mml:mtext><mml:mi mathvariant=\"normal\">K</mml:mi><mml:mo>−</mml:mo><mml:mtext> factor </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">%</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Fish weight </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:msup><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>fish length</mml:mtext><mml:mo>,</mml:mo><mml:mtext> cm</mml:mtext></mml:mrow></mml:mfenced><mml:mn>3</mml:mn></mml:msup></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq7\"><label>(7)</label><mml:math id=\"M7\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Hepatosomatic index </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>HSI</mml:mtext><mml:mo>,</mml:mo><mml:mtext> </mml:mtext><mml:mi mathvariant=\"normal\">%</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Liver weight </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mtext>body weight </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>", "<disp-formula id=\"EEq8\"><label>(8)</label><mml:math id=\"M8\" overflow=\"scroll\"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Viscerosomatic index </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>VSI</mml:mtext><mml:mo>,</mml:mo><mml:mtext> </mml:mtext><mml:mi mathvariant=\"normal\">%</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mtext>Viscera weight </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mtext>body weight </mml:mtext><mml:mfenced open=\"(\" close=\")\" separators=\"|\"><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>×</mml:mo><mml:mn>100.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>" ]
[]
[]
[]
[]
[ "<supplementary-material id=\"supp-1\" position=\"float\" content-type=\"local-data\"><label>Supplementary 1</label><caption><p>Table S1: per sample sequence count changes over analysis stages. Table S2: total and named (does not have uncultured, unknown, etc… as part of name or is a repeat of the name of at the previous taxonomic level) taxa at each taxonomic level for digesta samples. Table S3: permutational analysis of variance results.</p></caption></supplementary-material>", "<supplementary-material id=\"supp-2\" position=\"float\" content-type=\"local-data\"><label>Supplementary 2</label><caption><p>Figure S1: stacked bar graphs showing the phyla, which are represented by color, that had a mean relative abundance greater than 1% across all samples summarized by experimental treatment.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn><p> <sup><italic>∗</italic></sup>Source: Ÿnsect France. WMM, whole mealworm meal (<italic>Alphitobius diaperinus</italic>); DMM, defatted mealworm meal (<italic>Tenebrio molitor</italic>).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>\n<sup>1</sup>Extruded diets were produced at the USDA—ARS facility in Bozeman, MT, USA.  <sup><italic>∗</italic></sup>All substitutions were done on a crude protein basis. FM, fishmeal; DMM, defatted mealworm meal (<italic>Tenebrio molitor</italic>); WMM, whole mealworm meal (<italic>Alphitobius diaperinus</italic>).  <sup><italic>∗∗</italic></sup>SeaPro<sup>TM</sup> 75 is a deboned, high-protein (75% CP), low-ash fishmeal derived from fresh cuttings of marine whitefish; it provides a supplemental level of omega-3 fatty acids including DHA and EPA; and it is stabilized with natural antioxidants (BioOregon Protein Inc., Warrenton, OR, USA).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Analyses (except crude protein and ash) were carried out by the Experiment Station Chemical Laboratories (ESCL), University of Missouri, Columbia, MO, USA. Crude protein analysis was conducted at USDA—ARS facility, Franklin, ME, USA. W/W% = g per 100 g of sample. Crude protein <sup><italic>∗</italic></sup> = %<italic>N</italic> × 6.25. <sup>§</sup>Nonproteinogenic amino acids. Results are expressed on an “as is” basis.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Analyses were carried out by the Experiment Station Chemical Laboratories (ESCL), University of Missouri, Columbia, MO, USA. W/W% = g per 100 g of sample.  <sup><italic>∗</italic></sup>Omega-3 fatty acids are in bold type. Results are expressed on an “as is” basis unless.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>IGF-I, insulin-like growth factor-I; TIPRL, target of rapamycin signaling pathway regulator-like; IgM, immunoglobulin M; IgD, immunoglobulin D; IgT, immunoglobulin T; <italic>β</italic>-actin, beta-actin (reference gene). The primers of target genes were designed using online resources according to the partial cDNA sequences of the target genes using Atlantic salmon (<italic>Salmo salar</italic>) transcriptome analysis. All primers of the target genes and reference genes were synthesized by Integrated DNA Technologies (IDT, Morrisville, NC, USA).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>PER, protein efficiency ratio; K-factor, Fulton condition factor; VSI, viscerosomatic index; HSI, hepatosomatic index. Mean values within a row with different superscript letters were significantly different (<italic>P</italic> &lt; 0.05).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Analyses were carried out by the Experiment Station Chemical Laboratories (ESCL), University of Missouri, Columbia, MO, USA. W/W% = g per 100 g of sample. Crude protein <sup><italic>∗</italic></sup> = %<italic>N</italic> × 6.25. <sup>§</sup>Nonproteinogenic amino acids. Results are expressed on an “as is” basis. Mean values within a row with different superscript letters were significantly different (<italic>P</italic> &lt; 0.05).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Analyses were carried out by the Experiment Station Chemical Laboratories (ESCL), University of Missouri, Columbia, MO, USA. W/W% = g per 100 g of sample.  <sup><italic>∗</italic></sup>Omega-3 fatty acids are in bold type. Results are expressed on an “as is” basis. Mean values within a row with different superscript letters were significantly different (<italic>P</italic> &lt; 0.05).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; IgM, immunoglobulin M; TIBC, total iron-binding capacity; MDA, malondialdehyde; SOD superoxide dismutase; GPx, glutathione peroxidase. Mean values within a row with different superscript letters were significantly different (<italic>P</italic> &lt; 0.05).</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"ANU2024-6618117.001\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.002\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.003\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.004\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.005\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.006\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.007\" position=\"float\"/>", "<graphic xlink:href=\"ANU2024-6618117.008\" position=\"float\"/>" ]
[ "<media xlink:href=\"6618117.f1.pdf\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"6618117.f2.png\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["1"], "collab": ["FAO"], "source": ["\n"], "italic": ["The State of World Fisheries and Aquaculture: Towards Blue Transformation"], "year": ["2022"], "publisher-loc": ["Rome"], "publisher-name": ["FAO"], "pub-id": ["10.4060/cc0461en"]}, {"label": ["2"], "person-group": ["\n"], "surname": ["Veldkamp", "van Duinkerken", "van Huis"], "given-names": ["T.", "G.", "A."], "source": ["\n"], "italic": ["Insects as a Sustainable Feed Ingredient in Pig and Poultry Diets: A Feasibility Study"], "year": ["2012"], "publisher-loc": ["Wageningen, The Netherlands"], "publisher-name": ["Wageningen UR Livestock Production"]}, {"label": ["3"], "person-group": ["\n"], "surname": ["S\u00e1nchez-Muros", "Barroso", "Manzano-Agugliaro"], "given-names": ["M.-J.", "F. G.", "F."], "article-title": ["Insect meal as renewable source of food for animal feeding: a review"], "source": ["\n"], "italic": ["Journal of Cleaner Production"], "year": ["2014"], "volume": ["65"], "fpage": ["16"], "lpage": ["27"], "pub-id": ["10.1016/j.jclepro.2013.11.068", "2-s2.0-84893764778"]}, {"label": ["4"], "person-group": ["\n"], "surname": ["Tran", "Heuz\u00e9", "Makkar"], "given-names": ["G.", "V.", "H. P. S."], "article-title": ["Insects in fish diets"], "source": ["\n"], "italic": ["Animal Frontiers"], "year": ["2015"], "volume": ["5"], "issue": ["2"], "fpage": ["37"], "lpage": ["44"]}, {"label": ["5"], "person-group": ["\n"], "surname": ["Hua", "Cobcroft", "Cole"], "given-names": ["K.", "J. M.", "A."], "article-title": ["The future of aquatic protein: implications for protein sources in aquaculture diets"], "source": ["\n"], "italic": ["One Earth"], "year": ["2019"], "volume": ["1"], "issue": ["3"], "fpage": ["316"], "lpage": ["329"], "pub-id": ["10.1016/j.oneear.2019.10.018"]}, {"label": ["6"], "person-group": ["\n"], "surname": ["Alfiko", "Xie", "Astuti", "Wong", "Wang"], "given-names": ["Y.", "D.", "R. T.", "J.", "L."], "article-title": ["Insects as a feed ingredient for fish culture: status and trends"], "source": ["\n"], "italic": ["Aquaculture and Fisheries"], "year": ["2022"], "volume": ["7"], "issue": ["2"], "fpage": ["166"], "lpage": ["178"], "pub-id": ["10.1016/J.AAF.2021.10.004"]}, {"label": ["7"], "person-group": ["\n"], "surname": ["Sharifinia", "Bahmanbeigloo", "Keshavarzifard"], "given-names": ["M.", "Z. A.", "M."], "article-title": ["The effects of replacing fishmeal by mealworm ("], "italic": ["Tenebrio molitor", "Litopenaeus vannamei", "Annals of Animal Science"], "source": ["\n"], "year": ["2023"], "pub-id": ["10.2478/aoas-2022-0098"]}, {"label": ["8"], "person-group": ["\n"], "surname": ["Makkar", "Tran", "Heuz\u00e9", "Ankers"], "given-names": ["H. P. S.", "G.", "V.", "P."], "article-title": ["State-of-the-art on use of insects as animal feed"], "source": ["\n"], "italic": ["Animal Feed Science and Technology"], "year": ["2014"], "volume": ["197"], "fpage": ["1"], "lpage": ["33"], "pub-id": ["10.1016/j.anifeedsci.2014.07.008", "2-s2.0-85027932786"]}, {"label": ["9"], "person-group": ["\n", "\n"], "surname": ["Gasco", "Belforti", "Rotolo", "Vantomme", "Munke", "van Huis"], "given-names": ["L.", "M.", "L.", "P.", "C.", "A."], "article-title": ["Mealworm ("], "italic": ["Tenebrio molitor", "Oncorhynchus mykiss", "1st International Conference \u201cInsects to Feed the World\u201d"], "source": ["\n"], "year": ["2014"], "publisher-loc": ["Ede-Wageningen, The Netherlands"], "publisher-name": ["Wageningen University"]}, {"label": ["10"], "person-group": ["\n"], "surname": ["Belforti", "Gai", "Lussiana"], "given-names": ["M.", "F.", "C."], "article-title": ["\n"], "italic": ["Tenebrio molitor", "Oncorhynchus mykiss", "Italian Journal of Animal Science"], "source": ["\n"], "year": ["2015"], "volume": ["14"], "issue": ["4"], "fpage": ["670"], "lpage": ["676"], "pub-id": ["10.4081/ijas.2015.4170", "2-s2.0-84949970831"]}, {"label": ["11"], "person-group": ["\n"], "surname": ["Dumas", "Raggi", "Barkhouse", "Lewis", "Weltzien"], "given-names": ["A.", "T.", "J.", "E.", "E."], "article-title": ["The oil fraction and partially defatted meal of black soldier fly larvae ("], "italic": ["Hermetia illucens", "Oncorhynchus mykiss", "Aquaculture"], "source": ["\n"], "year": ["2018"], "volume": ["492"], "fpage": ["24"], "lpage": ["34"], "pub-id": ["10.1016/j.aquaculture.2018.03.038", "2-s2.0-85045044404"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Rema", "Saravanan", "Armenjon", "Motte", "Dias"], "given-names": ["P.", "S.", "B.", "C.", "J."], "article-title": ["Graded incorporation of defatted yellow Mealworm ("], "italic": ["Tenebrio molitor", "Oncorhynchus mykiss", "Animals"], "source": ["\n"], "year": ["2019"], "volume": ["9"], "issue": ["4"], "pub-id": ["10.3390/ani9040187", "2-s2.0-85070285432", "187"]}, {"label": ["13"], "person-group": ["\n"], "surname": ["Gasco", "Henry", "Piccolo"], "given-names": ["L.", "M.", "G."], "article-title": ["\n"], "italic": ["Tenebrio molitor", "Dicentrarchus labrax", "in vivo", "Animal Feed Science and Technology"], "source": ["\n"], "year": ["2016"], "volume": ["220"], "fpage": ["34"], "lpage": ["45"], "pub-id": ["10.1016/j.anifeedsci.2016.07.003", "2-s2.0-84995467750"]}, {"label": ["15"], "person-group": ["\n", "\n"], "surname": ["Piccolo", "Marono", "Gasco", "Iannaccone", "Bovera", "Nizza", "Vantomme", "Munke", "van Huis"], "given-names": ["G.", "S.", "L.", "F.", "F.", "A.", "P.", "C.", "A."], "article-title": ["Use of "], "italic": ["Tenebrio molitor", "Sparus aurata", "1st International Conference \u201cInsects to Feed the World\u201d"], "source": ["\n"], "year": ["2014"], "publisher-loc": ["Ede-Wageningen, The Netherlands"], "publisher-name": ["Wageningen University"]}, {"label": ["16"], "person-group": ["\n"], "surname": ["Ng", "Liew", "Ang", "Wong"], "given-names": ["W.-K.", "F.-L.", "L.-P.", "K.-W."], "article-title": ["Potential of mealworm ("], "italic": ["Tenebrio molitor", "Clarias gariepinus", "Aquaculture Research"], "source": ["\n"], "year": ["2001"], "volume": ["32"], "fpage": ["273"], "lpage": ["280"], "pub-id": ["10.1046/j.1355-557x.2001.00024.x", "2-s2.0-28644434553"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Gebremichael", "Szab\u00f3", "S\u00e1ndor", "Nagy", "Ali", "Kucska"], "given-names": ["A.", "A.", "Z. J.", "Z.", "O.", "B."], "article-title": ["Chemical and physical properties of African Catfish ("], "italic": ["Clarias gariepinus", "Aquaculture Nutrition"], "source": ["\n"], "year": ["2023"], "volume": ["2023"], "fpage": ["9"], "pub-id": ["10.1155/2023/6080387", "6080387"]}, {"label": ["18"], "person-group": ["\n", "\n"], "surname": ["Roncarati", "Gasco", "Parisi", "Terova", "Vantomme", "Munke", "van Huis"], "given-names": ["A.", "L.", "G.", "G.", "P.", "C.", "A."], "article-title": ["Growth performance of common catfish ("], "italic": ["Ameiurus melas", "1st International Conference \u201cInsects to Feed the World\u201d"], "source": ["\n"], "year": ["2014"], "publisher-loc": ["Ede-Wageningen, The Netherlands"], "publisher-name": ["Wageningen University"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["de Haro", "Barroso", "Hidalgo", "Sanchez-Muros", "Ferriz"], "given-names": ["C.", "F. G.", "L.", "M. J.", "M."], "article-title": ["Evaluation of "], "italic": ["Tenebrio mollitor", "Oreochromis nilotica", "Aquaculture Europe Mediterranean Aquaculture 2020"], "source": ["\n"], "year": ["2011"], "publisher-loc": ["Rhodes, Greece"], "publisher-name": ["European Aquaculture Society"]}, {"label": ["22"], "person-group": ["\n"], "surname": ["Sharifinia", "Bahmanbeigloo", "Keshavarzifard"], "given-names": ["M.", "Z. A.", "M."], "article-title": ["Fishmeal replacement by mealworm ("], "italic": ["Tenebrio molitor", "Litopenaeus vannamei", "Aquatic Living Resources"], "source": ["\n"], "year": ["2023"], "volume": ["36"], "pub-id": ["10.1051/alr/2023013", "19"]}, {"label": ["23"], "person-group": ["\n"], "surname": ["Ji", "Zhang", "Huang", "Cheng", "Liu"], "given-names": ["H.", "J.-L.", "J.-Q.", "X.-F.", "C."], "article-title": ["Effect of replacement of dietary fish meal with silkworm pupae meal on growth performance, body composition, intestinal protease activity and health status in juvenile Jian carp ("], "italic": ["Cyprinus carpio", "Aquaculture Research"], "source": ["\n"], "year": ["2015"], "volume": ["46"], "issue": ["5"], "fpage": ["1209"], "lpage": ["1221"], "pub-id": ["10.1111/are.12276", "2-s2.0-84926418613"]}, {"label": ["24"], "person-group": ["\n"], "surname": ["Sandnes", "Lie", "Waagb\u00f8"], "given-names": ["K.", "\u00d8.", "R."], "article-title": ["Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, "], "italic": ["Salmo salar", "Journal of Fish Biology"], "source": ["\n"], "year": ["1988"], "volume": ["32"], "issue": ["1"], "fpage": ["129"], "lpage": ["136"], "pub-id": ["10.1111/j.1095-8649.1988.tb05341.x", "2-s2.0-0000231740"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Wilhelm Filho", "Tribess", "G\u00e1spari", "Claudio", "Torres", "Magalh\u00e3es"], "given-names": ["D.", "T.", "C.", "F. D.", "M. A.", "A. R. M."], "article-title": ["Seasonal changes in antioxidant defenses of the digestive gland of the brown mussel ("], "italic": ["Perna perna", "Aquaculture"], "source": ["\n"], "year": ["2001"], "volume": ["203"], "issue": ["1-2"], "fpage": ["149"], "lpage": ["158"], "pub-id": ["10.1016/s0044-8486(01)00599-3", "2-s2.0-0035956246"]}, {"label": ["27"], "person-group": ["\n"], "surname": ["Kader", "Koshio", "Ishikawa", "Yokoyama", "Bulbul"], "given-names": ["M. A.", "S.", "M.", "S.", "M."], "article-title": ["Supplemental effects of some crude ingredients in improving nutritive values of low fishmeal diets for red sea bream, "], "italic": ["Pagrus major", "Aquaculture"], "source": ["\n"], "year": ["2010"], "volume": ["308"], "issue": ["3-4"], "fpage": ["136"], "lpage": ["144"], "pub-id": ["10.1016/J.AQUACULTURE.2010.07.037", "2-s2.0-77957345132"]}, {"label": ["28"], "person-group": ["\n"], "surname": ["Yadav", "Rossi", "Habte-Tsion", "Kumar"], "given-names": ["A. K.", "W.", "H.-M.", "V."], "suffix": ["Jr"], "article-title": ["Impacts of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) level and ratio on the growth, fatty acids composition and hepatic-antioxidant status of largemouth bass ("], "italic": ["Micropterus salmoides", "Aquaculture"], "source": ["\n"], "year": ["2020"], "volume": ["529"], "pub-id": ["10.1016/j.aquaculture.2020.735683", "735683"]}, {"label": ["29"], "person-group": ["\n"], "surname": ["Miller", "Rossi", "Habte-Tsion"], "given-names": ["K. K.", "W.", "H.-M."], "suffix": ["Jr"], "article-title": ["Assessment of total dietary phosphorus requirement of juvenile largemouth bass, "], "italic": ["Micropterus salmoides", "Aquaculture Nutrition"], "source": ["\n"], "year": ["2021"], "volume": ["27"], "issue": ["1"], "fpage": ["116"], "lpage": ["128"], "pub-id": ["10.1111/anu.13169"]}, {"label": ["30"], "person-group": ["\n"], "surname": ["Habte-Tsion", "Ren", "Ge"], "given-names": ["H.-M.", "M.-C.", "X.-P."], "article-title": ["Adequate dietary protein level enhances stress resistance and immune status of blunt snout bream ("], "italic": ["Megalobrama amblycephala", "Journal of Applied Ichthyology"], "source": ["\n"], "year": ["2017"], "volume": ["33"], "issue": ["1"], "fpage": ["75"], "lpage": ["83"], "pub-id": ["10.1111/jai.13219", "2-s2.0-84996484050"]}, {"label": ["31"], "person-group": ["\n"], "surname": ["Habte-Tsion", "Kolimadu", "Rossi", "Filer", "Kumar"], "given-names": ["H.-M.", "G. D.", "W.", "K.", "V."], "suffix": ["Jr."], "article-title": ["Effects of Schizochytrium and micro-minerals on immune, antioxidant, inflammatory and lipid-metabolism status of "], "italic": ["Micropterus salmoides", "Scientific Reports"], "source": ["\n"], "year": ["2020"], "volume": ["10"], "pub-id": ["10.1038/s41598-020-64286-9", "7457"]}, {"label": ["32"], "person-group": ["\n"], "surname": ["Habte-Tsion", "Riche", "Mejri"], "given-names": ["H.-M.", "M.", "S."], "article-title": ["The effects of fish meal substitution by clam meal on the growth and health of Florida pompano ("], "italic": ["Trachinotus carolinus", "Scientific Reports"], "source": ["\n"], "year": ["2022"], "volume": ["12"], "pub-id": ["10.1038/s41598-022-11675-x", "7696"]}, {"label": ["33"], "person-group": ["\n"], "surname": ["Jenberie", "Thim", "Sunyer", "Skj\u00f8dt", "Jensen", "J\u00f8rgensen"], "given-names": ["S.", "H. L.", "J. O.", "K.", "I.", "J. B."], "article-title": ["Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression"], "source": ["\n"], "italic": ["Scientific Reports"], "year": ["2018"], "volume": ["8"], "pub-id": ["10.1038/s41598-018-21895-9", "2-s2.0-85042531390", "3565"]}, {"label": ["34"], "person-group": ["\n"], "surname": ["Pe\u00f1aranda", "Jensen", "Tollersrud", "Bruun", "J\u00f8rgensen"], "given-names": ["M. M. D.", "I.", "L. G.", "J.-A.", "J. B."], "article-title": ["Profiling the Atlantic Salmon IgM+ B cell surface proteome: novel information on teleost fish B cell protein repertoire and identification of potential B cell markers"], "source": ["\n"], "italic": ["Frontiers in Immunology"], "year": ["2019"], "volume": ["10"], "pub-id": ["10.3389/fimmu.2019.00037", "2-s2.0-85060975524", "37"]}, {"label": ["36"], "person-group": ["\n"], "surname": ["Habte-Tsion"], "given-names": ["H.-M."], "article-title": ["A review on fish immuno-nutritional response to indispensable amino acids in relation to TOR, NF-"], "italic": ["\u03ba", "Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology"], "source": ["\n"], "year": ["2020"], "volume": ["241"], "pub-id": ["10.1016/j.cbpb.2019.110389", "110389"]}, {"label": ["37"], "person-group": ["\n"], "surname": ["Ring\u00f8", "Zhou", "Vecino"], "given-names": ["E.", "Z.", "J. L. G."], "article-title": ["Effect of dietary components on the gut microbiota of aquatic animals. A never-ending story?"], "source": ["\n"], "italic": ["Aquaculture Nutrition"], "year": ["2016"], "volume": ["22"], "issue": ["2"], "fpage": ["219"], "lpage": ["282"], "pub-id": ["10.1111/anu.12346", "2-s2.0-84959477063"]}, {"label": ["38"], "person-group": ["\n"], "surname": ["Spilsbury", "Foysal", "Tay", "Gagnon"], "given-names": ["F.", "M. J.", "A.", "M. M."], "article-title": ["Gut microbiome as a potential biomarker in fish: dietary exposure to petroleum hydrocarbons and metals, metabolic functions and cytokine expression in juvenile "], "italic": ["Lates calcarifer", "Frontiers in Microbiology"], "source": ["\n"], "year": ["2022"], "volume": ["13"], "pub-id": ["10.3389/fmicb.2022.827371"]}, {"label": ["39"], "person-group": ["\n"], "surname": ["Luan", "Li", "Zhou"], "given-names": ["Y.", "M.", "W."], "article-title": ["The fish microbiota: research progress and potential applications"], "source": ["\n"], "italic": ["Engineering"], "year": ["2023"], "comment": ["In Press"], "pub-id": ["10.1016/j.eng.2022.12.011"]}, {"label": ["40"], "person-group": ["\n"], "surname": ["Antonopoulou", "Nikouli", "Piccolo"], "given-names": ["E.", "E.", "G."], "article-title": ["Reshaping gut bacterial communities after dietary Tenebrio molitor larvae meal supplementation in three fish species"], "source": ["\n"], "italic": ["Aquaculture"], "year": ["2019"], "volume": ["503"], "fpage": ["628"], "lpage": ["635"], "pub-id": ["10.1016/J.AQUACULTURE.2018.12.013", "2-s2.0-85059578593"]}, {"label": ["42"], "person-group": ["\n"], "surname": ["Schumann", "Brinker"], "given-names": ["M.", "A."], "article-title": ["Understanding and managing suspended solids in intensive salmonid aquaculture: a review"], "source": ["\n"], "italic": ["Reviews in Aquaculture"], "year": ["2020"], "volume": ["12"], "issue": ["4"], "fpage": ["2109"], "lpage": ["2139"], "pub-id": ["10.1111/raq.12425"]}, {"label": ["43"], "person-group": ["\n"], "surname": ["Brinker"], "given-names": ["A."], "article-title": ["Guar gum in rainbow trout ("], "italic": ["Oncorhynchus mykiss", "Aquaculture"], "source": ["\n"], "year": ["2007"], "volume": ["267"], "issue": ["1\u20134"], "fpage": ["315"], "lpage": ["327"], "pub-id": ["10.1016/j.aquaculture.2007.02.037", "2-s2.0-34250212120"]}, {"label": ["44"], "collab": ["AOAC"], "source": ["\n"], "italic": ["Official Methods of Analysis of the Association of Official Analytical Chemists"], "year": ["2003"], "edition": ["15th"], "publisher-loc": ["Arlington, VA, USA"], "publisher-name": ["Association of Official Analytical Chemists, Inc."]}, {"label": ["46"], "person-group": ["\n"], "surname": ["Hoffman", "Siddiqui", "Fields"], "given-names": ["C.", "N. Y.", "I."], "article-title": ["Species-level resolution of female bladder microbiota from 16S rRNA amplicon sequencing"], "source": ["\n"], "italic": ["Msystems"], "year": ["2021"], "volume": ["6"], "issue": ["5"], "pub-id": ["10.1128/mSystems.00518-21", "e00518-21"]}, {"label": ["47"], "person-group": ["\n"], "surname": ["Gordon", "Hannon"], "given-names": ["A.", "G. J."], "article-title": ["Fastx-toolkit. FASTQ/A short-reads pre-processing tools"], "year": ["2010"]}, {"label": ["49"], "person-group": ["\n"], "surname": ["Bolyen", "Rideout", "Dillon"], "given-names": ["E.", "J. R.", "M. R."], "article-title": ["Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2"], "source": ["\n"], "italic": ["Nature Biotechnology"], "year": ["2019"], "volume": ["37"], "issue": ["8"], "fpage": ["852"], "lpage": ["857"], "pub-id": ["10.1038/s41587-019-0209-9", "2-s2.0-85069676260"]}, {"label": ["50"], "person-group": ["\n"], "surname": ["Bradshaw", "Dickens", "Trefry", "McCarthy"], "given-names": ["D. J.", "N. J.", "J. H.", "P. J."], "suffix": ["II"], "article-title": ["Defining the sediment prokaryotic communities of the Indian River Lagoon, FL, USA, an estuary of national significance"], "source": ["\n"], "italic": ["PLOS ONE"], "year": ["2020"], "volume": ["15"], "pub-id": ["10.1371/journal.pone.0236305", "e0236305"]}, {"label": ["51"], "person-group": ["\n"], "surname": ["Krueger"], "given-names": ["F."], "article-title": ["Trim Galore. Babraham Bioinformatics"], "year": ["2020"], "comment": ["\n"], "ext-link": ["https://github.com/FelixKrueger/TrimGalore"]}, {"label": ["55"], "person-group": ["\n"], "surname": ["Rognes", "Flouri", "Nichols", "Quince", "Mah\u00e9"], "given-names": ["T.", "T.", "B.", "C.", "F."], "article-title": ["VSEARCH: a versatile open source tool for metagenomics"], "source": ["\n"], "italic": ["PeerJ"], "year": ["2016"], "volume": ["4"], "issue": ["10"], "pub-id": ["10.7717/PEERJ.2584", "2-s2.0-84994444665", "e2584"]}, {"label": ["56"], "person-group": ["\n"], "surname": ["Bokulich", "Kaehler", "Rideout"], "given-names": ["N. A.", "B. D.", "J. R."], "article-title": ["Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2\u2019s q2-feature-classifier plugin"], "source": ["\n"], "italic": ["Microbiome"], "year": ["2018"], "volume": ["6"], "pub-id": ["10.1186/S40168-018-0470-Z", "2-s2.0-85052651259", "90"]}, {"label": ["57"], "person-group": ["\n"], "surname": ["Robeson", "O\u2019Rourke", "Kaehler"], "given-names": ["M. S.", "D. R.", "B. D."], "suffix": ["II"], "article-title": ["RESCRIPt: reproducible sequence taxonomy reference database management for the masses"], "source": ["\n"], "italic": ["BioRxiv"], "year": ["2020"], "comment": ["Preprint"], "pub-id": ["10.1101/2020.10.05.326504"]}, {"label": ["59"], "person-group": ["\n"], "surname": ["Olsvik", "Torstensen", "Hemre", "Sanden", "Waagb\u00f8"], "given-names": ["P. A.", "B. E.", "G. I.", "M.", "R."], "article-title": ["Hepatic oxidative stress in Atlantic salmon ("], "italic": ["Salmo salar", "Aquaculture Nutrition"], "source": ["\n"], "year": ["2011"], "volume": ["17"], "issue": ["2"], "fpage": ["e424"], "lpage": ["e436"], "pub-id": ["10.1111/j.1365-2095.2010.00778.x", "2-s2.0-79952558813"]}, {"label": ["61"], "person-group": ["\n"], "surname": ["McMurdie", "Holmes"], "given-names": ["P. J.", "S."], "article-title": ["Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data"], "source": ["\n"], "italic": ["PLoS ONE"], "year": ["2013"], "volume": ["8"], "issue": ["4"], "pub-id": ["10.1371/JOURNAL.PONE.0061217", "2-s2.0-84876427223", "e61217"]}, {"label": ["62"], "collab": ["RStudio Team"], "article-title": ["RStudio: integrated development environment for R"], "year": ["2020"], "comment": ["\n"], "ext-link": ["http://www.rstudio.com/"]}, {"label": ["63"], "person-group": ["\n"], "surname": ["Lin", "Peddada"], "given-names": ["H.", "S. D."], "article-title": ["Analysis of compositions of microbiomes with bias correction"], "source": ["\n"], "italic": ["Nature Communications"], "year": ["2020"], "volume": ["11"], "pub-id": ["10.1038/s41467-020-17041-7", "3514"]}, {"label": ["64"], "person-group": ["\n"], "surname": ["Wickham"], "given-names": ["H."], "article-title": ["Tidyverse: easily install and load the \u2018Tidyverse\u2019"], "year": ["2021"], "comment": ["\n"], "ext-link": ["https://CRAN.R-project.org/package=tidyverse"]}, {"label": ["65"], "collab": ["R Core Team"], "article-title": ["R: a language and environment for statistical computing"], "year": ["2022"], "comment": ["\n"], "ext-link": ["https://www.R-project.org/"]}, {"label": ["66"], "person-group": ["\n"], "surname": ["Kassambara"], "given-names": ["A."], "article-title": ["rstatix: pipe-friendly framework for basic statistical tests"], "year": ["2022"], "comment": ["\n"], "ext-link": ["https://CRAN.R-project.org/package=rstatix"]}, {"label": ["68"], "person-group": ["\n"], "surname": ["Clarke", "Gorley"], "given-names": ["K. R.", "R. N."], "article-title": ["Getting started with PRIMER v7 plymouth routines in: multivariate ecological research"], "year": ["2015"], "comment": ["\n"], "ext-link": ["https://www.primer-e.com"]}, {"label": ["69"], "person-group": ["\n"], "surname": ["Bruno", "Munro"], "given-names": ["D. W.", "A. L. S."], "article-title": ["Haematological assessment of rainbow trout, "], "italic": ["Salmo gairdneri", "Salmo salar", "Renibacterium salmoninarum", "Journal of Fish Diseases"], "source": ["\n"], "year": ["1986"], "volume": ["9"], "issue": ["3"], "fpage": ["195"], "lpage": ["204"], "pub-id": ["10.1111/J.1365-2761.1986.TB01004.X", "2-s2.0-84987593488"]}, {"label": ["71"], "person-group": ["\n"], "surname": ["Colquhoun", "Duodu"], "given-names": ["D. J.", "S."], "article-title": ["Francisella infections in farmed and wild aquatic organisms"], "source": ["\n"], "italic": ["Veterinary Research"], "year": ["2011"], "volume": ["42"], "pub-id": ["10.1186/1297-9716-42-47", "2-s2.0-80051988391", "47"]}, {"label": ["72"], "person-group": ["\n"], "surname": ["Higuera", "Bast\u00edas", "Tsertsvadze", "Romero", "Espejo"], "given-names": ["G.", "R.", "G.", "J.", "R. T."], "article-title": ["Recently discovered "], "italic": ["Vibrio anguillarum", "Salmo salar", "Aquaculture"], "source": ["\n"], "year": ["2013"], "volume": ["392\u2013395"], "fpage": ["128"], "lpage": ["133"], "pub-id": ["10.1016/J.AQUACULTURE.2013.02.013", "2-s2.0-84874940969"]}, {"label": ["73"], "person-group": ["\n"], "surname": ["Nowlan", "Britney", "Lumsden", "Russell"], "given-names": ["J. P.", "S. R.", "J. S.", "S."], "article-title": ["Experimental induction of Tenacibaculosis in Atlantic Salmon ("], "italic": ["Salmo salar", "Tenacibaculum maritimum", "T. dicentrarchi", "T. finnmarkense", "Pathogens"], "source": ["\n"], "year": ["2021"], "volume": ["10"], "issue": ["11"], "pub-id": ["10.3390/PATHOGENS10111439", "1439"]}, {"label": ["74"], "person-group": ["\n"], "surname": ["Boerlage", "Ashby", "Herrero", "Reeves", "Gunn", "Rodger"], "given-names": ["A. S.", "A.", "A.", "A.", "G. J.", "H. D."], "article-title": ["Epidemiology of marine gill diseases in Atlantic salmon ("], "italic": ["Salmo salar", "Reviews in Aquaculture"], "source": ["\n"], "year": ["2020"], "volume": ["12"], "issue": ["4"], "fpage": ["2140"], "lpage": ["2159"], "pub-id": ["10.1111/RAQ.12426"]}, {"label": ["75"], "person-group": ["\n"], "surname": ["Ring\u00f8", "Gatesoupe"], "given-names": ["E.", "F. J."], "article-title": ["Lactic acid bacteria in fish: a review"], "source": ["\n"], "italic": ["Aquaculture"], "year": ["1998"], "volume": ["160"], "issue": ["3-4"], "fpage": ["177"], "lpage": ["203"], "pub-id": ["10.1016/S0044-8486(97)00299-8", "2-s2.0-0032579167"]}, {"label": ["76"], "person-group": ["\n"], "surname": ["Henry", "Gasco", "Piccolo", "Fountoulaki"], "given-names": ["M.", "L.", "G.", "E."], "article-title": ["Review on the use of insects in the diet of farmed fish: past and future"], "source": ["\n"], "italic": ["Animal Feed Science and Technology"], "year": ["2015"], "volume": ["203"], "fpage": ["1"], "lpage": ["22"], "pub-id": ["10.1016/j.anifeedsci.2015.03.001", "2-s2.0-84928339785"]}, {"label": ["77"], "person-group": ["\n"], "surname": ["Gasco", "Biasato", "Dabbou", "Schiavone", "Gai"], "given-names": ["L.", "I.", "S.", "A.", "F."], "article-title": ["Animals fed insect-based diets: state-of-the-art on digestibility, performance and product quality"], "source": ["\n"], "italic": ["Animals"], "year": ["2019"], "volume": ["9"], "issue": ["4"], "pub-id": ["10.3390/ani9040170", "2-s2.0-85066850326", "170"]}, {"label": ["78"], "person-group": ["\n"], "surname": ["Nogales-M\u00e9rida", "Gobbi", "J\u00f3zefiak"], "given-names": ["S.", "P.", "D."], "article-title": ["Insect meals in fish nutrition"], "source": ["\n"], "italic": ["Reviews in Aquaculture"], "year": ["2019"], "volume": ["11"], "issue": ["4"], "fpage": ["1080"], "lpage": ["1103"], "pub-id": ["10.1111/raq.12281", "2-s2.0-85052929237"]}, {"label": ["79"], "person-group": ["\n"], "surname": ["Hua"], "given-names": ["K."], "article-title": ["A meta-analysis of the effects of replacing fish meals with insect meals on growth performance of fish"], "source": ["\n"], "italic": ["Aquaculture"], "year": ["2021"], "volume": ["530"], "pub-id": ["10.1016/j.aquaculture.2020.735732", "735732"]}, {"label": ["80"], "person-group": ["\n"], "surname": ["Mejri", "Tremblay", "Audet", "Wills", "Riche"], "given-names": ["S. C.", "R.", "C.", "P. S.", "M."], "article-title": ["Essential fatty acid requirements in tropical and cold-water marine fish larvae and juveniles"], "source": ["\n"], "italic": ["Frontiers in Marine Science"], "year": ["2021"], "volume": ["8"], "pub-id": ["10.3389/fmars.2021.680003", "557"]}, {"label": ["81"], "person-group": ["\n"], "surname": ["Habte-Tsion", "Liu", "Ge"], "given-names": ["H.-M.", "B.", "X."], "article-title": ["Effects of dietary protein level on growth performance, muscle composition, blood composition, and digestive enzyme activity of Wuchang bream ("], "italic": ["Megalobrama amblycephala", "The Israeli Journal of Aquaculture\u2014Bamidgeh"], "source": ["\n"], "year": ["2013"], "volume": ["65"], "pub-id": ["925"]}, {"label": ["82"], "person-group": ["\n"], "surname": ["Rossi", "Allen", "Habte-Tsion", "Meesala"], "given-names": ["W.", "K. M.", "H.-M.", "K.-M."], "suffix": ["Jr"], "article-title": ["Supplementation of glycine, prebiotic, and nucleotides in soybean meal-based diets for largemouth bass ("], "italic": ["Micropterus salmoides", "Aquaculture"], "source": ["\n"], "year": ["2021"], "volume": ["532"], "pub-id": ["10.1016/j.aquaculture.2020.736031", "736031"]}, {"label": ["83"], "person-group": ["\n"], "surname": ["Lundstedt", "Melo", "Santos-Neto", "Moraes"], "given-names": ["L. M.", "J. F. B.", "C.", "G."], "article-title": ["Diet influences proteolytic enzyme profile of the South American catfish Rhamdia quelen"], "conf-name": ["International Congress on the Biology of Fish, Biochemistry and Physiology Advances in Finfish Aquaculture"], "conf-date": ["2002"], "conf-loc": ["Vancouver, Canada"], "publisher-name": ["Peches et Oceans Canada"], "fpage": ["65"], "lpage": ["71"]}, {"label": ["84"], "person-group": ["\n"], "surname": ["Abdel-Tawwab", "Ahmad", "Khattab", "Shalaby"], "given-names": ["M.", "M. H.", "Y. A. E.", "A. M. E."], "article-title": ["Effect of dietary protein level, initial body weight, and their interaction on the growth, feed utilization, and physiological alterations of Nile tilapia, "], "italic": ["Oreochromis niloticus", "Aquaculture"], "source": ["\n"], "year": ["2010"], "volume": ["298"], "issue": ["3-4"], "fpage": ["267"], "lpage": ["274"], "pub-id": ["10.1016/j.aquaculture.2009.10.027", "2-s2.0-71949085383"]}, {"label": ["85"], "person-group": ["\n"], "surname": ["Severini", "Aliberti", "Di Giovannandrea"], "given-names": ["G.", "L. M.", "R."], "article-title": ["Diagnostic aspects of alkaline phosphatase: separation of isoenzymes in normal and pathological human serum by high-performance liquid chromatography"], "source": ["\n"], "italic": ["Journal of Chromatography B: Biomedical Sciences and Applications"], "year": ["1991"], "volume": ["563"], "issue": ["1"], "fpage": ["147"], "lpage": ["152"], "pub-id": ["10.1016/0378-4347(91)80287-m", "2-s2.0-0026067712"]}, {"label": ["87"], "person-group": ["\n", "\n"], "surname": ["Yamamoto", "Egami"], "given-names": ["Y.", "N."], "article-title": ["Determination of toxicity by biochemical method"], "source": ["\n"], "italic": ["Fishes as Laboratory"], "year": ["1981"], "publisher-loc": ["Tokyo"], "publisher-name": ["Soft Science"], "fpage": ["568"], "lpage": ["574"]}, {"label": ["88"], "person-group": ["\n"], "surname": ["Magnadottir"], "given-names": ["B."], "article-title": ["Comparison of immunoglobulin (IgM) from four fish species"], "source": ["\n"], "italic": ["Icelandic Agricultural Sciences"], "year": ["1998"], "volume": ["12"], "fpage": ["47"], "lpage": ["59"]}, {"label": ["89"], "person-group": ["\n"], "surname": ["Bayir", "Sirkecioglu", "Bayir", "Haliloglu", "Kocaman", "Aras"], "given-names": ["A.", "A. N.", "M.", "H. I.", "E. M.", "N. M."], "article-title": ["Metabolic responses to prolonged starvation, food restriction, and refeeding in the brown trout, "], "italic": ["Salmo trutta", "Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology"], "source": ["\n"], "year": ["2011"], "volume": ["159"], "issue": ["4"], "fpage": ["191"], "lpage": ["196"], "pub-id": ["10.1016/j.cbpb.2011.04.008", "2-s2.0-79958820975"]}, {"label": ["91"], "person-group": ["\n"], "surname": ["Mart\u00ednez-\u00c1lvarez", "Morales", "Sanz"], "given-names": ["R. M.", "A. E.", "A."], "article-title": ["Antioxidant defenses in fish: biotic and abiotic factors"], "source": ["\n"], "italic": ["Reviews in Fish Biology and Fisheries"], "year": ["2005"], "volume": ["15"], "fpage": ["75"], "lpage": ["88"], "pub-id": ["10.1007/s11160-005-7846-4", "2-s2.0-29544452595"]}, {"label": ["93"], "person-group": ["\n"], "surname": ["Livingstone"], "given-names": ["D. R."], "article-title": ["Oxidative stress in aquatic organisms in relation to pollution and aquaculture"], "source": ["\n"], "italic": ["Revue de Medecine Veterinaire"], "year": ["2003"], "volume": ["154"], "issue": ["6"], "fpage": ["427"], "lpage": ["430"]}, {"label": ["94"], "person-group": ["\n"], "surname": ["Zhou", "Wang", "Wang", "Tan"], "given-names": ["Q.-C.", "Y.-L.", "H.-L.", "B.-P."], "article-title": ["Dietary threonine requirements of juvenile Pacific white shrimp, "], "italic": ["Litopenaeus vannamei", "Aquaculture"], "source": ["\n"], "year": ["2013"], "volume": ["392\u2013395"], "fpage": ["142"], "lpage": ["147"], "pub-id": ["10.1016/j.aquaculture.2013.01.026", "2-s2.0-84874941535"]}, {"label": ["95"], "person-group": ["\n"], "surname": ["Gajardo", "Rodiles", "Kortner"], "given-names": ["K.", "A.", "T. M."], "article-title": ["A high-resolution map of the gut microbiota in Atlantic salmon ("], "italic": ["Salmo salar", "Scientific Reports"], "source": ["\n"], "year": ["2016"], "volume": ["6"], "pub-id": ["10.1038/srep30893", "2-s2.0-84981328143", "30893"]}, {"label": ["97"], "person-group": ["\n"], "surname": ["Egerton", "Culloty", "Whooley", "Stanton", "Ross"], "given-names": ["S.", "S.", "J.", "C.", "R. P."], "article-title": ["The gut microbiota of marine fish"], "source": ["\n"], "italic": ["Frontiers in Microbiology"], "year": ["2018"], "volume": ["9"], "pub-id": ["10.3389/fmicb.2018.00873", "2-s2.0-85046675602", "873"]}, {"label": ["98"], "person-group": ["\n"], "surname": ["Minich", "Poore", "Jantawongsri"], "given-names": ["J. J.", "G. D.", "K."], "article-title": ["Microbial ecology of Atlantic Salmon ("], "italic": ["Salmo salar", "Applied and Environmental Microbiology"], "source": ["\n"], "year": ["2020"], "volume": ["86"], "issue": ["12"], "pub-id": ["10.1128/AEM.00411-20"]}, {"label": ["99"], "person-group": ["\n"], "surname": ["Kim", "Shin", "Lee"], "given-names": ["P. S.", "N.-R.", "J.-B."], "article-title": ["Host habitat is the major determinant of the gut microbiome of fish"], "source": ["\n"], "italic": ["Microbiome"], "year": ["2021"], "volume": ["9"], "pub-id": ["10.1186/s40168-021-01113-x", "166"]}, {"label": ["100"], "person-group": ["\n"], "surname": ["Song", "Wen", "Liu"], "given-names": ["C.", "H.", "G."], "article-title": ["Gut microbes reveal Pseudomonas medicates ingestion preference via protein utilization and cellular homeostasis under feed domestication in freshwater drum, "], "italic": ["Aplodinotus grunniens", "Frontiers in Microbiology"], "source": ["\n"], "year": ["2022"], "volume": ["13"], "pub-id": ["10.3389/fmicb.2022.861705", "861705"]}, {"label": ["101"], "person-group": ["\n"], "surname": ["Wiklund"], "given-names": ["T."], "article-title": ["\n"], "italic": ["Pseudomonas anguilliseptica", "Microbiology Australia"], "source": ["\n"], "year": ["2016"], "volume": ["37"], "issue": ["3"], "fpage": ["135"], "lpage": ["136"], "pub-id": ["10.1071/MA16046"]}, {"label": ["103"], "person-group": ["\n"], "surname": ["Taoka", "Maeda", "Jo"], "given-names": ["Y.", "H.", "J.-Y."], "article-title": ["Growth, stress tolerance and non-specific immune response of Japanese flounder "], "italic": ["Paralichthys olivaceus", "Fisheries Science"], "source": ["\n"], "year": ["2006"], "volume": ["72"], "fpage": ["310"], "lpage": ["321"], "pub-id": ["10.1111/j.1444-2906.2006.01152.x", "2-s2.0-33645749879"]}, {"label": ["104"], "person-group": ["\n"], "surname": ["Li", "Zhou", "Ling"], "given-names": ["H.", "Y.", "H."], "article-title": ["The effect of dietary supplementation with "], "italic": ["Clostridium butyricum", "Oreochromis niloticus", "PLoS One"], "source": ["\n"], "year": ["2019"], "volume": ["14"], "issue": ["12"], "pub-id": ["10.1371/journal.pone.0223428", "e0223428"]}, {"label": ["106"], "person-group": ["\n"], "surname": ["Tran", "Proke\u0161ov\u00e1", "Zare"], "given-names": ["H. Q.", "M.", "M."], "article-title": ["Production performance, nutrient digestibility, serum biochemistry, fillet composition, intestinal microbiota and environmental impacts of European perch ("], "italic": ["Perca fluviatilis", "Tenebrio molitor", "Aquaculture"], "source": ["\n"], "year": ["2022"], "volume": ["547"], "pub-id": ["10.1016/J.AQUACULTURE.2021.737499", "737499"]}, {"label": ["107"], "person-group": ["\n"], "surname": ["Huyben", "Vidakovi\u0107", "Werner Hallgren", "Langeland"], "given-names": ["D.", "A.", "S.", "M."], "article-title": ["High-throughput sequencing of gut microbiota in rainbow trout ("], "italic": ["Oncorhynchus mykiss", "Hermetia illucens", "Aquaculture"], "source": ["\n"], "year": ["2019"], "volume": ["500"], "fpage": ["485"], "lpage": ["491"], "pub-id": ["10.1016/J.AQUACULTURE.2018.10.034", "2-s2.0-85055655611"]}, {"label": ["108"], "person-group": ["\n"], "surname": ["Forquin", "Weimer"], "given-names": ["M. P.", "B. C."], "article-title": ["Brevibacterium"], "source": ["\n"], "italic": ["Encyclopedia of Food Microbiology"], "year": ["2014"], "edition": ["Second"], "fpage": ["324"], "lpage": ["330"], "pub-id": ["10.1016/B978-0-12-384730-0.00047-1", "2-s2.0-85021390726"]}, {"label": ["109"], "person-group": ["\n"], "surname": ["P\u00e9rez-S\u00e1nchez", "Ruiz-Zarzuela", "de Blas", "Balc\u00e1zar"], "given-names": ["T.", "I.", "I.", "J. L."], "article-title": ["Probiotics in aquaculture: a current assessment"], "source": ["\n"], "italic": ["Reviews in Aquaculture"], "year": ["2014"], "volume": ["6"], "issue": ["3"], "fpage": ["133"], "lpage": ["146"], "pub-id": ["10.1111/RAQ.12033", "2-s2.0-84927523378"]}, {"label": ["110"], "person-group": ["\n"], "surname": ["Collins", "Brown", "Jones"], "given-names": ["M. D.", "J.", "D."], "article-title": ["\n"], "italic": ["Brachybacterium faecium", "International Journal of Systematic and Evolutionary Microbiology"], "source": ["\n"], "year": ["1988"], "volume": ["38"], "issue": ["1"], "fpage": ["45"], "lpage": ["48"], "pub-id": ["10.1099/00207713-38-1-45"]}, {"label": ["112"], "person-group": ["\n"], "surname": ["Orsod", "Joseph", "Huyop"], "given-names": ["M.", "M.", "F."], "article-title": ["Characterization of exopolysaccharides produced by "], "italic": ["Bacillus cereus", "Brachybacterium", "Lates calcarifer", "Malaysian Journal of Microbiology"], "source": ["\n"], "year": ["2012"], "volume": ["8"], "issue": ["3"], "fpage": ["170"], "lpage": ["174"], "pub-id": ["10.21161/mjm.04412"]}, {"label": ["115"], "person-group": ["\n"], "surname": ["Picha", "Turano", "Beckman", "Borski"], "given-names": ["M. E.", "M. J.", "B. R.", "R. J."], "article-title": ["Endocrine biomarkers of growth and applications to aquaculture: a minireview of growth hormone, insulin-like growth factor (IGF)-I, and IGF-binding proteins as potential growth indicators in fish"], "source": ["\n"], "italic": ["North American Journal of Aquaculture"], "year": ["2008"], "volume": ["70"], "issue": ["2"], "fpage": ["196"], "lpage": ["211"], "pub-id": ["10.1577/A07-038.1", "2-s2.0-43049091863"]}, {"label": ["116"], "person-group": ["\n"], "surname": ["Fillatreau", "Six", "Magadan", "Castro", "Sunyer", "Boudinot"], "given-names": ["S.", "A.", "S.", "R.", "J. O.", "P."], "article-title": ["The astonishing diversity of Ig classes and B cell repertoires in teleost fish"], "source": ["\n"], "italic": ["Frontiers in Immunology"], "year": ["2013"], "volume": ["4"], "pub-id": ["10.3389/fimmu.2013.00028", "2-s2.0-84883659680", "28"]}, {"label": ["117"], "person-group": ["\n"], "surname": ["Bakke", "Bj\u00f8rgen", "Koppang"], "given-names": ["A. F.", "H.", "E. O."], "article-title": ["IgM+ and IgT+ B cell traffic to the heart during SAV infection in Atlantic Salmon"], "source": ["\n"], "italic": ["Vaccines"], "year": ["2020"], "volume": ["8"], "issue": ["3"], "pub-id": ["10.3390/vaccines8030493", "493"]}]
{ "acronym": [], "definition": [] }
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oa_package/5e/42/PMC10787657.tar.gz
PMC10787658
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[ "<title>1. Introduction</title>", "<p>Uric acid is a catabolic product of purine metabolism in humans and higher primates (chimpanzees, orangutans, gibbons, and new world apes) who have lost urate oxidase activity [##REF##1556746##1##, ##REF##817107##2##]. This water-soluble organic compound has a physiological role as an antioxidant, and it is thought that increased plasma urate levels (uricemia) caused an acceleration of brain development during evolution; furthermore, uric acid may help the immune system in recognizing injured or damaged cells [##UREF##0##3##–##REF##14520412##5##]. In addition to its physiological roles, uric acid also has clinical significance. Increased plasma urate concentration (hyperuricemia) can cause cardiovascular disease [##REF##18946066##6##], high blood pressure [##REF##11711505##7##], and renal disease [##REF##33678312##8##]. Hyperuricemia and gout have a significant impact on lipid metabolism, the most significant glycerophospholipid dysregulation was recently found in early onset patients ≤40 years [##UREF##1##9##]. Prevalence in the European adult population is estimated to 11.9%–25.0% for hyperuricemia and 0.3%–4.7% for gout, respectively [##UREF##2##10##]. Over the past decade, genome-wide association studies and meta-analyses have led to a massive increase in our knowledge of the common genetic variants that influence serum UA concentrations. However, knowledge of the extent to which genetic variants predict serum UA concentrations and the functional pathways through which they may influence serum UA levels remains limited.</p>", "<p>Urate excretion or reabsorption in the proximal tubule of the nephron is managed by various transporters [##UREF##3##11##]—multimolecular complex “transportosome” that probably involves cooperation between multiple transporters, ##FIG##0##Figure 1##. For urate excretion in the proximal tubule seems to be most important ABCG2 transporter (BCRP), which is localized on an apical membrane of human proximal tubule [##UREF##4##12##, ##REF##17978814##13##]. It was shown that single nucleotide polymorphism (SNP) such as p.Q141K is associated with hyperuricemia and/or gout [##REF##20421215##14##]. This mutation caused impaired transport function [##UREF##5##15##]. Similarly, p.Q126X allelic variant in this transporter abolished urate excretion [##UREF##6##16##]. Moreover, further SNPs were discovered in <italic>ABCG2</italic> which change the transport capacity for urate [##UREF##7##17##]. Other variants associated with hyperuricemia and gout were found in the genes encoding the transporters GLUT9 and URAT1 [##REF##26500098##18##]. These SNPs are associated with hyperuricemia[##REF##18327257##19##–##UREF##8##21##] and even more often with renal hypouricemia [##REF##19026395##22##, ##REF##19926891##23##]. URAT1 is urate transporter localized on the apical membrane of proximal tubular cells [##REF##12024214##24##]. The association of p.C850G with gout has been described [##REF##16837472##25##], but similarly as in the GLUT9, allelic variants seem to be more frequently associated with hypouricemia [##REF##23043931##26##, ##UREF##9##27##]. In addition to these well-known and well-studied transporters in the renal epithelial cells, there are other proteins involved in urate handling in the kidney. In our last paper, we examinated rare variants in <italic>SLC22A6</italic> (OAT1) and <italic>SLC22A8</italic> (OAT3) and in this work we focus on another less examined urate transport proteins [##UREF##10##28##]. In ##TAB##0##Table 1##, we present an overview of the influence of identified allelic variants in the urate trasportosome, which we published in our previous paper.</p>", "<title>1.1. <italic>SLC22A11</italic> (OAT4)</title>", "<p>Organic anion transporter 4 (OAT4) is a 550 amino acid protein encoded by the <italic>SLC22A11</italic> gene. The OAT4 protein is expressed in the apical membrane of the proximal tubules in the kidney as well as in the placenta [##REF##10660625##32##, ##REF##24309898##33##]. This protein functions as an organic anion/dicarboxylate exchanger [##REF##15037815##34##] and as a low-affinity urate transporter [##REF##17229912##35##]. Some studies have reported polymorphisms in <italic>SLC22A11</italic> associated with serum urate concentrations and gout [##UREF##13##36##–##REF##33832965##39##]. Single amino acid substitutions, i.e., p.L29P and p.H469R, both of which decrease urate transport by the OAT4 transporter, were found in the healthy individuals [##REF##20668102##40##].</p>", "<title>1.2. <italic>SLC22A13</italic> (OAT10)</title>", "<p>Organic anion transporter 10 (OAT10) is a 551 amino acid protein encoded by the <italic>SLC22A13</italic> gene. OAT10 was first discovered as the orphan transporter hORCTL3; its sequence was similar to the organic cation transporter family [##UREF##14##41##]. Protein OAT10 is highly expressed in the apical membrane of the proximal tubules in the kidneys, where it mediates urate reabsorption [##REF##18411268##42##, ##UREF##15##43##]. This protein transports organic anions, such as urate, nicotinate, orotate, or para-aminohippurate. Urate is exchanged for OH-anions or organic anions such as lactate, pyrazinoate, or nicotinate [##REF##18411268##42##, ##UREF##16##44##]. To date, there are no known variants of the <italic>SLC22A13</italic> gene associated with the risk of gout; on the other hand, the polymorphism rs117371763 (p.R377C) is associated with a reduction of gout risk [##REF##31780526##45##].</p>", "<title>1.3. <italic>SLC17A1</italic> (NPT1)</title>", "<p>Sodium-dependent phosphate cotransporter type 1 (NPT1) is a 467 amino acid protein encoded by the <italic>SLC17A1</italic> gene. The NPT1 protein is expressed mainly in the kidneys and liver. NPT1 was initially discovered as a sodium phosphate cotransporter in the cortex of human kidneys [##REF##7826357##46##]. NPT1 has also been shown to transport para-aminohyppurate, estradiol-17- <italic>β</italic>-glucuronide, and urate. The transporter is chloride sensitive and is located on the apical side of epithelial cells [##REF##10733936##47##, ##REF##25252215##48##]. The transport is dependent on the membrane polarization and is responsible for the urate excretion from cells [##REF##20566650##49##]. The variant rs1165196 (p.I269T) in <italic>SLC17A1</italic> causes an increase in urate transport via NPT1 and, therefore, significantly reduces the risk of gout linked to renal underexcretion. This variant increases the urate transport capacity in uptake assays with <italic>Xenopus</italic> oocytes in a high-potassium buffer [##REF##25252215##48##, ##UREF##17##50##]. In addition, the intronic variant rs1183201 in <italic>SLC17A1</italic> has been described as a variant significantly associated with gout [##REF##25636897##51##].</p>", "<p>This study examines six rare nonsynonymous variants in the <italic>SLC22A11</italic>, <italic>SLC22A13</italic>, and <italic>SLC17A1</italic> genes found in a cohort of 150 Czech patients of Caucasian origin with primary hyperuricemia and gout. This study finished the complex analysis concerning the identification of genetic variants, their influence, and mutual interactions of individual variants of urate transportosome on serum UA concentrations encompassing the detailed characterized cohort suffered from the primary hyperuricemia/gout.</p>" ]
[ "<title>2. Material and Methods</title>", "<title>2.1. Subject</title>", "<p>The primary cohort consisted of 36 patients with primary hyperuricemia (21 males/15 females) and 114 individuals with primary gout (100 males/14 females). We also analyzed a cohort of 34 pediatric patients with hyperuricemia and gout, 10 of whom were included in the primary cohort. We previously described this cohort in our publication focusing on pediatric patients [##REF##30606217##52##]. Primary hyperuricemia was defined as serum uric acid levels (SUA) &gt; 420 <italic>μ</italic>mol/L (7.0 mg/dL) in men and &gt;360 <italic>μ</italic>mol/L (6.0 mg/dL) in women and children under 15 years. Primary hyperuricemia was confirmed with a second measurement at least 4 weeks after the first. Gouty arthritis was diagnosed according to the criteria of the American College of Rheumatology [##REF##856219##53##]. The control group consisted of 115 normouricemic subjects. All patient samples were collected from the Biobank of the Institute of Rheumatology in Prague. Patients with secondary gout and other disorders of purine metabolism manifesting as pathological SUA concentrations were excluded. All tests were conducted according to standards set by the institutional ethics committee. All procedures were performed in accordance with the Declaration of Helsinki.</p>", "<title>2.2. Material</title>", "<p>Plasmid DNA with cloned genes, turboGFP mouse monoclonal antibody (cat. TA150041, clone OTI2H8) was purchased from OriGene Technologies, Inc, USA. The following vectors were used: <italic>SLC17A1</italic> (cat. RG211006), <italic>SLC22A11</italic> (cat. RG206469), and <italic>SLC22A13</italic> (cat.RG222116) cloned in pCMV6-AC-GFP with C-terminal turbo GFP tag. The HEK293A cell line was purchased from ThermoFisher Scientific, USA (cat. R70507). DMEM, fetal bovine serum (FBS), and other cultivation media and supplements were also from the ThermoFisher Scientific. The secondary antibody conjugated with HRP (cat.A90-117P) was purchased from Bethyl-Fortis Life Science, USA. The <italic>β</italic>-actin primary antibody (clone 8H10D10) was purchased from Cell Signaling, USA. Cultivation plastic for cells was purchased from VWR. Radiolabeled uric acid (MC-1394) was purchased from the Hartman Analytic GmbH, Germany. Western blot material and all common chemicals were from Merck KGaA, Germany, or Penta s.r.o, Czech Republic. Primers for sequencing and mutagenesis were synthesized by Generi biotech, Czech Republic. For measurement of radioactivity, we used liquid scintillant Ultima Gold and a TriCarb 2900TR scintillation counter (USA).</p>", "<title>2.3. PCR Amplification and Sequencing Analysis</title>", "<p>Genomic DNA was isolated from EDTA-anticoagulated whole blood using GeneAll Exgene Blood SV mini kits (GeneAll, South Korea). All coding exons and exon/intron borders of the <italic>SLC22A11</italic>, <italic>SLC22A13</italic>, and <italic>SLC17A1</italic> genes were PCR amplified. The PCR products were confirmed using gel electrophoresis in 2% agarose gel and purified using PCR Cleanup Kits (Geneaid, Taiwan). Subsequent Sanger sequencing was performed using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA) and CentriPure Dye Terminator purification 96-well plates (Gennaxon bioscience, Germany). DNA sequencing was carried out using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA). The resulting sequences were evaluated using SeqMan Pro software and DNASTAR Lasergene (DNASTAR, Inc., USA).</p>", "<title>2.4. Site-Directed Mutagenesis and Plasmid Preparation</title>", "<p>First, we performed site-directed mutagenesis with a GENEART Site-directed mutagenesis system (Invitrogen). We used the standard protocol recommended in the kit manual. We used the primers listed in Table <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>. The plasmids were multiplied in DH5<italic>α</italic> bacteria. For the preparation of pure plasmids, we used mini and maxi kits provided by Qiagen, Germany. Before transfection, each plasmid was sequenced to evaluate the correct sequences.</p>", "<title>2.5. Cell Maintenance and Transfection</title>", "<p>HEK293-A cells were maintained according to the usual protocol. The cells were cultivated in Dulbecco Minimal Eagle Medium (Gibco, 11965092), supplemented with 10% FBS (Gibco, 10270106), 1.0 mM pyruvate, 1.0% nonessential amino acids solution, and gentamycin 0.04 mg/mL. We passaged the cells with a 1% trypsin-EDTA solution when the cells grew to ca. 90% confluency. After thawing the aliquot, which had been stored in liquid nitrogen, we allowed the cells to grow for two passages and then used them in uptake assays.</p>", "<title>2.6. Microscopy</title>", "<p>We prepared cover glasses with 0.01% poly-L-lysine (Merck, Germany) in 12-well dishes. After this step, we seeded the cells with 0.05× 10<sup>6</sup> cells per well. We transfected the cells at ca. 20% confluency, and after 24 hr, we fixed the cells with 4% paraformaldehyde for 10 min (after longer fixation, the tGFP signal was quenched, data not shown). We mounted the cover glasses with cells in Mowiol with DAPI 0.1 <italic>µ</italic>g/mL. The cells were observed using a Leica DM-6 fluorescence microscope.</p>", "<title>2.7. Immunoblotting</title>", "<p>We performed western blotting to detect the transfection efficiency of the different variants of each transporter with same method as in our previous paper [##UREF##10##28##]. We prepared a transiently transfected cell monolayer, as shown above. We collected the cells with a scraper into phosphate-buffered saline (PBS, pH = 7.4) and centrifuged them at 150x <italic>g</italic> for 5 min. After this step, we lysed the cells with a 10% SDS and 9% protease inhibitory cocktail (Merck, cat I3786). We resuspended cells in lysis solution immediately, and then we sonicated the samples with a UP50H (Hielscher) ultrasound homogenizer on ice (25 impulses per sample at 60% amplitude). Afterward, we mixed the samples with 2x Laemmli loading buffer (Merck, cat. S34701) and denatured them at 56°C for 60 min. The samples were loaded on an SDS-PAGE acrylamide gel (5% focusing and 10% separating gel). After separation and blotting on a PVDF membrane, we blocked the membrane with 5% nonfat milk for 1.5 hr and then incubated it with the primary antibody overnight at 4°C. We used the anti-tGFP antibody 1 : 1,000 and anti-CapZ antibody 1 : 1,000. Then, we incubated it with the secondary, HRP conjugated antibody 1 : 7,000 and detected the signal from the SuperSignal West Pico Plus Chemiluminescence substrate (Thermo Scientific, USA). The chemiluminogram was taken using a BioRad Chemidoc touch imaging system (Biorad, USA).</p>", "<title>2.8. Uptake Assay</title>", "<p>Cells from the storage bottle were passaged with 1% trypsin-EDTA solution, dissociated cells were added to fresh medium, and this suspension was centrifuged at 150x <italic>g</italic> for 5 min. After this step, cell pellets were diluted with fresh medium, and cells were plated in 12-well dishes at a concentration of 0.2 × 10<sup>6</sup> cells per well. The cells were further cultivated for 48 hr and transfected with the polyethyleneimine (PEI) method after reaching about 75% confluency. For each 1.0 <italic>µ</italic>g of DNA, we used 2.6 <italic>µ</italic>g of PEI. The cells were further cultivated for 48 hr, followed by uptake assays. We used cell cultivation dishes covered with 0.01% poly-L-lysine (Merck, Germany). The uptake assay was performed according to our previous study and other published work [##UREF##10##28##, ##REF##14749323##54##, ##REF##18818201##55##]. First, we flushed out the cultivation medium and washed the monolayer twice with Hankʼs balanced salt solution (HBSS, NaCl 138.0 mM, KCl 5.0 mM; CaCl<sub>2</sub> 1.0 mM, MgCl<sub>2</sub> 0.5 mM, MgSO<sub>4</sub> 0.4 mM; KH<sub>2</sub>PO<sub>4</sub> 0.4 mM; NaHCO<sub>3</sub> 4.0 mM; Na<sub>2</sub>HPO<sub>4</sub> 0.3 mM; glucose 5.6 mM, pH = 7.4). After this step, cells were preincubated with HBSS for 15 min at 37°C. Subsequently, the HBSS was removed, and 30 <italic>µ</italic>M 14C radiolabeled uric acid dissolved in HBSS was added to three wells from each variant and cultivated at 37°C. We used different cultivation times for the isotopes—5 min for SLC22A13, 20 min for SLC22A11, and 30 min for SLC17A1. The fourth well from each variant was used for measuring protein concentrations and was treated the same way but not with radiolabeled uric acid. After incubation, the radioactive HBSS was removed, and the washed cell monolayer was kept on ice and rinsed three times with ice-cold HBSS. After washing the cells, they were lysed in 0.15 M NaOH for 2 hr with mild shaking; the cultivation dishes were kept on ice. The cells transfected with <italic>SLC17A1</italic> were treated the same, but we used depolarizing HBSS (HBSS 142 mM potassium gluconate; 5.0 mM sodium gluconate; 1.8 mM CaCl<sub>2</sub>; 1.0 mM MgCl<sub>2</sub>; 5.0 mM HEPES; pH = 7.4) instead of HBSS. After completion of lysis, the lysate was collected into scintillation vials, neutralized with HCl, and Ultima Gold liquid scintillant was added. After measuring isotope activity with a TriCarb 2900 TR (1 min of premeasuring and 5 min of measuring), protein samples were stored at −80°C. For further measurement of these protein samples, we used Bradford assay with a Biorad Start Bradford dye reagent (Biorad, USA).</p>", "<title>2.9. Evolutionary Alignment</title>", "<p>We used an Uniprot alignment tool. In this application, we used protein sequences obtained from an Uniprot database [##REF##33237286##56##]. In Figure <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>, we marked evolutionary fully conserved amino acid residues with gray color and similar amino acid residues with black rectangles without fill.</p>", "<title>2.10. Statistical Analysis</title>", "<p>The scintillation counter provided data in counts per minute, which we converted to disintegrations per minute (DPM) with a quenching correction. Uric acid uptake was in pmol per mg of protein per minute. Finally, we expressed the uptake as a percentage of the wild-type protein variant since this form is better for understanding, and the results were not affected by the current condition of the cells. We repeated the uptake assay and protein concentration measurements three times independently; the cells were from different frozen aliquots. In each repetition, we have three wells for uptake measurement (each with 2.0 × 10<sup>6</sup> cells) for each group, i.e., control, wild type, and allelic variants. Then, we obtained three values of CPM for each of this variant. From all these measurements, we calculated the mean and the standard deviation. We used the Studentʼs <italic>t</italic>-test for two samples with different variances for statistical analysis. We plotted the data into graphs, where the statistical significance expressed as <italic>p</italic>-values are marked by asterisks ( <sup><italic>∗</italic></sup> for <italic>p</italic> ≤ 0.05 and  <sup><italic>∗∗</italic></sup> for <italic>p</italic> ≤ 0.01). The statistical analysis and graph plotting was performed in Microsoft Excel 2019 MSO (Microsoft, USA).</p>" ]
[ "<title>3. Results</title>", "<title>3.1. Subjects</title>", "<p>The cohort used for this study was identical to our previous study. The main demographic and biochemical parameters of the patients are summarized in ##TAB##0##Table 1## in our previous paper [##UREF##10##28##]. The cohort consisted of 150 patients—114 with gout (100 men and 14 women) and 36 with primary hyperuricemia (21 men and 15 women). The familial occurrence was 30.7% in gout and 19.4% in hyperuricemia. The median age/age of onset was 59/45 years for gout patients and 55.5/48 years for hyperuricemia patients. The median of serum uric acid values and fractional excretion of uric acid at the time of examination was 371 <italic>µ</italic>mol/L and 3.4 for gout patients and 411 <italic>µ</italic>mol/L, and 3.5 for hyperuricemia patients.</p>", "<title>3.2. Sequencing Analysis</title>", "<p>In the <italic>SLC22A11</italic> gene (##TAB##1##Table 2##), we found missense variants p.V202M (rs201209258), p.R343L (rs75933978), and p.P519L (rs144573306). Furthermore, in this gene, we detected two rare synonymous variants p.T110T (rs774860411) and p.L496L (rs753269187). In analyzed intron regions, we discovered c.497 + 85A &gt; G (rs2277312), c.1058 + 53A &gt; C (rs71456318), and c.1589 + 54T &gt; A (rs185640375). In the <italic>SLC22A13</italic> gene (##TAB##1##Table 2##), we also identified two missense variants, p.R16H (rs72542450) and p.R102H (rs113229654), and further two rare synonymous variants, p.A53A (rs9842091) and p.P186P (rs146083340). Besides these variants, we described several variants in analyzed intron regions c.1022 + 31C &gt; G (rs41285121), c.1346 + 86A &gt; G, c.1346 + 107G &gt; A (rs551131182), c.1346 + 139C &gt; T (rs2236631), c.1346 + 164G &gt; A (rs1456539831), and c.1346 + 208C &gt; T (rs181912533). In the <italic>SLC17A1</italic> gene (##TAB##1##Table 2##), we discovered one rare nonsynonymous variant, p.W75C (rs149708935), with minor allele frequency (MAF) &lt;0.1% in Europeans, and one common nonsynonymous variant, p.T269I (rs1165196), with MAF 56.5% in Europeans. In addition, we detected seven variants in analyzed intron regions c.207 + 115C &gt; T (rs115398536), c.207 + 40C &gt; T (rs373732735), c.208− 14C &gt; T (rs200114666), c.898− 71T &gt; C (rs10498730), c.1269 + 61A &gt; G (rs1165210), c.1179 − 111C &gt; T (rs1165209), and c. <sup><italic>∗</italic></sup> 2 + 79C &gt; T (rs1165215). We used some <italic>in silico</italic> model (SIFT, PolyPhen, CADD, REVEL, MetaLR, and MutationAssesor) for predicting impact of examined allelic variants [##REF##36318249##57##]. We obtained deleterious or damaging in case of p.P519L (OAT4), p.W75C (NPT1), and p.V202M (OAT4). Surprisingly for p.R16H, we did not obtain any damaging prediction (Table <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S2</xref>).</p>", "<title>3.3. Functional Study</title>", "<title>3.3.1. <italic>SLC22A11</italic> (OAT4)</title>", "<p>We identified three allelic variants of the OAT4 transporter—p.V202M, p.R343L, and p.P519L (##TAB##1##Table 2##). We performed an uptake assay with 30 <italic>µ</italic>M 14C labeled uric acid with 20 min of incubation at 37°C in HBSS buffer (##FIG##1##Figure 2(d)##). As a baseline, we used uric acid uptake by the OAT4 wild-type protein (100%). The uptake of urate by the negative control (MOCK, cells transfected only with water instead of the plasmid with the transporter gene) was 39.4% of the wild type. We think this uptake was caused by endogenously expressed transporters and a channel in the HEK293A line. Substitution with p.P519L in our uptake assay decreased urate uptake to 69.4% of the wild-type uptake. This decrease in uptake was significant, with a <italic>p</italic>-value &lt;5%. Substitution with p.V202M produced a nonsignificant increase to 108.1% of the wild type. Because the standard deviation of the value of this transporter is relatively high, we suspect that the transport activity of the transporter with this variant is very similar to the wild type. Finally, p.R343L nonsignificantly increased the uptake to 105.5% of the wild type. As with the p.V202M variant, the standard deviation of this value implies that the uptake is probably similar to the wild type. Further, we analyzed the membrane localization using microscopy (##FIG##1##Figure 2(a)##), and based on this we suspect that all the variants of this transporter have a cell membrane localization. We also analyzed protein expression using western blot (##FIG##1##Figure 2(b)##). In MOCK, we did not obtain any band for the tGFP tag. In the wild type and p.P519L, we obtained a band with a similar density to p.V202M and p.R343L, which had a stronger band than the wild type and p.P519L. Both variants (p.V202M and p.R343L) had a concurrently denser band of loading control (<italic>β</italic>-actin) than the other two variants (wild type and p.P519L). This result suggests that all transporter variants are expressed at similar levels. The p.R343 is evolutionarily conserved among the representative models in higher primates (<italic>Homo sapiens</italic> and <italic>Pan troglodytes</italic>), ruminants (<italic>Bos taurus</italic>), artiodactyla (<italic>Sus scrofa</italic>), and birds (<italic>Gallus gallus</italic>). Similarly, the p.F519 is conserved in all mammalian models, and birds but not in the ruminantia, where it is substituted with leucine. The p.V202 is conserved only in higher primate models, but in ruminantia, artiodactyla, and bird models, it is substituted with leucine (Figure <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>).</p>", "<title>3.3.2. <italic>SLC22A13</italic> (OAT10)</title>", "<p>In OAT10, we identified two allelic variants—p.R16H and p.R102H (##TAB##1##Table 2##). We performed the uptake assay using the same conditions as the OAT4 transporter and its variants but with different incubation times—5 min with the uric acid isotope (##FIG##2##Figure 3(d)##). Similarly, we chose wild-type uptake of uric acid as the baseline. The uptake of urate by the negative control (MOCK) was 44.3% of the uptake by the OAT10 wild type. The uptake by p.R16H significantly decreased to 81.8% of the wild type with a <italic>p</italic>-value &lt;5%. Finally, p.R102H nonsignificantly decreased the uptake to 98.8% of the wild type, but based on the standard deviation, we suspect that transport activity was not affected. We performed fluorescent microscopy of protein localization on the cell membrane and concluded that all variants are localized on the cell membrane (##FIG##2##Figure 3(a)##). Similarly, as in the previous transporter, we examined the protein expression of the transporter using western blot (##FIG##2##Figure 3(b)##). In MOCK, we did not detect any tGFP signal (only loading control <italic>β</italic>-actin). We observed a similar band density in the wild type and p.R16H. In p.R102H, we observed a lower density, which corresponded to the density of the loading control (<italic>β</italic>-actin). We concluded that all variants were expressed in cells at similar levels. Subsequently, we performed an evolutionary analysis of the protein sequence of the transporter (Figure <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>). Arginine 16 is conserved in all mammalian model species but not in the model representative of birds—chicken (<italic>Gallus gallus</italic>); in this species we found p.R16P and p.R102G substitutions. Surprisingly, we identify R102H substitution in rhesus macaques (<italic>Macaca mulatta</italic>).</p>", "<title>3.3.3. <italic>SLC17A1</italic> (NPT1)</title>", "<p>Finally, we performed an uptake assay of the allelic variants in the NPT1 transporter (##TAB##1##Table 2##). We incubated the cells with 30 <italic>µ</italic>M 14C labeled uric acid for 30 min at 37°C in depolarizing HBSS buffer (##FIG##3##Figure 4(d)##). We chose urate uptake in the wild type (100%) as a baseline. Uptake by the negative control (MOCK) was 68.9% of the wild type, and as in the other two cases, we suspect that it is caused by endogenous urate transporters or passive flow across the cell membrane. We identified only one allelic variant, p.W75C in this transporter. We found that uric acid transport by NPT1 with this substitution was significantly decreased to 63.9% of the wild type with a <italic>p</italic>-value &lt;1%. This value is similar to an uptake value of the negative control (MOCK). Subsequently, we performed a fluorescent microscopy analysis of protein localization (##FIG##3##Figure 4(a)##). We observed mainly a cytoplasmic membrane localization in the wild type and p.W75C variant. Western blot analysis of expression (##FIG##3##Figure 4(b)##) shows that the wild type and p.W75C are expressed in a similar amount of protein, and in the MOCK, no signal was observed. Finally, we examined the evolutionary conservation of p.W75, and we found that tryptophan at this position is conserved among model species of primates, ruminants, artiodactyla, and birds (Figure <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>).</p>" ]
[ "<title>4. Discussion</title>", "<p>Many experimental or clinical works have been published which identify allelic variants in urate transporters in kidney. Most important (and simultaneously well researched) seem to be polymorphisms in transporters ABCG2 [##REF##20421215##14##, ##UREF##6##16##, ##UREF##7##17##], GLUT9 [##REF##18327257##19##–##UREF##8##21##], and URAT1[##REF##16837472##25##–##UREF##9##27##].Therefore in our previous study, we examined novelty SNP in <italic>SLC22A6</italic> (OAT1) and <italic>SLC22A8</italic> (OAT3) transporters [##UREF##10##28##]. Now, we focused on new variant identification in not too much researched transporters OAT4, OAT10, and NPT1. We found allelic variant p.P519L in Patient 1 (##TAB##2##Table 3##), significantly decreased urate transport capacity (##FIG##1##Figure 2(d)##). This patient was homozygous at ABCG2 (p.Q141K) and SLC2A9 (p.P350L). The p.Q141K variant in the ABCG2 transporter significantly increases the risk of gout[##UREF##6##16##, ##REF##24857923##59##–##REF##28968913##61##]. It was shown that this substitution significantly decreases urate efflux from cells[##UREF##5##15##]. We suspect that decreased urate excretion by ABCG2 p.Q141K in Patient 1 (##TAB##2##Table 3##) may cause gout despite reuptake transporter OAT4 with p.P519L being decreased. The homozygous mutation p.P350L in GLUT9 (<italic>SLC2A9</italic>) probably has little or no effect on urate uptake or efflux, respectively [##UREF##12##31##]. We found that the p.V202M substitution in OAT4 had no effect in our <italic>in vitro</italic> uptake assay. This variant was present in Patient 2 (##TAB##2##Table 3##) with hyperuricemia; they were homozygous for p.G25R in GLUT9a (long isoform, ##TAB##2##Table 3##). This GLUT9 variant did not significantly decrease urate transport, nor did it significantly affect gout risk [##UREF##12##31##, ##UREF##18##62##]. These facts may explain why the p.V202M variant caused only hyperuricemia and not gout. A similar situation appeared with a p.R343L variant in OAT4, which did not affect transport in our uptake assay. Patient 3 with this variant was homozygous for p.Q141K in the ABCG2, which decreased urate efflux [##UREF##5##15##]. Two patients with the p.R16H variant in OAT10 have gout; reuptake by this transporter was significantly decreased. It is possible that the main reuptake transporter URAT1 is upregulated when the other reuptake transporters OAT4 or OAT10 have allelic variants, which decrease transport activity such as p.P519L (OAT4) or p.R16H (OAT10). This hypothesis is supported by the decreased FEUA (##TAB##1##Table 2##) in patients with mutations that significantly decreased transport (Patients 1, 4, and 5; ##TAB##2##Table 3##). We suggest that the scaffold protein PDZK1 may play a regulatory role. PDZK1 interacts with URAT1 at its C-terminus and affects urate transport [##REF##15304510##63##]. The same scaffold protein interacts with the OAT4 transporter and modulates its activity similarly [##REF##16236806##64##]. Direct evidence of an interaction between PDZK1 and OAT10 is currently unavailable. We found the PDZK binding motif at the C-terminus of NPT1 transporter (peptide position 465-TRL-467), but we have no experimental evidence, that this motif really have role in regulation by PDZK1 protein (Figure <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>). However, we suspect this interaction is possible because the sequence homology between OAT10 and OAT4 is 32.4% (35.1% for URAT1; data not shown). Allelic variant p.R102H had a negligible effect on urate transport by OAT10, so we suspect that the hyperuricemia in Patient 6 was caused predominantly by heterozygous mutation p.Q141K in ABCG2. Substitution of p.W75C in NPT1 significantly decreased urate transport in our study (##FIG##3##Figure 4(d)##). It is not surprising that Patient 7 with this allelic variant had higher SUA and hyperuricemia but did not have gout because ABCG2 probably compensated for the efflux of urate. This is supported by a mild decrease in FEUA levels (##TAB##1##Table 2##). The variant p.I269T in <italic>SLC17A1</italic> increased urate transport and reduced the risk of gout, which shows the effect of this transporter on SUA [##REF##25252215##48##]. Other possible explanation is influence of intestinal uric acid transporters such as <italic>ABCC4</italic> or <italic>SLC17A4</italic> [##REF##22460716##65##, ##REF##15210835##66##]. Patient 7 is homozygous for three allelic variant in this transporter, which are expressed in intestinal epithelial cells. Similarly, intestinal urate transporter <italic>SLC17A4</italic> can affect SUA. We speculate that Patient 7 should have some mutation in intestinal transporter (<italic>SLC17A4</italic> or other), which caused hyperuricemia without change of FEUA.</p>", "<p>We next discuss the impact of our described allelic variant on the molecular function of the transporters. We found that substitution of p.P519L significantly decreased urate uptake into HEK293A cells that transiently expressed OAT4 with this variant. The expression and localization of the protein were not changed. P519 is evolutionarily conserved in apes and pigs. We speculate that this mutation, which caused the insertion of leucine (instead of proline) between p.L518 and p.L520, causes a conformation change. Deleting the whole C-terminus of the OAT1 transporter (OAT1 is 42.1% identical with OAT4, data not shown) completely abolishes para-aminohippurate transport. Substitution of p.L512 in OAT1 leads to loss of its function (this leucine is analogical to L520 in OAT4) [##REF##16920720##67##]. Therefore, it is possible that p.P519 can have a similarly important role in human OAT4.</p>", "<p>We did not observe any change in protein localization and processing, so we suspect that P519 could function in attaining the correct conformation. We suspect that the function of the C-terminal region of OAT4 will need to be described in more detail. Substitution of p.V202M did not affect our transport assay. In rats, the fourth transmembrane helix in the OCT1 (organic cation transporter <italic>SLC22A1</italic>, 30.5% identity) transporter was shown to be necessary for substrate transport [##REF##15662044##68##]. However, it seems that a valine by methionine (amino acids with similar chemical properties) substitution did not affect the conformation of the helix or the interaction with urate as a substrate.</p>", "<p>Similarly, substituting a positively charged p.R343 with a nonpolar leucine did not affect urate transport. The amino acid in this position is located at the carboxy end of a cytoplasmic loop. It seems that p.R343 is not critical for urate handling. In homologous OAT1, a substitution of p.R293W a cytoplasmic loop did not affect para-aminohippurate transport [##REF##15864112##69##]. In OAT10, we identified that the substitution of p.R16H affected uric acid transport (81.8% of wild-type level). R16 is located in the N-terminus of OAT10 (##FIG##2##Figure 3(c)##) and is conserved in mammal orthologues (data not shown). In human OAT1, the substitution of p.F16Q or p.Q17A decreases the transport of para-aminohyppurate [##REF##15145940##70##]. p.F16 and p.Q17 are conserved in OAT1 and OAT10 (data not shown). The substitution of p.V13M in human OAT1 does not affect estrone sulfate transport [##REF##20015291##71##]. We suspect R16 in OAT10 affects urate binding at the N-terminus. Substitution of p.R102H had an insignificant effect on urate transport. Substitution of amino acids with similar properties also did not affect urate transport. R102 is located in an extracellular loop. Mutation p.P104L in the same loop in OAT1 did not affect para-aminohyppurate transport [##REF##15864112##69##]. Substitution of p.W75C completely abolished NPT1 transporter function. Tryptophan is conserved among ape, mammal, and bird models (Figure <xref rid=\"supplementary-material-1\" ref-type=\"sec\">S1</xref>). This substitution is located in an extracellular loop of the protein. In the Glut 9 transporter, W110 is crucial for urate transport [##REF##25922070##72##]. We hypothesize that p.W75 in NPT1 may be an analog playing the same role in urate transport. We detected five allelic variants in <italic>SLC22A12</italic> (URAT1) transporter, but all these variants are synonymous in protein sequence, so we supposed that these variants are no protective effect for hyperuricemia or gout (##TAB##2##Table 3##). Further detailed studies concerning urate transporters and their interactions could clarify genetic and molecular background of renal urate transport. In future work, it will be beneficial to study a more comprehensive <italic>in vitro</italic> model to study the interactions of transporters and their variants with other cellular proteins and regulatory elements specific to epithelial cells of the proximal tubule of the human kidney.</p>" ]
[ "<title>5. Conclusions</title>", "<p>In summary, our findings will deepen our understanding of urate transport-related gout/hyperuricemia risk and the biochemical characteristics of the OAT4, OAT10, and NPT1 transporters. Our identification and functional characterization of rare variants provide a better understanding of renal urate handling systems and support the “Common Disease, Multiple Common, and Rare variant” hypothesis [##REF##25005607##73##], which argues that genetic susceptibility to common diseases, such as gout, does not reside in common genetic variants but rather in a multiplicity of individual rare genetic variations each with relatively high penetrance.</p>" ]
[ "<p>Academic Editor: Nashwa El-Khazragy</p>", "<p>Genetic variations in urate transporters play a significant role in determining human urate levels and have been implicated in developing hyperuricemia or gout. Polymorphism in the key urate transporters, such as ABCG2, URAT1, or GLUT9 was well-documented in the literature. Therefore in this study, our objective was to determine the frequency and effect of rare nonsynonymous allelic variants of <italic>SLC22A11</italic>, <italic>SLC22A13</italic>, and <italic>SLC17A1</italic> on urate transport. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined all coding regions and exon–intron boundaries of <italic>SLC22A11</italic>, <italic>SLC22A13</italic>, and <italic>SLC17A1</italic> using PCR amplification and Sanger sequencing. For comparison, we used a control group consisting of 115 normouricemic subjects. To examine the effects of the rare allelic nonsynonymous variants on the expression, intracellular processing, and urate transporter protein function, we performed a functional characterization using the HEK293A cell line, immunoblotting, fluorescent microscopy, and site directed mutagenesis for preparing variants <italic>in vitro</italic>. Variants p.V202M (rs201209258), p.R343L (rs75933978), and p.P519L (rs144573306) were identified in the <italic>SLC22A11</italic> gene (OAT4 transporter); variants p.R16H (rs72542450), and p.R102H (rs113229654) in the <italic>SLC22A13</italic> gene (OAT10 transporter); and the p.W75C variant in the <italic>SLC17A1</italic> gene (NPT1 transporter). All variants minimally affected protein levels and cytoplasmic/plasma membrane localization. The functional <italic>in vitro</italic> assay revealed that contrary to the native proteins, variants p.P519L in OAT4 (<italic>p</italic> ≤ 0.05), p.R16H in OAT10 (<italic>p</italic> ≤ 0.05), and p.W75C in the NPT1 transporter (<italic>p</italic> ≤ 0.01) significantly limited urate transport activity. Our findings contribute to a better understanding of (1) the risk of urate transporter-related hyperuricemia/gout and (2) uric acid handling in the kidneys.</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>The authors are grateful to all the patients who kindly took part in this study, as well as our colleagues at the Institute of Rheumatology and Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and the General University Hospital in Prague, for their help in recruiting patients for the study. The authors would like to thank Vladimir Krylov (Department of Cell Biology, Faculty of Science, Charles University) for his council. The study was supported by grants from the Czech Republic Ministry of Health NU22-01-00465 and DRO (Institute of Rheumatology, 00023728 and VFN, 64165), the Ministry of Education Youth and Sport BBMRICZ LM2023033.</p>", "<title>Data Availability</title>", "<p>The data used to support the findings of this study are available from the corresponding author upon request.</p>", "<title>Additional Points</title>", "<p>\n<italic>Limitations</italic>. Although the size of the cohort is sufficient, rare, and extra-rare variants that impact the development of hyperuricemia/gout may still be missed. Our uptake assays were limited by the use of the HEK293A cell line, which was transiently transfected by study genes. In future studies, it would be better to use a cell line derived from renal epithelial cells. We have no model for study the interaction of uric acid transporter and its allelic variation. For examining the interaction we would need an epithelial cell line (such as primary renal proximal tubule epithelial cells, RPTEC or Madin–Darby canine kidney cell, MDCK) which will be transfected by the examined transporters with certain allelic variants and with a combination of different variant. It will be very difficult to prepare this experimental model, which will be very artificial and will be loaded by big experimental error. From similar reasons, it would be difficult and artificial to use some <italic>in vivo</italic> model, such as mouse or rat with inhibited uricase activity. The mutual interaction of transporters exceeds the main goal of our paper (bring the first view on new allelic variants and its clinical phenotype).</p>", "<title>Ethical Approval</title>", "<p>This study was approved by the Ethics Committee of the Institute of Rheumatology (project no. 6181/2015 and 6484/2020). All patients and healthy controls were fully informed of the aim of the study.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from all participants.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that they have no conflicts of interest.</p>", "<title>Authors' Contributions</title>", "<p>BS and JV contributed to the study conception and design of the study. LH and EB contributed to the clinical observation of the study. BS, KP, JM, JV, AV, and AM contributed to the acquisition of data of the study. KP and JM contributed to the sequencing analysis. JV, AV, and AM contributed to the functional characterization analysis. All authors were involved in drafting the manuscript or revising the content. All authors approved the final version for publication.</p>", "<title>Supplementary Materials</title>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Scheme of renal tubular epithelial cells. Transporters OAT4 and OAT10 provide reuptake of urate from the luminal side of proximal tubules, while NPT1 is responsible for urate excretion across the luminal membrane of tubules into the urine. Created with BioRender.com.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>(a) Expression of OAT4 (<italic>SLC22A11</italic>) and its allelic variants in the HEK293A cell line. Wild-type protein was tagged with a C-terminal tGFP tag, and cells were transiently transfected by polyethyleneimine lipofection. Samples were fixed with 4% paraformaldehyde, and pictures were taken with a Leica DM6 microscope at 400x magnification. Plasma membrane localization was observed in all variants. Cell nuclei were stained with DAPI (blue). (b) Western blot of OAT4 allelic variants. Expression of the protein was detected with tGFP antibody and anti-<italic>β</italic>-actin antibody as a loading control. (c) Predicted structure of OAT4 protein visualized in Protter [##REF##24162465##58##]. Allelic variants are marked with red dots. (d) Uptake study with 30 <italic>µ</italic>M 14C uric acid with 20-min incubation. The intracellular uptake of urate is expressed as a % of the wild-type uptake, <italic>n</italic> = 3,  <sup><italic>∗</italic></sup>means <italic>p</italic> ≤ 0.05 (Students <italic>t</italic>-test).</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>(a) Expression of OAT10 (<italic>SLC22A13</italic>) and its allelic variants in HEK293A cells line. Wild-type protein was tagged with a C-terminal tGFP tag, and cells were transiently transfected by polyethyleneimine lipofection. Samples were fixed with 4% paraformaldehyde, and pictures were taken with a Leica DM6 microscope at 400x magnification. Plasma membrane localization was observed in all variants. Cell nuclei were stained with DAPI (blue). (b) Western blot of OAT10 allelic variants. Expression of the protein was detected with tGFP antibody and anti-<italic>β</italic>-actin antibody as a loading control. (c) Predicted structure of OAT4 protein visualized in Protter [##REF##24162465##58##]. Allelic variants are marked with red dots. (d) Uptake study with 30 <italic>µ</italic>M 14C uric acid with 5-min incubation. The intracellular uptake of urate is expressed as a % of the wild-type uptake, <italic>n</italic> = 3,  <sup><italic>∗</italic></sup>means <italic>p</italic> ≤ 0.05 (Student's <italic>t</italic>-test).</p></caption></fig>", "<fig position=\"float\" id=\"fig4\"><label>Figure 4</label><caption><p>(a) Expression of NPT1 (<italic>SLC17A1</italic>) and its allelic variants in HEK293A cells line. Wild-type protein was tagged with a C-terminal tGFP tag, and cells were transiently transfected by polyethyleneimine lipofection. Samples were fixed with 4% paraformaldehyde, and pictures were taken with a confocal microscope at 400x magnification. Plasma membrane localization was observed in all variants. Cell nuclei were stained with DAPI (blue). (b) Western blot of NPT1 allelic variants. Expression of the protein was detected with tGFP antibody and anti-<italic>β</italic>-actin antibody as a loading control. (c) Predicted structure of NPT1 protein visualized in Protter [##REF##24162465##58##]. Allelic variants are marked with red dots. (d) Uptake study with 30 <italic>µ</italic>M 14C uric acid with 30-min incubation. The intracellular uptake of urate is expressed as a % of the wild-type uptake, <italic>n</italic> = 3,  <sup><italic>∗∗</italic></sup>means <italic>p</italic> ≤ 0.01 (Student's <italic>t</italic>-test).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>Human kidney proximal tubule transporter and its allelic variants, which was previously published in our papers.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Gene</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Kidney transporter</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">SNP ref.</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Amino acid substitution</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Membrane localization</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Urate uptake</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"12\" colspan=\"1\">\n<italic>ABCG2</italic>\n</td><td align=\"center\" rowspan=\"12\" colspan=\"1\">ABCG2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs2231137</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V12M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs2231142</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.Q141K</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs372192400</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R147W</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs753759474</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.T153M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.I242T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##30635032##29##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs750972998</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.K360del</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs752626614</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.F373C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs199854112</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.T421A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs769734146</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.T434M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.S476P</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs200894058</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.S572R</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs34783571</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.D620N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##7##17##]</td></tr><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">\n<italic>SLC22A12</italic>\n</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">URAT1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs144328876</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R92C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##26500098##18##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R203C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##26500098##18##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs150255373 <sup><italic>∗</italic></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.P325W</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##11##30##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC17A1</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NTP1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs149708935</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.W75C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"6\" colspan=\"1\">This paper</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<italic>SLC22A13</italic>\n</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">OAT10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs121908321</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R16H</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs113229654</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R102H</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">\n<italic>SLC22A11</italic>\n</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">OAT4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs201209258</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V202M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs75933978</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R343L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs144573306</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.P519L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<italic>SLC22A6</italic>\n</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">OAT1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs146282438</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.A190T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##10##28##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs11568627</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.P104L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##10##28##]</td></tr><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">\n<italic>SLC22A8</italic>\n</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">OAT3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs45566039</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R149C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##10##28##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs11568486</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V448F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##10##28##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs145474422</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R513Q</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##10##28##]</td></tr><tr><td align=\"left\" rowspan=\"12\" colspan=\"1\">\n<italic>SLC2A9</italic>\n</td><td align=\"center\" rowspan=\"12\" colspan=\"1\">GLUT9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs6820230</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p. A17T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs2276961</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.G25R</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.G72D</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##26500098##18##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.I118HfsX27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##26500098##18##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs144196049</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V169M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.G261R</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##26500098##18##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs112404957</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.T275M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs73225891</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.D281H</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs16890979</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V282I</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs3733591</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R294H</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.N333S</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##26500098##18##]</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs2280205</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.P350L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##12##31##]</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>Identified nonsynonymous <italic>SLC22A11</italic>, <italic>SLC22A12</italic>,and <italic>SLC17A1</italic> allelic variants, their mutant allele frequency (MAF) and overview of main biochemical parameters in the patient cohort.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Gene</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Reference SNP number</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Position CDS</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Position AA</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Variant allele hetero/homozygotes</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Allelic variant MAF</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Normo-uricemia control MAF</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">European MAF</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Gender (patient identification)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Diagnosis</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Familial occurrence</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Age at examination (years)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Gout/hyperuricemia onset (years)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">BMI at examination (−)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">SUA without medication (<italic>µ</italic>mol/L)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">SUA with medication (<italic>µ</italic>mol/L)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">FEUA without medication (%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">FEUA with medication (%)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A11</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs144573306</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">c.1556C &gt; T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.P519L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1/0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male (Patient 1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gout</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">655</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">300</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.64</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A11</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs201209258</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">c.604G &gt; A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V202M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1/0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.003</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male (Patient 2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hyperuricemia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A11</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs75933978</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">c.1028G &gt; T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R343L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1/0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.002</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male (Patient 3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hyperuricemia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">463</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.92</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<italic>SLC22A13</italic>\n</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">\n<italic>rs72542450</italic>\n</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">c.47G &gt; A</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">p.R16H</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">2/0</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">0.007</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">0.009</td><td align=\"center\" rowspan=\"2\" colspan=\"1\">0.008</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Female (Patient 4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gout</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">606</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male (Patient 5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gout</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">387</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">332</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.02</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A13</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs113229654</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">c.305G &gt; A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R102H</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1/0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.003</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.008</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male (Patient 6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hyperuricemia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">467</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">483</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.14</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC17A1</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">rs149708935</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">c.225G &gt; T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.W75C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1/0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male (Patient 7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hyperuricemia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">433</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>Current occurrence of allelic variants in different transporters in the patient cohort.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"3\" colspan=\"1\">Variants in other genes</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Gene</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>ABCG2</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC2A9</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC2A9</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC2A9</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC2A9</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC17A1</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC17A3</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC17A3</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A8</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>ABCC4</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>ABCC4</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>ABCC4</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>ABCC4</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A12</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A12</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A12</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A12</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SLC22A12</italic>\n</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Reference SNP number</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs2231142</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs2276961</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs16890979</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs3733591</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs2280205</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs1165196</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs1165165</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs56027330</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs45566039</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs11568658</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs2274406</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs1678339</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs1751034</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs3825017</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs3825016</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs11231825</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs1630320</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">rs7932775</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">AA change</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.Q141K</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.G25R</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.V282I</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.R294H</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.P350L</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.T269I</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.A100T</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.G279R</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.R149C</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.G187W</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.R317S</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.L904F</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.K1116N</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.N82 = </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.H86 = </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.H142 = </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.A416 = </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">p.L437 = </th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"7\" colspan=\"1\">Variants in <italic>SLC22A11</italic>, <italic>SLC22A13</italic>, and <italic>SLC17A1</italic></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.P519L (Patient 1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.V202M (Patient 2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R343L (Patient 3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R16H (Patient 4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HM</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R16H (Patient 5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.R102H (Patient 6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">p.W75C (Patient 7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">wt</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>wt</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HM</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>HT</bold>\n</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"supp-1\" position=\"float\" content-type=\"local-data\"><label>Supplementary Materials</label><caption><p>Table S1: sequences of primers used for site-directed mutagenesis. Table S2: prediction using different models for the allelic variants in the patient cohort. Figure S1: evolutionary comparison of variants in transporters.</p></caption></supplementary-material>" ]
[ "<table-wrap-foot><fn><p>Correct membrane localization is marked with the symbol “+”, impaired localization is marked with “-”, unexamined localization is marked with “?”. Transport capacity for urate without significant change is marked with “+”, changed capacity is marked with “−”. Unknown influence on localization or transport is marked with “?”.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Only in this, patients were detected new allelic variants. <italic>Abbreviations</italic>: BMI, body mass index; SUA, serum uric acid level (<italic>µ</italic>mol/L); FEUA, fraction excretion of uric acid (%).</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Only in this, seven patients were detected this new allelic variants, but some other allelic variants in other urate transport proteins, which was described in the previous works. Wt, wildtype; HM, homozygous; and HT, heterozygous.</p></fn></table-wrap-foot>" ]
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[ "<media xlink:href=\"5930566.f1.docx\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["3"], "person-group": ["\n"], "surname": ["Ames", "Cathcart", "Schwiers", "Hochstein"], "given-names": ["B. N.", "R.", "E.", "P."], "article-title": ["Uric acid provides an antioxidant defense in humans against oxidant-and radical-caused aging and cancer: a hypothesis"], "source": ["\n"], "italic": ["Proceedings of the National Academy of Sciences"], "year": ["1981"], "volume": ["78"], "issue": ["11"], "fpage": ["6858"], "lpage": ["6862"], "pub-id": ["10.1073/pnas.78.11.6858"]}, {"label": ["9"], "person-group": ["\n"], "surname": ["Kvasni\u010dka", "Friedeck\u00fd", "Brumarov\u00e1"], "given-names": ["A.", "D.", "R."], "article-title": ["Alterations in lipidome profiles distinguish early-onset hyperuricemia, gout, and the effect of urate-lowering treatment"], "source": ["\n"], "italic": ["Arthritis Research & Therapy"], "year": ["2023"], "volume": ["25"], "pub-id": ["10.1186/s13075-023-03204-6", "234"]}, {"label": ["10"], "person-group": ["\n"], "surname": ["Butler", "Alghubayshi", "Roman"], "given-names": ["F.", "A.", "Y."], "article-title": ["The epidemiology and genetics of hyperuricemia and gout across major racial groups: a literature review and population genetics secondary database analysis"], "source": ["\n"], "italic": ["Journal of Personalized Medicine"], "year": ["2021"], "volume": ["11"], "issue": ["3"], "fpage": ["1"], "lpage": ["15"], "pub-id": ["10.3390/jpm11030231"]}, {"label": ["11"], "person-group": ["\n"], "surname": ["Sun", "Wu", "Bian"], "given-names": ["H.-L.", "Y.-W.", "H.-G."], "article-title": ["Function of uric acid transporters and their inhibitors in hyperuricaemia"], "source": ["\n"], "italic": ["Frontiers in Pharmacology"], "year": ["2021"], "volume": ["12"], "pub-id": ["10.3389/fphar.2021.667753"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Dehghan", "K\u00f6ttgen", "Yang"], "given-names": ["A.", "A.", "Q."], "article-title": ["Association of three genetic loci with uric acid concentration and risk of gout: a genome-wide association study"], "source": ["\n"], "italic": ["The Lancet"], "year": ["2008"], "volume": ["372"], "issue": ["9654"], "fpage": ["1953"], "lpage": ["1961"], "pub-id": ["10.1016/S0140-6736(08)61343-4", "2-s2.0-57049083981"]}, {"label": ["15"], "person-group": ["\n"], "surname": ["Woodward", "K\u00f6ttgen", "Coresh", "Boerwinkle", "Guggino", "K\u00f6ttgen"], "given-names": ["O. M.", "A.", "J.", "E.", "W. B.", "M."], "article-title": ["Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout"], "source": ["\n"], "italic": ["Proceedings of the National Academy of Sciences"], "year": ["2009"], "volume": ["106"], "issue": ["25"], "fpage": ["10338"], "lpage": ["10342"], "pub-id": ["10.1073/pnas.0901249106", "2-s2.0-67649886415"]}, {"label": ["16"], "person-group": ["\n"], "surname": ["Matsuo", "Takada", "Ichida"], "given-names": ["H.", "T.", "K."], "article-title": ["Common defects of ABCG2, a high-capacity urate exporter, cause gout: a function-based genetic analysis in a Japanese population"], "source": ["\n"], "italic": ["Science Translational Medicine"], "year": ["2009"], "volume": ["1"], "issue": ["5"], "pub-id": ["10.1126/scitranslmed.3000237", "2-s2.0-77952845866"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Toyoda", "Man\u010d\u00edkov\u00e1", "Krylov"], "given-names": ["Y.", "A.", "V."], "article-title": ["Functional characterization of clinically-relevant rare variants in ABCG2 identified in a gout and hyperuricemia cohort"], "source": ["\n"], "italic": ["Cells"], "year": ["2019"], "volume": ["8"], "issue": ["4"], "pub-id": ["10.3390/cells8040363", "363"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Li", "Sanna", "Maschio"], "given-names": ["S.", "S.", "A."], "article-title": ["The GLUT9 gene is associated with serum uric acid levels in Sardinia and chianti cohorts"], "source": ["\n"], "italic": ["PLoS Genetics"], "year": ["2007"], "volume": ["3"], "issue": ["11"], "pub-id": ["10.1371/journal.pgen.0030194", "2-s2.0-37349008492", "e194"]}, {"label": ["27"], "person-group": ["\n"], "surname": ["Stiburkova", "Bohat\u00e1", "Pavelcov\u00e1"], "given-names": ["B.", "J.", "K."], "article-title": ["Renal hypouricemia 1: rare disorder as common disease in Eastern Slovakia roma population"], "source": ["\n"], "italic": ["Biomedicines"], "year": ["2021"], "volume": ["9"], "issue": ["11"], "fpage": ["1"], "lpage": ["10"], "pub-id": ["10.3390/biomedicines9111607"]}, {"label": ["28"], "person-group": ["\n"], "surname": ["V\u00e1vra", "Man\u010d\u00edkov\u00e1", "Pavelcov\u00e1", "Has\u00edkov\u00e1", "Bohat\u00e1", "Stib\u016frkov\u00e1"], "given-names": ["J.", "A.", "K.", "L.", "J.", "B."], "article-title": ["Functional characterization of rare variants in OAT1/"], "italic": ["SLC22A6", "SLC22A8", "Cells"], "source": ["\n"], "year": ["2022"], "volume": ["11"], "issue": ["7"], "fpage": ["1"], "lpage": ["19"], "pub-id": ["10.3390/cells11071063"]}, {"label": ["30"], "person-group": ["\n"], "surname": ["Stiburkova", "Bohata", "Minarikova"], "given-names": ["B.", "J.", "I."], "article-title": ["Clinical and functional characterization of a novel URAT1 dysfunctional variant in a pediatric patient with renal hypouricemia"], "source": ["\n"], "italic": ["Applied Sciences"], "year": ["2019"], "volume": ["9"], "issue": ["17"], "fpage": ["10"], "lpage": ["17"], "pub-id": ["10.3390/app9173479", "2-s2.0-85072245054", "3479"]}, {"label": ["31"], "person-group": ["\n"], "surname": ["Hurba", "Mancikova", "Krylov"], "given-names": ["O.", "A.", "V."], "article-title": ["Complex analysis of urate transporters "], "italic": ["SLC2A9", "SLC22A12", "PLoS ONE"], "source": ["\n"], "year": ["2014"], "volume": ["9"], "issue": ["9"], "pub-id": ["10.1371/journal.pone.0107902", "2-s2.0-84907487137", "e107902"]}, {"label": ["36"], "person-group": ["\n"], "surname": ["Flynn", "Phipps-Green", "Hollis-Moffatt"], "given-names": ["T. J.", "A.", "J. E."], "article-title": ["Association analysis of the "], "italic": ["SLC22A11", "SLC22A12", "Arthritis Research & Therapy"], "source": ["\n"], "year": ["2013"], "volume": ["15"], "issue": ["6"], "pub-id": ["10.1186/ar4417", "2-s2.0-84890764128"]}, {"label": ["41"], "person-group": ["\n"], "surname": ["Nishiwaki", "Daigo", "Tamari", "Fujii", "Nakamura"], "given-names": ["T.", "Y.", "M.", "Y.", "Y."], "article-title": ["Molecular cloning, mapping, and characterization of two novel human genes, ORCTL3 and ORCTL4, bearing homology to organic-cation transporters"], "source": ["\n"], "italic": ["Cytogenetic and Genome Research"], "year": ["1998"], "volume": ["83"], "issue": ["3-4"], "fpage": ["251"], "lpage": ["255"], "pub-id": ["10.1159/000015197"]}, {"label": ["43"], "person-group": ["\n"], "surname": ["Toyoda", "Kawamura", "Nakayama"], "given-names": ["Y.", "Y.", "A."], "article-title": ["OAT10/SLC22A13 acts as a renal urate re-absorber: clinico-genetic and functional analyses with pharmacological impacts"], "source": ["\n"], "italic": ["Frontiers in Pharmacology"], "year": ["2022"], "volume": ["13"], "fpage": ["1"], "lpage": ["13"], "pub-id": ["10.3389/fphar.2022.842717"]}, {"label": ["44"], "person-group": ["\n"], "surname": ["Shinoda", "Yamashiro", "Hosooka", "Yasujima", "Yuasa"], "given-names": ["Y.", "T.", "A.", "T.", "H."], "article-title": ["Functional characterization of human organic anion transporter 10 (OAT10/SLC22A13) as an orotate transporter"], "source": ["\n"], "italic": ["Drug Metabolism and Pharmacokinetics"], "year": ["2022"], "volume": ["43"], "pub-id": ["10.1016/j.dmpk.2021.100443", "100443"]}, {"label": ["50"], "person-group": ["\n"], "surname": ["Sakiyama", "Matsuo", "Nagamori"], "given-names": ["M.", "H.", "S."], "article-title": ["Expression of a human NPT1/SLC17A1 missense variant which increases urate export"], "source": ["\n"], "italic": ["Nucleosides, Nucleotides & Nucleic Acids"], "year": ["2016"], "volume": ["35"], "issue": ["10\u201312"], "fpage": ["536"], "lpage": ["542"], "pub-id": ["10.1080/15257770.2016.1149192", "2-s2.0-85000428181"]}, {"label": ["62"], "person-group": ["\n"], "surname": ["Pavelcova", "Bohata", "Pavlikova", "Bubenikova", "Pavelka", "Stiburkova"], "given-names": ["K.", "J.", "M.", "E.", "K.", "B."], "article-title": ["Evaluation of the influence of genetic variants of "], "italic": ["SLC2A9", "SLC22A12", "Journal of Clinical Medicine"], "source": ["\n"], "year": ["2020"], "volume": ["9"], "issue": ["8"], "fpage": ["1"], "lpage": ["21"], "pub-id": ["10.3390/jcm9082510", "2510"]}]
{ "acronym": [ "FEUA:", "HBSS:", "HEK293-A:", "NPT:", "OAT:", "PEI:", "SLC:", "SNP:", "SUA:", "RPTEC:", "MDCK:" ], "definition": [ "Fraction excretion of uric acid", "Hankʼs balanced salt solution", "Human embryonic kidney cell line", "Sodium-dependent phosphate transport protein", "Organic anion transporter", "Polyethyleneimine", "Solute carrier", "Single nucleotide polymorphism", "Serum uric acid level", "Primary renal proximal tubule epithelial cells", "Madin–Darby canine kidney cells." ] }
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Dis Markers. 2024 Jan 6; 2024:5930566
oa_package/ba/7f/PMC10787658.tar.gz
PMC10787659
0
[ "<title>1. Introduction</title>", "<p>Mild cognitive impairment (MCI) is characterized by a subtle decline in cognitive abilities that surpasses normal age-related changes but does not significantly impact daily functioning. It serves as an intermediary stage between typical cognitive aging and dementia. Although individuals with MCI may experience difficulties in memory, attention, language, executive functions, or visuospatial skills, their impairment is not severe enough to warrant classification as dementia [##REF##33440197##1##]. It is noteworthy that not all cases of MCI progress to dementia, some may stabilize or even revert to normal cognitive function over time. However, individuals with MCI are at a higher risk of developing dementia compared to those without it [##REF##35964184##2##]. Various factors contribute to the development of MCI, including vascular issues, depression, anxiety, and sleep disorders [##REF##32289790##3##, ##REF##34939171##4##].</p>", "<p>Quality sleep is essential for optimal cognitive health, as it supports attention, problem-solving abilities, creativity, and decision-making processes. During sleep, the brain undergoes crucial processes such as waste elimination, cellular repair, and energy replenishment that enable it to function at its best [##REF##33829232##5##]. Dyssomnias, such as chronic sleep deprivation, fragmented sleep, and excessive daytime sleepiness, can significantly impact the quality of life and impair cognitive functions like memory and attention. Research suggests that sleep disorders not only exacerbate cognitive and affective symptoms associated with neurodegeneration but also contribute to disease progression [##REF##33179197##6##]. Dyssomnias have been identified as a significant risk factor for MCI, with a prevalence of approximately 70.1% in individuals with MCI compared to around 56.5% in those who are cognitively healthy [##REF##36320021##7##–##REF##32640633##9##]. Furthermore, the severity of dyssomnias tends to be higher in individuals with MCI. MCI is frequently accompanied by sleep and circadian rhythm disorders, which significantly contribute to cognitive decline [##UREF##0##10##]. Different subtypes of MCI exhibit distinct patterns of sleep disorders, with patients diagnosed with amnestic mild cognitive impairment (aMCI) exhibiting a lower arousal index and those diagnosed with nonamnestic mild cognitive impairment (naMCI) showing longer total sleep time but also a lower arousal index [##REF##28455768##11##]. Understanding the relationship between MCI and sleep disorders, exploring their underlying mechanisms, and developing effective interventions are crucial for optimizing outcomes in individuals with MCI and dyssomnias.</p>", "<p>Scientometrics is a discipline that employs quantitative analysis of scientific publications, citations, and bibliometric data to comprehend various aspects of scientific research. It involves examining patterns, relationships, and trends within the scientific literature to gain insights into the structure and dynamics of scientific knowledge [##REF##36415118##12##, ##REF##35798128##13##]. However, bibliometric analyses of research on MCI with dyssomnias are currently lacking. In this study, we utilized CiteSpace 6.1.R6 (64 bits) and VOSviewer 1.6.18 for scientometric literature visualization and analysis to explore and identify the current research hotspots in this field over the past two decades, as well as predict future trends.</p>" ]
[ "<title>2. Materials and Methods</title>", "<title>2.1. Data Sources and Search Strategy</title>", "<p>The Web of Science Core Collection (WoSCC) was utilized to retrieve studies pertaining to MCI with dyssomnia research from January 1, 2003, to April 30, 2023. To obtain relevant literature on MCI with dyssomnia research in the past two decades, we employed the following search strategy (see ##FIG##0##Figure 1##): “mild cognitive impairment” (Topic) AND “sleep” (Topic) and Article or Review Article (Document Types) and English (Languages).</p>", "<p>The WoS tool was utilized to convert all data into plain text format, which were subsequently subjected to further bibliometric analysis using CiteSpace 6.1.R6, 64-bit (Drexel University, Philadelphia, PA, USA) and VOSviewer 1.6.18 (Leiden University, Van Eck NJ).</p>", "<title>2.2. Analysis Methods</title>", "<p>We employ CiteSpace and VOSviewer to discern collaborations among countries/territories, institutions, and authors, as well as to identify keywords, journals, major cocited journals, and references, thereby constructing pertinent visualization networks. Both CiteSpace and VOSviewer facilitate researchers in visually exploring and analysing scientific literature by identifying clusters of interconnected research areas, citation networks, emerging trends, and collaborative efforts.</p>", "<p>CiteSpace, developed by Chaomei Chen, is a software tool specifically designed for the visual analysis of citation networks in scientific literature [##REF##33870064##14##]. It generates maps and timelines that highlight significant citation clusters, emerging trends, influential papers, and research frontiers. Each node represents individual items such as papers, authors, or research topics with node size indicating their importance or impact. The coloured circular ring of a node is called the tree ring history, representing the citation history of a specific article. The overall size of the ring reflects the number of times the paper has been cited, while its colour represents the corresponding time period in which it was cited. Additionally, the thickness of a ring is directly proportional to the number of citations within that specific time period. Relationships between items are depicted through connecting lines, with the thickness of the lines indicating citation strength or frequency. The centrality measures such as degree centrality and betweenness centrality can help identify influential nodes. The outermost purple circle represents node centrality, with thicker circles denoting higher levels of centrality. Higher centrality values indicate greater prominence or influence, revealing key papers, authors, or research topics with significant impact or serving as bridges between clusters, thus highlighting their crucial role in the scholarly network. The CiteSpace analysis image generation process utilized the default parameters. The time period from 2003 to 2023 was divided into 20 intervals for analysis, and the unit of time slicing is one year. The default selection was based on the g-index and a scaling factor <italic>k</italic> of 25 (<italic>k</italic> = 25). The link retaining factor (LRF) was set at 3, and the maximum links per node (L/N) parameter was set to 10. The look back years (LBY) parameter was set to 5. In the equation TopN = {n|f(n) &gt; = e}, the value of <italic>e</italic> was set to 1. Additionally, we applied Pathfinder network scaling within the Pruning panel.</p>", "<p>VOSviewer, a widely employed visualization tool for scientometric analyses [##REF##37088947##15##], facilitates the generation of maps that visually represent relationships among various elements, such as authors, keywords, journals, or research institutions, based on patterns of cooccurrence or cocitation. The cooperation network diagram visualizes collaboration patterns. By analysing the cooperation network diagram, researchers can identify items, such as important journals, countries, or territories that have significant collaborative activity within the analysed literature.</p>" ]
[ "<title>3. Results</title>", "<title>3.1. Annual Growth Trends and Citation Trends</title>", "<p>A total of 546 articles related to MCI with dyssomnias were identified in the WoSCC database. ##FIG##1## Figure 2## presents the publication and citation counts from 2003 to 2023, where publications are represented by blue bars and citations by a yellow line. The quantity of publications and citations within this particular field has exhibited a noticeable upward trend over the past two decades. The field of MCI with dyssomnias has attracted significant attention, resulting in a total of 13,957 citations with an average of 25.56 citations per paper. Initially, there were only a limited number of publications annually; however, since 2010, substantial growth has been observed, culminating in the highest number of publications in 2021. Although there was a slight decline in 2022, the citation count continues to rise.</p>", "<title>3.2. Research Areas and Most Cited Articles</title>", "<p>The research topic covered a wide range of 58 distinct research fields related to MCI with dyssomnias, with Neurosciences and Clinical Neurology occupying a prominent position, followed by Geriatrics Gerontology ranking third.</p>", "<p>The top 10 most cited articles and their primary findings are presented in ##TAB##0##Table 1##. Among these articles, six specifically highlight the correlation between sleep disorders and the susceptibility to neurodegenerative diseases. Additionally, the highly cited articles not only elucidated the underlying pathological mechanisms linking neuropsychiatric symptoms, such as sleep disorders, with neurodegenerative diseases but also encompassed comprehensive discussions on the screening, diagnosis, and treatment of sleep disorders associated with cognitive impairment in elderly individuals.</p>", "<title>3.3. Analysis of Author and Coauthor Collaboration</title>", "<p>Between 2003 and 2023, a total of 3,466 authors contributed to research on MCI with dyssomnias. The node size and colour in the map correspond to the number of citations and a single time slice, respectively. The author and cocited author collaboration network map provide valuable insights into potential collaborators, facilitating the establishment of collaborative relationships among researchers (see Figures ##FIG##2##3(a)## and ##FIG##2##3(b)##). The cooperative network map constructed by the author revealed a significant number of researchers (478 nodes) and a strong collaborative relationship (887 lines) among them. Six major research teams have emerged in this field, including Sharon L Naismith, Simon J G Lewis, Postuma Ronald B, Gagnon Jean-Francois, Arnaldi Dario, and Luigi Ferini-Strambi. Each team demonstrates strong internal connections, fostering effective interteam collaboration. Among the cocited authors, Ronald C Petersen, Marshal F Folstein, and Ronald B Postuma emerge as the top three most frequently cited researchers. In terms of centrality measures, Donald L Bliwise, Sonia Ancoli-Israel, and Marshal F Folstein exhibit the highest values.</p>", "<title>3.4. Major Research Institutions and Most Productive Countries/Territories</title>", "<p>A total of 2,520 institutions contributed articles on MCI with dyssomnias. Among them, the University of Sydney, Université de Montréal, and McGill University ranked as the top three institutions in terms of publication volume. Regarding citations, Université de Montréal, Mayo Clinic, and Hôpital du Sacré-Cœur de Montréal emerged as the leading institutions. Notably, the Université de Montréal exhibited exceptional productivity and citation impact, indicating a high-quality research output. Using CiteSpace, ##FIG##3##Figure 4(a)## visualizes the network of institutions comprising 372 nodes and 534 lines. The figure reveals distinct collaborative clusters among regional institutions; however, the level of cooperation between these clusters is not extensive enough.</p>", "<p>A total of 227 countries/territories have contributed to the literature on MCI with dyssomnias, demonstrating international collaboration in this field. ##FIG##3## Figure 4(b)## depicts the collaborative networks and publication density of each country/territory, while ##TAB##1##Table 2## highlights the top 10 most productive nations in terms of published literature. The United States ranks first in terms of publication volume, closely followed by China as the second-largest contributor, and Italy follows suit. In terms of centrality, Sweden exhibits the highest level, while the United Kingdom and Northern Ireland rank second and third, respectively. The top three countries in citation frequency are the United States, Canada, and Spain; these countries also demonstrate high levels of centrality and citation rates. Overall, these countries have played a significant role in the research field of MCI with dyssomnias and have made substantial contributions to this topic.</p>", "<title>3.5. Analysis of Journal Collaboration and Volume of Publications</title>", "<p>The data analysis encompassed a total of 194 journals specializing in MCI with dyssomnias. Figures ##FIG##4##5(a)## and ##FIG##4##5(b)## depict the citation network and cocitation network maps, respectively, of these journals, which were generated using VOSviewer. In these visual representations, larger nodes indicate journals with higher publication volumes. ##TAB##2## Table 3## provides an overview of the top 10 most cited journals and cocited journals based on both publication quantity and citation frequency. The <italic>Journal of Alzheimer's Disease</italic> exhibits the highest publication volume, while <italic>Sleep Medicine</italic> demonstrates the most frequent journal citation frequency, thereby emphasizing their paramount significance within the field. <italic>Neurology</italic>, <italic>Sleep</italic>, and <italic>Movement Disorders</italic> emerge as the top three cocited journals, thus endorsing their authoritative status as trusted sources in this domain. These esteemed journals hold substantial importance and wield significant influence, indicating that the literature they publish surpasses the average standard.</p>", "<p>\n##FIG##4##Figure 5(c)## presents an overlay diagram of dual graphs generated by CiteSpace, depicting the distribution of journals pertaining to MCI with dyssomnias. This diagram facilitates researchers in comprehending the knowledge flow and frontier hotspots across diverse disciplines. The left side of the overlay atlas represents the fields of citing literature, while the right side signifies those of cited literature. The figure illustrates five primary reference paths. The left side corresponds to the citation map, while the right side represents the cited journal map. Molecular/biology/immunology journals are depicted by the orange path, Neurology/Sports/Ophthalmology journals are represented by the pink path, and Psychology/Education/Health journals are indicated by the blue path in relation to Psychology/Education/Social areas. Furthermore, both the orange and pink paths are also cited within the Molecular/Biology/Genetics domains.</p>", "<title>3.6. Analysis of Cocited References</title>", "<p>Reference cocitation analysis is a crucial tool for trend analysis and hotspot identification in specific fields. ##FIG##5## Figure 6(a)## presents the CiteSpace visual map of cocited references in the field of MCI with dyssomnias over the past two decades. Among the top ten cocited articles (refer to ##TAB##3##Table 4##), there are two consensus reports, one animal experiment article, three clinical trial articles, three meta-analyses, and one review. These articles underscore the considerable attention devoted by researchers to investigating the impact of dyssomnias on cognitive disorders. The identification of dyssomnias as potential biomarkers and therapeutic targets for these cognitive disorders represents a pivotal focus in this field. Furthermore, these cited studies represent seminal publications that establish the groundwork for future research.</p>", "<p>The cluster analysis results of cocited references are illustrated in ##FIG##5##Figure 6(b)##. The distinct colour blocks correspond to separate clusters in various regions. ##FIG##5## Figure 6(c)## presents the results of a cluster analysis of cocited references in the form of a timeline view. The size of the nodes corresponds to the total number of citations, while colour indicates specific time periods. The cocitations between two articles are visually represented by lines of varying colours that connect the nodes. This visualization enhances understanding of current research topics and provides insights into future directions in MCI research with dyssomnias. The graph depicts the top 15 reference clusters ranked by publication count, consisting of 735 nodes and 1716 edges. In 2022, obstructive sleep apnea (OSA) (#1) emerged as the largest active cluster with a size of 56. It was followed by possible mediating pathways (#4) with a size of 46 and isolated rapid eye movement (REM) sleep behaviour disorder (#11) with a size of 29. It is evident that significant attention has been devoted to OSA or REM behaviour disorder combined with MCI among dyssomnias. Furthermore, researchers are keen on exploring potential links between MCI and dyssomnias.</p>", "<p>\n##FIG##5##Figure 6(d)## presents the top 15 references exhibiting the highest number and intensity of cited bursts, with red horizontal stripes indicating years with frequent publications and blue horizontal stripes indicating years with infrequent publications. Citation bursts refer to frequently cited references over time, and their identification can highlight hotspots within a specific time period based on reference topics. The study by Gagnon, “Mild cognitive impairment in rapid eye movement sleep behaviour disorder and Parkinson's disease”, with a burst rate of 10.05 (2010-2014), highlights the association between idiopathic REM sleep behaviour disorder and both Parkinson's disease (PD) and its idiopathic form, serving as a significant risk factor for MCI [##REF##19670440##26##].</p>", "<title>3.7. Analysis of Keywords</title>", "<p>The keyword cooccurrence analysis function of CiteSpace serves as an invaluable tool for researchers to acquire a comprehensive understanding of the knowledge landscape within a specific domain by unveiling significant associations among keywords in academic literature.</p>", "<p>The keyword cooccurrence network map displayed in ##FIG##6##Figure 7(a)## was generated by CiteSpace and includes a total of 2257 keywords. The top five most frequently occurring keywords, in addition to the search term MCI, are Alzheimer's disease (AD), dementia, risk factor, and PD. ##FIG##6## Figure 7(b)## presents the top 20 keywords with the strongest citation bursts as indicated on the graph. Notably, PD, neurodegenerative disease, and Lewy body emerge as the keywords with the most intense citation bursts.</p>", "<p>Further cluster analysis of the keywords based on their cooccurrence enables the generation of a keyword cluster map and a clustering timeline view (Figures ##FIG##6##7(c)## and ##FIG##6##7(d)##). These maps provide additional insights into the relationships and patterns among the keywords. The node at the beginning of the horizontal axis represents the initial appearance of the reference. The size of each node in the network is proportional to the number of citations for its corresponding keyword, while lines connecting nodes indicate cocited relationships. Our analysis has identified a total of 24 distinct clusters, out of which only the first 17 larger clusters are displayed by the software. These visually represented clusters are distinguished by different colours and consist of a combined total of 491 nodes and 1,124 lines. The Modularity Q value was calculated as 0.7513, indicating well-defined network clusters as it exceeds the threshold of 0.5. The mean silhouette <italic>S</italic> value was calculated to be 0.8867, indicating a satisfactory level of homogeneity within the clusters, surpassing the threshold of 0.5 as well [##REF##36016904##34##].</p>", "<p>During the clustering process, elements with similar characteristics were grouped together into independent clusters, each labelled to represent its contents. The resulting clusters are as follows: #0 cerebrospinal fluid (CSF), #1 circadian rhythm, #2 memory consolidation, #3 PD, #4 MCI, #5 dementia with Lewy body, #6 preventive medicine, #7 subjective cognitive impairment, #8 correlates, #9 REM sleep behaviour disorder, #10 Lewy body disease, #11 AD, #12 neuropsychiatric symptoms, #13 dietary patterns, #14 quantitative electroencephalogram (EEG) analysis, #15 OSA, and #16 bright light therapy.</p>" ]
[ "<title>4. Discussion</title>", "<p>This study represents the first bibliometric analysis of research articles on MCI with dyssomnias worldwide over the past two decades, utilizing the WOSCC database, VOSviewer, and CiteSpace to explore research trends and hotspots in this field. A total of 546 articles and review papers published between 2003 and 2023 were searched and analysed.</p>", "<p>Over the course of two decades, there has been a consistent upward trend in the annual publication count, capturing the attention of researchers in Neurosciences, Clinical Neurology, Geriatrics Gerontology, and other related fields toward exploring the association between sleep disorders and MCI. From 2003 to 2009, the growth rate exhibited a relatively gradual pace with a predominant focus on clinical research resulting in less than ten publications per year. However, following 2010, there was a notable surge in development, with a steady increase in the number of published literatures until 2018. Throughout this period, clinical research expanded, and researchers commenced investigating the intrinsic correlation and pathological mechanisms between MCI, its neuropsychiatric symptoms, and various sleep disorders. Since 2019, there has been a substantial upswing in the growth rate of literature within this field, accompanied by several high-impact meta-analyses and systematic reviews. The spectrum of neurodegenerative disorders associated with MCI has also expanded. Researchers are currently investigating effective strategies for early prevention, screening, and treatment of MCI accompanied by dyssomnias, including interventions such as bright light therapy and dietary patterns.</p>", "<p>Among the 227 countries analysed in this study, the United States has emerged as the foremost contributor in terms of both publications (<italic>n</italic> = 161) and citations (<italic>n</italic> = 5138), underscoring its significant impact on research related to MCI with dyssomnias over the past two decades. Among the top 10 most productive institutions, six are situated in North America, two in Europe, one in Oceania, and one in Asia. This observation implies that economically advanced regions have made greater advancements in research related to MCI with dyssomnias.</p>", "<p>In terms of individual authors, Naismith Sharon L (<italic>n</italic> = 23), Gagnon Jean-Francois (<italic>n</italic> = 17), and Postuma Ronald B (<italic>n</italic> = 15) emerged as the top three contributors based on publication count. Notably, an article authored by Naismith et al. from Australia [##REF##20354239##35##], was the first to establish a link between objectively measured sleep disturbances and cognitive function in elderly patients with MCI. This study also investigated various subtypes of MCI with psychiatric, vascular, and neurological features. Additionally, a significant impact was observed from a large multicentre prospective cohort study published in Brain [##REF##30789229##16##], which involved the collaboration of Gagnon Jean-Francois and Postuma Ronald B from Canada and received a relatively high number of citations (<italic>n</italic> = 402). The study confirmed the heightened risk of developing PD, dementia with Lewy bodies, and multiple system atrophy in individuals with idiopathic REM sleep behaviour disorder (iRBD). Furthermore, it demonstrated a significant increase in the conversion rate from iRBD to overt neurodegenerative syndrome due to MCI. These findings have substantial implications for potential prevention and early treatment of synucleinopathies associated with neurodegeneration.</p>", "<p>The <italic>Journal of Alzheimer's Disease</italic> and <italic>Sleep Medicine</italic> are distinguished by their high publication volume and citation rates, respectively. <italic>Neurology</italic> is recognized as the most frequently cocited journal based on citations. In recent years, these journals have devoted significant attention to investigating the neural correlations and pathological mechanisms underlying the relationship between MCI and abnormal sleep patterns. Their research is focused on developing strategies for the prevention and treatment of MCI in its early stages, with the aim of halting cognitive decline and mitigating neuropsychiatric symptoms. For instance, a clinical study published in the <italic>Journal of Alzheimer's Disease</italic> compared patients diagnosed with iRBD and MCI (MCI-RBD) to matched patients diagnosed with MCI caused by AD (MCI-AD) [##REF##36155519##36##]. This study has identified distinct neuropsychological and brain metabolic characteristics between MCI-RBD and MCI-AD, which can facilitate the diagnosis and prediction of MCI conversion to dementia. A recent review in <italic>Sleep Medicine</italic> has highlighted significant micro- and macrostructural changes in sleep patterns associated with aging [##REF##32912799##37##], discussing their impact on cognitive function, quality of life, and the brain. Altered sleep patterns are recognized as fundamental components of both MCI and AD, with abnormal sleep accelerating the progression of AD and promoting the buildup of amyloid-<italic>β</italic> (A<italic>β</italic>) and phosphorylated tau. The article also explores potential therapeutic strategies for addressing sleep disruption, emphasizing the urgent need for testing new interventions. Moreover, Neurology articles in this research field predominantly concentrate on exploring the intrinsic pathological features of MCI with dyssomnias at a neurobiological level. Notably, a prospective cohort study investigating patients with iRBD demonstrated that MCI in individuals with iRBD was linked to functional and metabolic alterations in specific brain regions [##REF##35437260##38##]. Hypometabolism within these regions served as a predictive factor for the phenotypic transition to Parkinson's disease or dementia with Lewy bodies.</p>", "<p>Reference cocitation analysis, a bibliometric approach utilized to explore the interrelationships among articles based on their shared references, reveals the frequency of joint citations by other publications for any given pair of articles. Notably, among the top ten highly cited articles, the inaugural publication in <italic>Sleep Medicine Reviews</italic> [##REF##28890168##27##] stands out prominently. These findings imply that sleep disturbances may serve as an indicative factor for an elevated risk of developing dementia. Insomnia was found to be specifically associated with incident AD, while sleep-disordered breathing was identified as a risk factor for all-cause dementia, AD, and vascular dementia. This article has garnered significant attention due to its ability to establish a clear link between sleep disorders and dementia, both of which are important health concerns among older adults. The insights provided can aid in identifying individuals at risk for developing dementia and optimizing early prevention strategies.</p>", "<p>The analysis of overlay maps provides a visually intuitive approach to explore the intellectual structure, interdisciplinary connections, and evolution of research topics within a field. By examining the cooccurrence of keywords across articles, it is possible to identify areas of overlap between different disciplines and track the flow of knowledge. In the dual-map overlay of journals related to MCI with dyssomnia research, the majority of citing publications are found in the fields of Molecular Biology, Immunology, Neurology, Sports, and Ophthalmology. Meanwhile, cited articles primarily originate from the fields of Molecular Biology, Genetics, Psychology, Education, and Social Sciences.</p>", "<p>The CiteSpace software employs keyword cluster analysis to group closely related keywords based on patterns of literature cooccurrence, thereby facilitating the identification of shared research themes. The timeline view provides a visual representation of cluster evolution, enabling researchers to track the emergence, popularity, decline, and transformation of research topics over time. The solid line on the timeline represents the duration of clustering research, while the dashed line indicates a lack of relevant literature during that period. The significance of a cluster domain increases with the inclusion of more literature. Analysing the keyword clustering timeline view (##FIG##6##Figure 7(d)##) based on research time and document count reveals a total of 8 clusters (#1, #3, #4, #5, #7, #10, #11, and #13) with a research span extending until 2023. These 8 clusters consist of an average of 140 documents each, surpassing the overall mean for all clusters (86 documents). Therefore, it is predicted that these eight clusters will remain active throughout 2023 and possess a certain degree of influence. Cluster #1, centered on circadian rhythm, encompasses keywords such as sleep disorders and diseases. Studies have indicated that disruptions in daytime activity, sleep patterns, and circadian rhythm indicators may serve as predictors for the onset of MCI or AD [##REF##34520319##39##, ##REF##33642368##40##]. Cluster #3, which focuses on PD, includes keywords such as OSA and slow wave sleep. Research has suggested that enhancing the quality of sleep, particularly slow wave sleep, may improve cognitive abilities [##REF##32251840##41##]. Additionally, OSA is linked to an increased risk of MCI, AD, and PD [##UREF##2##42##]. Cluster #4, centered on MCI, encompasses keywords such as REM sleep, neurodegenerative disease, and cognitive impairment. A noteworthy study within this cluster is Boeve et al.'s research published in <italic>Sleep Medicine</italic> [##REF##23474058##17##], which further corroborates the significant association between REM sleep behaviour disorder and synucleinopathy. Cluster #5, known as dementia with Lewy body, encompasses keywords such as Dementia, risk factor, and PD. A significant clinical study within this cluster revealed comparable rates of sleep disorders in AD and MCI patients. Sleep-disordered breathing is more prevalent in vascular dementia while REM behaviour disorder is frequently observed in Lewy bodies or PD dementia [##REF##22415141##23##]. The seventh cluster, namely, subjective cognitive impairment (SCI), encompasses keywords such as performance, duration, and quality of life. Existing research indicates that individuals with sleep disorders often perceive subjective cognitive impairment despite having similar objective cognitive levels [##UREF##3##43##]. Sleep disorders are prevalent among the elderly population and have been associated with an increased risk of future MCI and dementia. Sleep disturbances are frequently observed in individuals with subjective cognitive impairment, highlighting the importance of sleep screening and improving sleep quality. Cluster #10, associated with Lewy body disease, is linked to the primary keyword “sleep behaviour disorder”. Clinical studies have compared neuropsychiatric symptoms (NPS) in patients with MCI-Lewy body (MCI-LB) and those with MCI-AD. The results indicate a higher prevalence and severity of NPS, visual hallucinations, and RBD in patients with MCI-LB [##REF##32896168##44##]. Cognitive, parkinsonian, neuropsychiatric, sleep, and autonomic symptoms were significantly more common in patients with MCI-LB than those with MCI-AD [##REF##36912421##45##]. Cluster #11 AD is focused on the keyword “sleep disturbance”. A systematic review has investigated the association between sleep disorders and APOE <italic>ε</italic>4 status in individuals with MCI and AD [##REF##35439097##46##]. The results suggest that APOE <italic>ε</italic>4 is associated with poorer sleep quality, including factors such as total sleep time, REM sleep, sleep efficiency, latency, and wakefulness after sleep onset. Other studies have demonstrated age-related disruptions in sleep patterns among individuals with MCI and AD, which can contribute to cognitive decline irrespective of underlying pathology. Sleep parameters commonly affected include REM sleep, sleep efficiency, latency, and duration [##UREF##0##10##], thereby emphasizing the association between sleep disturbances and cognitive impairment. Cluster #13, dietary patterns, focuses on the concept of “quality”. This section delves into the impact of dietary patterns on sleep and cognitive performance. The Mediterranean diet has been linked to favourable effects on inflammation, oxidative stress, metabolic disorders, aging-related decline in cognitive function, and overall health. Adherence to this dietary regimen is associated with adequate sleep duration and enhanced sleep quality [##REF##35889954##47##]. Furthermore, an additional study underscores the significance of incorporating dietary and sleep interventions as preventive measures against cognitive decline [##REF##34180411##48##].</p>", "<p>However, it is important to acknowledge the limitations of this study. Firstly, the accuracy and reliability of bibliometric analysis are contingent upon data quality and sources; as such, our reliance solely on the WoSCC database may have resulted in relevant studies being overlooked. Secondly, our restriction to English-language articles introduces a potential language bias that may exclude valuable research conducted in other languages. Therefore, further investigation across multiple databases and languages is necessary.</p>" ]
[ "<title>5. Conclusions</title>", "<p>By utilizing CiteSpace, VOSviewer, and WOS analysis tools, we have gained valuable insights into the advancements, emerging areas, and future directions of research in the field of MCI with dyssomnias. It is crucial that future research continues to prioritize exploring the neural mechanisms underlying the intricate relationship between MCI and dyssomnias. Moreover, the development of more efficacious intervention programs targeting dyssomnias in individuals with MCI is imperative. Continued investigation in these areas will not only enhance our understanding of this overlapping domain but also pave the way for potential therapeutic interventions.</p>" ]
[ "<p>Academic Editor: Yu En Gao</p>", "<title>Background</title>", "<p> Mild cognitive impairment (MCI), an intermediate stage between normal aging and dementia, has emerged as a prominent research area in geriatric care due to its heightened propensity for progressing toward dementia. Sleep plays a pivotal role in cognitive function, with dyssomnias not only exacerbating cognitive and affective symptoms associated with neurodegenerative diseases but also contributing to disease progression. </p>", "<title>Aim</title>", "<p> This bibliometric analysis investigates the global research on MCI with dyssomnias over the past two decades, aiming to discern key findings, research domains, and emerging trends in this field. </p>", "<title>Methods</title>", "<p> In this study, a bibliometric analysis was conducted using the search terms “MCI” and “sleep”. Data were extracted from the Web of Science Core Collection database, and visualization and collaborative analysis were performed using CiteSpace and VOSviewer. </p>", "<title>Results</title>", "<p> This study encompassed 546 publications from 2003 to 2023. The publication volume and citation rate consistently increased over time. Neurosciences, Clinical Neurology, and Geriatrics Gerontology emerged as the top three research fields. The <italic>Journal of Alzheimer's Disease</italic> had the highest publication count, while <italic>Sleep Medicine</italic> received the most citations. USA, China, and Italy led in publication output. Collaborative clusters among authors and institutions were identified, but cooperation between clusters was limited. Active cocited reference clusters included “obstructive sleep apnea”, “possible mediating pathways”, and “isolated rapid eye movement sleep behaviour disorder”. The top frequently mentioned keywords, besides “MCI”, were “Alzheimer's disease”, “dementia”, “risk factor”, and “Parkinson's Disease”. Notable keyword clusters spanned circadian rhythm, Parkinson's disease, MCI, dementia with Lewy body, subjective cognitive impairment, Lewy body disease, Alzheimer's disease, and dietary patterns. </p>", "<title>Conclusion</title>", "<p> The field of MCI with dyssomnias is rapidly expanding, encompassing a wide range of neurodegenerative disorders and sleep disturbances. Current research endeavors are primarily focused on elucidating the underlying pathogenesis, predicting disease progression, and developing innovative treatment strategies for individuals affected by MCI with dyssomnias.</p>" ]
[]
[ "<title>Acknowledgments</title>", "<p>This study was supported by the National Natural Science Foundation of China (No. 81973922), the Shenzhen Science and Technology Plan (Nos. JCYJ20190809151013581, JCYJ20210324110809025, and JCYJ20220530142806014), and the Shenzhen Bao'an Traditional Chinese Medicine Development Foundation (2022KJCX-ZJZL-14).</p>", "<title>Data Availability</title>", "<p>The bibliometric data supporting the findings of this study are available upon request from the corresponding author, in accordance with established academic practices.</p>", "<title>Conflicts of Interest</title>", "<p>The authors declare that there are no conflicts of interest regarding the publication of this paper.</p>" ]
[ "<fig position=\"float\" id=\"fig1\"><label>Figure 1</label><caption><p>Flowchart for literature screening.</p></caption></fig>", "<fig position=\"float\" id=\"fig2\"><label>Figure 2</label><caption><p>The annual number of publications and citations on MCI with dyssomnias has been recorded from 2003 to 2023.</p></caption></fig>", "<fig position=\"float\" id=\"fig3\"><label>Figure 3</label><caption><p>The visualization map displays participating authors (a) and cocited authors (b) for MCI with dyssomnias.</p></caption></fig>", "<fig position=\"float\" id=\"fig4\"><label>Figure 4</label><caption><p>The visualization map displays institutions (a) and countries/territories (b).</p></caption></fig>", "<fig position=\"float\" id=\"fig5\"><label>Figure 5</label><caption><p>The visualization maps of citation networks for journals (a) and cocitation networks for journals (b), along with the dual-map overlay of journals (c).</p></caption></fig>", "<fig position=\"float\" id=\"fig6\"><label>Figure 6</label><caption><p>The visualization map of the cocited reference network (a). The visualization map of the cluster analysis for cocited references (b). The timeline view of clusters formed by cocited references (c). The top 15 references with the strongest citation bursts (d).</p></caption></fig>", "<fig position=\"float\" id=\"fig7\"><label>Figure 7</label><caption><p>The visualization map of the keywords (a). The top 20 keywords with the strongest citation bursts (b). The visualization map of the cluster analysis for keywords (c). The timeline view of the cluster analysis for keywords (d).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"tab1\"><label>Table 1</label><caption><p>The top 10 most cited articles.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Rank</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">First author</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Title</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Journal</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Cited</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Highlight</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ronald B Postuma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Risk and Predictors of Dementia and Parkinsonism in Idiopathic REM Sleep Behaviour Disorder: A Multicentre Study</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Brain</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">402</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The study has confirmed a high risk of phenoconversion to overt neurodegenerative disease in individuals with rapid eye movement sleep behaviour disorder (RBD) and has identified several predictors of phenoconversion.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##30789229##16##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Bradley F Boeve</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Clinicopathologic Correlations in 172 Cases of Rapid Eye Movement Sleep Behavior Disorder with or without a Coexisting Neurologic Disorder</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep Medicine</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">225</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The study demonstrates that RBD typically manifests before cognitive impairment, parkinsonism, or autonomic dysfunction in individuals with an underlying neurodegenerative disorder. These findings provide strong evidence for the association between RBD and synucleinopathies.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##23474058##17##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Bradley F Boeve</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Melatonin for Treatment of REM Sleep Behavior Disorder in Neurologic Disorders: Results in 14 Patients</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep Medicine</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">225</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The findings of this study suggest that melatonin may be a viable option as either a standalone or adjunctive therapy for patients with RBD who present various neurological symptoms and disorders.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##14592300##18##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yonas E Geda</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Neuropsychiatric Symptoms in Alzheimer's Disease: Past Progress and Anticipation of the Future</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Alzheimers &amp; Dementia</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">224</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The study proposes four potential mechanisms that establish a connection between neuropsychiatric symptoms (including depression, apathy, sleep disturbances, agitation, and psychosis) and MCI or Alzheimer's disease dementia.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##23562430##19##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Jean Woo</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Frailty Screening in the Community Using the FRAIL Scale</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of The American Medical Directors Association</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">216</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Among individuals aged 65 years and older who are frail or prefrail, approximately 60% exhibit MCI along with suboptimal sleep quality. Additionally, they consume a higher number of medications, especially sleeping pills.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##25732832##20##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ronald B Postuma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Rapid Eye Movement Sleep Behavior Disorder and Risk of Dementia in Parkinson's Disease: A Prospective Study</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Movement Disorders</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">205</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">All participants enrolled in the study exhibited MCI; the findings revealed that a loss of baseline REM sleep atonia predicted subsequent dementia development, as well as the emergence of hallucinations and cognitive fluctuations.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##22322798##21##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Carmen E Westerberg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Concurrent Impairments in Sleep and Memory in Amnestic Mild Cognitive Impairment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of The International Neuropsychological Society</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">183</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The findings suggest that sleep disturbances in individuals with amnestic MCI contribute to memory deficits by disrupting the consolidation of memories dependent on sleep.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##22300710##22##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Biancamaria Guarnieri</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Prevalence of Sleep Disturbances in Mild Cognitive Impairment and Dementing Disorders: A Multicenter Italian Clinical Cross-Sectional Study on 431 Patients</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Dementia and Geriatric Cognitive Disorders</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">183</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">More than 60% of individuals experienced one or more sleep disturbances, often occurring in conjunction with each other without any discernible pattern of cooccurrence. The frequency of any sleep disorder was similar between those with Alzheimer's disease and MCI.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##22415141##23##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Bradley F Boeve</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Validation of the Mayo Sleep Questionnaire to Screen for REM Sleep Behavior Disorder in an Aging and Dementia Cohort</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep Medicine</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">181</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">The study suggests that the Mayo Sleep Questionnaire demonstrates sufficient sensitivity and specificity to diagnose RBD in elderly individuals with cognitive impairment and/or parkinsonism.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##21349763##24##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Martijn L. T. M. Müller</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Cholinergic Dysfunction in Parkinson's Disease</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Current Neurology and Neuroscience Reports</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">159</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Given that RBD serves as an antecedent marker for cognitive impairment and dementia in Parkinson's Disease, these findings suggest the possibility of early degeneration in the cholinergic system in this condition.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##1##25##]</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab2\"><label>Table 2</label><caption><p>The top 10 Countries/territories with the highest number of publications.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Rank</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Countries/territories</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">First year</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Record count</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Centrality</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Citations</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">USA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2003</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">161</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5138</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2013</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1184</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Italy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2008</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2130</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Canada</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2009</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">55</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2490</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">England</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2010</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1606</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Australia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2010</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1809</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Spain</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2005</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2139</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">South Korea</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2011</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">725</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Germany</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2011</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1073</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">France</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2004</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1473</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab3\"><label>Table 3</label><caption><p>The top 10 cited and cocited journals with the highest publication and citation volumes.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Rank</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Cited journals</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Documents</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Citations</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Cocited journals</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Documents</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Citations</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of Alzheimers Disease</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">731</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Neurology</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">426</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1715</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep Medicine</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1233</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">338</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1520</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">639</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Movement Disorders</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">164</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">901</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Frontiers in Aging Neuroscience</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">384</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Sleep Medicine</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">263</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">654</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Frontiers in Neurology</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">117</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of Alzheimer's Disease</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">256</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">643</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>International Journal of Geriatric Psychiatry</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">447</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Alzheimers &amp; Dementia</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">255</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">599</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>International Psychogeriatrics</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">256</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of the American Geriatrics Society</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">288</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">559</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Parkinsonism &amp; Related Disorders</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">352</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Neurobiology of Aging</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">512</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of Geriatric Psychiatry and Neurology</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">213</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Brain</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">230</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">474</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Journal of Sleep Research</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">194</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Annals of Neurology</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">232</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">441</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tab4\"><label>Table 4</label><caption><p>The top 10 cocited references.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Rank</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Count</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">First author</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Year</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Title</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Le Shi</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sleep Disturbances Increase the Risk of Dementia: A Systematic Review and Meta-Analysis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##28890168##27##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yue Leng</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Association of Sleep-Disordered Breathing With Cognitive Function and Risk of Cognitive Impairment: A Systematic Review and Meta-Analysis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##28846764##28##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Bryce A Mander</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2016</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sleep: A Novel Mechanistic Pathway, Biomarker, and Treatment Target in the Pathology of Alzheimer's Disease?</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##27325209##29##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ian G McKeith</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Diagnosis and Management of Dementia with Lewy Bodies: Fourth Consensus Report of the DLB Consortium</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##28592453##30##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Omonigho M Bubu</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sleep, Cognitive Impairment, and Alzheimer's Disease: A Systematic Review and Meta-Analysis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##UREF##0##10##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ronald C Petersen</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Practice Guideline Update Summary: Mild Cognitive Impairment: Report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##29282327##31##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ronald B Postuma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2019</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Risk and Predictors of Dementia and Parkinsonism in Idiopathic REM Sleep Behaviour Disorder: A Multicentre Study</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##30789229##16##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ram A Sharma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Obstructive Sleep Apnea Severity Affects Amyloid Burden in Cognitively Normal Elderly. A Longitudinal Study</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##29125327##32##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Lulu Xie</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2013</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sleep Drives Metabolite Clearance from the Adult Brain</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##24136970##33##]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Jean-François Gagnon</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2009</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mild Cognitive Impairment in Rapid Eye Movement Sleep Behavior Disorder and Parkinson's Disease</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[##REF##19670440##26##]</td></tr></tbody></table></table-wrap>" ]
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[{"label": ["10"], "person-group": ["\n"], "surname": ["Casagrande", "Forte", "Favieri", "Corbo"], "given-names": ["M.", "G.", "F.", "I."], "article-title": ["Sleep quality and aging: a systematic review on healthy older people"], "source": ["\n"], "italic": ["International Journal of Environmental Research and Public Health"], "year": ["2022"], "volume": ["19"], "issue": ["14"], "fpage": ["p. 8457"], "pub-id": ["10.3390/ijerph19148457"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["M\u00fcller", "Bohnen"], "given-names": ["M. L. T. M.", "N. I."], "article-title": ["Cholinergic dysfunction in Parkinson\u2019s disease"], "source": ["\n"], "italic": ["Current Neurology and Neuroscience Reports"], "year": ["2013"], "volume": ["13"], "issue": ["9"], "fpage": ["p. 377"], "pub-id": ["10.1007/s11910-013-0377-9", "2-s2.0-84881298328"]}, {"label": ["42"], "person-group": ["\n"], "surname": ["Ferini-Strambi", "Lombardi", "Marelli", "Galbiati"], "given-names": ["L.", "G. E.", "S.", "A."], "article-title": ["Neurological deficits in obstructive sleep apnea"], "source": ["\n"], "italic": ["Current Treatment Options in Neurology"], "year": ["2017"], "volume": ["19"], "issue": ["4"], "fpage": ["p. 16"], "pub-id": ["10.1007/s11940-017-0451-8", "2-s2.0-85016995745"]}, {"label": ["43"], "person-group": ["\n"], "surname": ["Tsapanou", "Vlachos", "Cosentino"], "given-names": ["A.", "G. S.", "S."], "article-title": ["Sleep and subjective cognitive decline in cognitively healthy elderly: results from two cohorts"], "source": ["\n"], "italic": ["Journal of Sleep Research"], "year": ["2019"], "volume": ["28"], "issue": ["5, article e12759"], "pub-id": ["10.1111/jsr.12759", "2-s2.0-85054369126"]}]
{ "acronym": [], "definition": [] }
48
CC BY
no
2024-01-14 23:43:50
Behav Neurol. 2024 Jan 6; 2024:6622212
oa_package/f9/54/PMC10787659.tar.gz
PMC10787661
37634192
[ "<title>Introduction</title>", "<p>While coronary revascularization is intended to improve myocardial perfusion and reduce ischaemia, randomized revascularization trials have not demonstrated improved survival compared with medical treatment.<sup>##REF##22924622##1–3##</sup> The artery-specific map of coronary flow capacity (CFC) by quantitative rest-stress positron emission tomography (PET) perfusion imaging associates with or predicts survival probability in chronic coronary artery disease (CAD).<sup>##REF##30115688##4–12##</sup> Severely reduced CFC and associated low survival probability are significantly improved after revascularization compared to no revascularization for comparable severity in large non-randomized cohorts.<sup>##REF##30115688##4–6##</sup></p>", "<p>Conversely, non-severe CFC regions associated with low mortality risk show no improvement after revascularization.<sup>##REF##30806750##9##,##REF##34878102##10##</sup> Similarly, severe perfusion abnormalities with no improvement after revascularization have no improved survival.<sup>##REF##30115688##4–6##,##REF##34878102##10–12##</sup> In patients with severely reduced CFC and low survival probability that improves after revascularization, substantial unquantified residual CAD commonly remains with abnormal CFC and associated residual limited survival probability.<sup>##REF##30115688##4–6##,##REF##34878102##10–12##</sup> The quantitative burden of residual focal or diffuse CAD, incomplete or failed revascularization, and associated survival probability before and after revascularization remain undefined and unpredictable in individuals.</p>", "<p>Based on these previously reported observations, we tested the following hypothesis. Severely reduced CFC associates with or predicts low 10-year survival probability that is significantly improved after revascularization compared to no revascularization for comparable CFC severity. However, improvement may be heterogeneous or limited by residual diffuse or focal CAD that has not been previously quantified or virtually predicted before revascularization. The analysis requires three steps.</p>", "<p>First, in a cohort of 6979 routine diagnostic quantitative rest-stress PET cases followed over 12 years, extended data indicate that CFC maps of the size-severity range of stress perfusion in mL/min/g and coronary flow reserve (CFR) per pixel as % of left ventricle (LV) associate with or predict survival probability. As an extension of this observation, we hypothesized that low survival probability associated with severely reduced CFC might be substantially modified by the size-severity of surrounding normal or mildly reduced CFC regions reflecting diffuse disease or multiple mild to moderate stenosis in a continuum of relatively limited survival probability. Consequently, revascularization might improve survival probability to a greater or lesser extent depending on the range of residual CFC size-severity abnormalities reflecting residual focal or diffuse CAD to explain variable or limited revascularization effects on mortality. As a specific test of this hypothesis in individual patients, we analysed 283 pre- and post-revascularization PET pairs of CFC maps and associated survival probability for their pre- to post-revascularization changes observed for variable effects of revascularization in individual patients.</p>", "<p>Second, we hypothesized that <italic toggle=\"yes\">Virtual</italic> survival probability can be predicted for ideal, hypothetical, complete revascularization by replacing the % of severely reduced CFC pixels in the pre-revascularization CFC map with normal CFC severity pixels in the Cox regression modelling of survival probability. This CFC-derived <italic toggle=\"yes\">Virtual</italic> survival probability after hypothetical, ideal, complete revascularization can then be compared with CFC-associated <italic toggle=\"yes\">Observed</italic> survival probability after actual revascularization. Discordance between the CFC-associated <italic toggle=\"yes\">Observed</italic> survival probability after actual revascularization and the CFC <italic toggle=\"yes\">Virtually</italic> predicted post-revascularization survival probability would indicate suboptimal revascularization due to residual diffuse CAD, incomplete or failed revascularization, or progressive CAD. The <italic toggle=\"yes\">Virtual</italic> survival probability for the pre-revascularization CFC would therefore suggest the optimal potential effect of revascularization on survival probability in an individual patient before doing the procedure.</p>", "<p>Finally, in 2552 post-revascularization PET patients, we also hypothesized that CFC maps in patients with prior revascularization may have residual severe regional abnormalities associated with poor survival probability that improve after repeat revascularization. However, substantial residual limited survival probability may remain due to more residual CAD than patients without prior revascularization.</p>" ]
[ "<title>Methods</title>", "<p>At the Weatherhead PET Center, McGovern Medical School, University of Texas (UT) Health Science Center, Houston, 6979 routine diagnostic rest-stress, quantitative, myocardial perfusion PETs were done on consecutive patients after informed consent in prospective ongoing research with systematic follow-up over 12 years by trained, blinded research assistants as approved by our institutional Committee for Protection of Human Subjects (CPHS). Complete medical history is entered into a dedicated Health Insurance Portability and Accountability Act of 1996 (HIPAA)-compliant relational database.<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> For every PET, a blood sample is tested for caffeine. Systematic repeat PET after revascularization is done under a UT CPHS-approved protocol with written consent and funded by the UT Weatherhead Endowment.</p>", "<title>Cardiac positron emission tomography acquisition and analysis</title>", "<p>All patients are instructed to fast for 4 h and abstain from caffeine and cigarettes for 24 h. Cardiac PET-computed tomography (CT) was acquired as previously reported (Discovery ST 16-slice GE Healthcare PET-CT scanner in 2-dimensional mode, Waukesha, Wisconsin, or a United Imaging solid state 3-D PET-CT, Houston, TX) after intravenous 30–50 mCi (1110–1850 MBq) of Rb-82 (Bracco Diagnostics, Princeton, New Jersey).<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> In 196 paired PETs acquired in the same patient on same day, rest and stress myocardial perfusion in mL/min/g on these different scanners are equivalent for Rb-82 (R<sup>2</sup> = 0.99, coefficient of variance ±12%). Attenuation correction used cine CT tube current modulation with reduced radiation dose, co-registration, and validated region-of-interest for arterial input.<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> Only 0.7% failed to yield quantitative perfusion data due to equipment failure or subclavian vein occlusion precluding arterial input. Standard pharmacological stress used dipyridamole infusion (0.142 mg/kg/min) over 4 min with 4-min-wait to peak vasodilation for second infusion of Rb-82.<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup></p>", "<p>The regional CFC map (<italic toggle=\"yes\">##FIG##1##Figure 1##</italic>) of the LV combines regional pixel values of CFR and stress (mL/min/g).<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> The wide range of stress myocardial perfusion in mL/min/g and CFR values for each of 1344 radial pixels comprise vast numbers of possible stress perfusion and CFR pixel combinations that are compressed into the following objectively determined ranges of combined values for each regional pixel by ROC analysis for specific clinical groups as reported<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> summarized as follows:</p>", "<p>\n<italic toggle=\"yes\">Excellent</italic>, coloured red from healthy young volunteers (CFR &gt;2.9 and stress perfusion &gt;2.17 mL/min/g).</p>", "<p>\n<italic toggle=\"yes\">Adequate</italic>, coloured orange from asymptomatic patients with risk factors without known CAD (CFR &gt;2.38 to 2.9 and stress perfusion &gt;1.82 to 2.17).</p>", "<p>\n<italic toggle=\"yes\">Mildly reduced</italic>, yellow from patients with known CAD without symptoms of ischaemia (CFR &gt;1.6 to 2.38 and stress perfusion &gt;1.09 to 1.82).</p>", "<p>\n<italic toggle=\"yes\">Moderately reduced</italic>, coloured green from patients with either a regional stress defect or angina or ST depression ≥ 1 mm during dipyridamole stress (CFR &gt;1.27 to 1.6 and stress perfusion &gt;0.83 to 1.09).</p>", "<p>\n<italic toggle=\"yes\">Severely reduced</italic>, coloured blue from patients with two of these three manifestations of ischaemia (CFR 1.0 to 1.27 and stress perfusion ≤0.83). <italic toggle=\"yes\">Myocardial steal</italic>, coloured dark blue (defined as CFR &lt;1.0).<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup></p>", "<p>\n<italic toggle=\"yes\">Myocardial scar,</italic> coloured as grey hatch marks, defined as fixed resting and stress perfusion ≤0.3 mL/min/g as % of LV.</p>", "<p>Each colour-coded pixel is spatially mapped back onto its LV location with per cent of LV calculated for each range of combined both CFR and stress perfusion pixel values listed in the CFC colour histogram bar. The regional, colour-coded 1344 pixels provide integrated, size-severity quantification for each specific coronary artery distribution down to tertiary branches using Food and Drug Administration (FDA)-approved software (FDA K202679).<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> The coefficient of variance for mL/min/g is ±10% on serial rest–rest and stress–stress images in the same patient minutes apart and &lt;±1% on the Kolmogorov–Smirnov (KS) test of serial CFC histograms in the same patient.<sup>##REF##28017383##13##</sup></p>", "<title>\n<italic toggle=\"yes\">Observed</italic> survival probability associated with 6979 coronary flow capacity maps by positron emission tomography</title>", "<p>As previously reported, the <italic toggle=\"yes\">Observed</italic> 10-year survival probability associated with individual PET CFC maps was determined as a fraction of 1.0 by multi-variable Cox regression modelling of 6979 rest-stress PET with &gt;90-day follow-up for time to all-cause mortality in the prospective database with follow-up over 12 years.<sup>##REF##36599572##6##</sup> Covariates in the Cox model were the combined CFC size-severity as % of LV with CFCmild, CFCsevere (i.e. moderate and/or severe), and myocardial scar, using CFCnormal (i.e. normal and/or minimal) as the reference in the Cox model.<sup>##REF##36599572##6##</sup></p>", "<title>\n<italic toggle=\"yes\">Virtual</italic> survival probability after hypothetical, ideal, complete revascularization</title>", "<p>The <italic toggle=\"yes\">Virtual</italic> survival probability after optimal, ideal, complete, hypothetical revascularization was calculated in the Cox regression model by replacing the % of severely reduced CFC pixels in pre-revascularization CFC maps with % of LV as normal CFC pixels surrounding the severe CFC abnormality. Therefore, the <italic toggle=\"yes\">Optimal Virtual</italic> survival probability reflects the likelihood of improved survival probability after ideal complete revascularization compared to the <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability after actual revascularization to aid decision-making on revascularization before procedures were done. For frequent post-revascularization residual CAD or incomplete revascularization, a <italic toggle=\"yes\">Realistic Virtual survival probability</italic> was determined in the Cox model by replacing % LV with severely reduced CFC in the baseline pre-revascularization PET by the proportionate ratio of regional distribution of CFC mild pixels to normal CFC pixels outside the CFC severely reduced pixels while keeping constant the % of LV with mildly reduced CFC and scar.</p>", "<p>The <italic toggle=\"yes\">Optimal</italic> and <italic toggle=\"yes\">Realistic Virtual</italic> survival probabilities bracketed the range of expected post-revascularization survival probabilities. Discordances between the <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability after actual revascularization and the <italic toggle=\"yes\">Virtual</italic> survival probabilities indicate risk of residual diffuse narrowing, stenosis, or incomplete revascularization.</p>", "<title>Clinical follow-up</title>", "<p>A programmed, prospective follow-up consent form approved by our Committee for Protection of Human Patients was obtained after every PET. Blinded research assistants systematically and continuously record clinical events from clinic or hospital records, mailed questionnaires, phone calls, email, or web searches of newspaper obituaries as an ongoing monthly routine process, repeated three times for initial non-responders.<sup>##REF##30115688##4–6##,##REF##28017383##13##</sup> A team of cardiologists, experienced research nurses, and experienced research assistants blinded to PET data adjudicated outcomes. All-cause death was used as a definitive, hard outcome to avoid the well-recognized bias in determining cause of death or controversy in defining myocardial infarction (MI).</p>", "<title>Analysis schema for coronary flow capacity severity and survival probability (<italic toggle=\"yes\">Table ##TAB##0##1##</italic>)</title>", "<p>Data analyses were performed in the following steps to address study objectives outlined in the introduction.</p>", "<p>\n<bold>Step 1</bold>. Multi-variable Cox regression modelling for <italic toggle=\"yes\">Observed</italic> survival probability as a fraction of 1.0 over 12 years associated with 6979 CFC maps of routine rest-stress diagnostic quantitative PET with and without severely reduced CFC, with and without revascularization as previously reported.<sup>##REF##36599572##6##</sup> (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic>).</p>", "<p>\n<bold>Step 2.</bold> The CFC maps were compared from 283 pre- and post-revascularization CFC pairs. The range of changes in CFC maps is displayed in colour (<italic toggle=\"yes\">##FIG##3##Figure 3##</italic>) and CFC severity histogram plots were systematically compared using KS tests (<italic toggle=\"yes\">##FIG##4##Figure 4##</italic>). The <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability as a fraction of 1.0 for each pre- and post-revascularization CFC pair was better or worse after revascularization as a % of patients having pre-revascularization CFC that was severely reduced, not severely reduced, or normal for ≥90% of LV (<italic toggle=\"yes\">##FIG##4##Figure 4##</italic>).</p>", "<p>\n<bold>Step 3</bold>. The <italic toggle=\"yes\">Observed</italic> survival probabilities before and after revascularization of the 283 PET pairs were compared to % of LV with abnormal CFC or % myocardial scar (<italic toggle=\"yes\">##FIG##5##Figure 5##</italic>). <italic toggle=\"yes\">Observed</italic> and <italic toggle=\"yes\">Virtual</italic> survival probability were compared to assess the residual risk from residual diffuse CAD or incomplete revascularization that limits improvement of CFC or associated survival probability (<italic toggle=\"yes\">##FIG##6##Figure 6##</italic>).</p>", "<p>\n<bold>Step 4</bold>. Comparison of CFC severity histograms by KS test was undertaken for the following patient categories: Healthy young volunteers, patients with or at risk of CAD with no prior revascularization, patients with prior revascularization, the 283 pre-revascularization CFC maps of pre-post-revascularization CFC pairs to document comparable CFC severity of the latter two groups. (<italic toggle=\"yes\">##FIG##7##Figure 7##</italic>)</p>", "<p>\n<bold>Step 5.</bold> Multi-variable Cox regression modelling for observed survival probability over 12 years derived from 2552 CFC maps of patients with prior revascularization with and without severely reduced CFC, with and without repeat revascularization (<italic toggle=\"yes\">##FIG##8##Figure 8##</italic>).</p>", "<title>Statistical analysis</title>", "<p>For continuous variables, two-tailed tests with <italic toggle=\"yes\">P</italic> &lt; .05 were considered statistically significant for rejecting the null hypothesis. Chi-square was used for a significance of discrete variables. Unpaired <italic toggle=\"yes\">t</italic>-tests with unequal variance between groups were used for continuous variables. As previously reported,<sup>##REF##32227755##3–6##,##REF##28017383##13##,##UREF##0##14##</sup> SAS 9.4 was used for multiple variable Cox regression modelling of both severely reduced CFC and non-severe CFC with time-dependent covariates of revascularization after PET and time to all-cause-death after PET on per patient basis with and without post-PET percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) surgery including age and gender covariates. KS tests compared histogram distributions between groups in colour-coded ranges of relative regional CFC distribution as % of LV as previously reported.<sup>##REF##28017383##13##,##UREF##0##14##</sup></p>" ]
[ "<title>Results</title>", "<p>As a tertiary care academic centre with advanced quantitative perfusion imaging and complex revascularization services, our large referral cohort of 6979 routine, diagnostic, rest-stress quantitative PET perfusion images included high prevalence of known (40%), or suspected CAD, high-risk factor burden or coronary calcium (77%) scores as previously reported.<sup>##REF##32227755##3–6##,##REF##28017383##13##</sup> Over 60% are quantitatively not-normal, but only 21% had severely reduced CFC and only 12% were revascularized depending on clinical judgement, comorbidities, size, and proximal vs. distal stress defects. Severely reduced CFC was associated with low survival probability (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic> solid blue line) that was significantly improved after revascularization (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic> dashed blue line) (<italic toggle=\"yes\">P</italic> = .0015) over 12 years compared to no revascularization for comparable severity (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic>); average mortality was 21.4% without vs. 12.5% with revascularization for comparable pre-procedure CFC severity, a reduction of 42%. While improved after revascularization, the survival probability remained substantially reduced (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic> dashed blue lines) due to residual CFC abnormalities not defined or predicted before revascularization. Non-severe CFC was associated with higher survival probability (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic> solid black line) that was not changed by revascularization (<italic toggle=\"yes\">##FIG##2##Figure 2##</italic> dashed black line).</p>", "<title>Coronary flow capacity maps and associated survival probability before and after revascularization to explain and predict low survival probability after revascularization</title>", "<p>\n<italic toggle=\"yes\">\n##FIG##3##Figure 3##\n</italic> displays the range of quantitative regional severity-size of perfusion abnormalities and illustrative CFC ‘rainbow’ map examples in 283 cases from before and after revascularization (<italic toggle=\"yes\">##TAB##1##Table 2##</italic>). Each size-severity fraction of colour-coded CFC maps contributes cumulatively to different <italic toggle=\"yes\">Observed</italic> CFC-associated survival probabilities for each individual PET as listed beside each image.</p>", "<p>Such favourable outcomes as in <italic toggle=\"yes\">##FIG##3##Figure 3A##</italic> occurred only after complete revascularization of large regions of severely reduced CFC (blue) with surrounding regions of normal CFC (red), thereby indicating no severe diffuse flow limiting narrowing or multiple stenosis. Commonly, the revascularization only partially improved CFC due to residual abnormalities limiting improvement in survival probability (<italic toggle=\"yes\">##FIG##3##Figure 3B–D##</italic>). Flow-limiting stenosis superimposed on diffuse CAD improved with residual abnormalities and correspondingly limited improvement in survival probability (<italic toggle=\"yes\">##FIG##3##Figure 3E##</italic>). Predominant diffuse mildly reduced CFC (yellow) of diffuse CAD commonly did not improve after revascularization, with no change in low survival probability (<italic toggle=\"yes\">##FIG##3##Figure 3F##</italic>). Stent procedures done despite normal CFC commonly showed worsened CFC and reduced survival probability due to potential risks of procedures including jailed arterial branches that made CFC and survival probability worse (<italic toggle=\"yes\">##FIG##3##Figure 3G##</italic>).</p>", "<p>For 283 paired pre- and post-revascularization PET pairs, the KS plots in <italic toggle=\"yes\">##FIG##4##Figure 4##</italic> compare CFC size-severity cumulative histograms of PET that were better (<italic toggle=\"yes\">##FIG##4##Figure 4A##</italic>), unchanged (<italic toggle=\"yes\">##FIG##4##Figure 4B##</italic>), or worse (<italic toggle=\"yes\">##FIG##4##Figure 4C##</italic>) after revascularization. The CFC-associated post-revascularization survival probability was improved or worse for baseline pre-revascularization CFC severe (<italic toggle=\"yes\">##FIG##4##Figure 4D##</italic>), for non-severe CFC (<italic toggle=\"yes\">##FIG##4##Figure 4E##</italic>), and for baseline normal CFC (<italic toggle=\"yes\">##FIG##4##Figure 4F##</italic>).</p>", "<p>For 184 of 283 (65%) paired PET with CFCsevere in pre-revascularization PET, the CFC histogram significantly improved in post-revascularization PET (<italic toggle=\"yes\">##FIG##4##Figure 4A##</italic>) (KS = 0.14, <italic toggle=\"yes\">P</italic> &lt; .001) with correspondingly improved average survival probability (<italic toggle=\"yes\">##FIG##4##Figure 4D##</italic>) (<italic toggle=\"yes\">P</italic> = .00006). For the remaining 99 of 283 (35%) paired PET scans with no severely reduced CFC in pre-revascularization PET, the CFC histogram did not improve (<italic toggle=\"yes\">##FIG##4##Figure 4B##</italic>) (KS = 0.05, <italic toggle=\"yes\">P</italic> = .14), had unchanged or worsened survival probability (<italic toggle=\"yes\">##FIG##4##Figure 4E##</italic>) with 40/99 (40%) better and 59/99 (60%) worse (<italic toggle=\"yes\">P</italic> = .00006). For 34 of 283 PET images with normal CFC in ≥90% of the LV (12%), average CFC histogram was significantly worse on post-revascularization PET (<italic toggle=\"yes\">##FIG##4##Figure 4C##</italic>) (KS = 0.24, <italic toggle=\"yes\">P</italic> &lt; .00001), had worse survival probability (<italic toggle=\"yes\">##FIG##4##Figure 4F##</italic>) with 7/34 (21%) better and 27/34 (79%) worse.</p>", "<p>Of 283 pre-post revascularization PET pairs, only 5.7% with CFC severe normalized CFC with improved survival probability of +Δ0.4 or a 10-year survival probability of ≥0.9 after revascularization. The average improved, no different, or worse changes in the CFC histograms and associated survival probabilities for different severity thresholds were due to different proportions of individual cases with binary better or worse CFC and associated better or worse survival probabilities, reflecting variable effectiveness of revascularization.</p>", "<p>Of 283 pre- and post-revascularization PET pairs with severely reduced CFC that improved after revascularization, the survival probability improved by 12% (0.59 ± 0.19 to 0.66 ± 0.22, <italic toggle=\"yes\">P</italic> &lt; .001). However, for pre-revascularization PET with no severely reduced CFC that did not improve on post-revascularization PET, survival probability also showed no improvement (0.83 ± 0.11 to 0.81 ± 0.13, <italic toggle=\"yes\">P</italic> = .14). For all 283 pre- and post-revascularization PET pairs, the net effect was the average of these discordant outcomes, with 160/283 (57%) having better and 123/283 (43%) worse CFC with improved average survival probability from 0.67 ± 0.2 to 0.71 ± 0.2 (<italic toggle=\"yes\">P</italic> &lt; .001). For pre-revascularization PET with normal CFC for ≥90% of LV and survival probability of 0.92 ± 0.01, revascularization was done in a subset of 34/283 patients (12%) associated with worsening CFC and reduced survival probability to 0.85 ± 0.11 (<italic toggle=\"yes\">P</italic> = .00001).</p>", "<title>Microvascular dysfunction, gender, and ethnicity</title>", "<p>Severe or moderate transmural CFC abnormalities characterized this population of pre- and post-revascularization PET-pairs. As previously reported, in the absence of regional flow-limiting stenosis or diffuse CAD (no regional severely blue or moderately green reduced CFC), microvascular dysfunction limits coronary blood flow to mildly reduced CFC (yellow).<sup>##REF##36599572##6##,##UREF##1##15##,##REF##30466522##16##</sup> This flow limitation due to microvascular disease maintains coronary pressure and thereby maintains normal subendocardial perfusion and relative transmural perfusion gradient. In contrast, diffuse epicardial CAD without focal flow-limiting stenosis decreases hyperaemic coronary pressure and hence subendocardial perfusion quantified as % of LV with reduced relative subendocardial perfusion outside ± 2SD of 125 healthy young volunteers separate from the study participants.<sup>##REF##36599572##6##,##UREF##1##15##,##REF##30466522##16##</sup></p>", "<p>For diffusely reduced CFC or CFR, reduced relative subendocardial perfusion or transmural perfusion gradient during vasodilatory stress distinguishes diffuse epicardial CAD (reduced subendocardial relative transmural perfusion gradient) from microvascular dysfunction (no relative transmural perfusion gradient).<sup>##REF##36599572##6##,##UREF##1##15##,##REF##30466522##16##</sup></p>", "<p>For the 283 pre- and post-revascularization PET pairs, the great majority of PET scans had either transmural or subendocardial perfusion abnormalities outside ± 2SD of healthy volunteers indicating flow-limiting stenosis or diffuse CAD. Nine (3%) had microvascular dysfunction limiting perfusion in the absence of moderate or severe stress regional defects or reduced subendocardial perfusion either before or after revascularization (see ##SUPPL##0##Supplementary data online##, <italic toggle=\"yes\">##SUPPL##0##Table S2##</italic>). These findings are consistent with diffuse epicardial CAD and reduced subendocardial perfusion explaining over 95% of no-stenosis angina.<sup>##REF##36599572##6##,##UREF##1##15##,##REF##30466522##16##</sup></p>", "<p>No gender differences were observed for % of LV with moderately or severely reduced CFC, % of participants with myocardial scar ≥10% of LV, % of participants with microvascular dysfunction, or for survival probability (see ##SUPPL##0##Supplementary data online##, <italic toggle=\"yes\">##SUPPL##0##Table S2##</italic>). Ethnic distribution included Caucasian 70%, Asian 11%, Hispanic 9%, Black 8% and other 2%.</p>", "<title>Coronary bypass surgery vs. percutaneous coronary intervention</title>", "<p>Significant improvement was observed in CFC maps (<italic toggle=\"yes\">P</italic> = .001) and <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability (<italic toggle=\"yes\">P</italic> &lt; .00001) from before to after coronary artery bypass surgery (<italic toggle=\"yes\">P</italic> &lt; .03) but not comparably after PCI (<italic toggle=\"yes\">P</italic> = .3) (see ##SUPPL##0##Supplementary data online##, <italic toggle=\"yes\">##SUPPL##0##Figure S1##</italic>).</p>", "<title>Observed coronary flow capacity-associated survival probability after actual revascularization vs. <italic toggle=\"yes\">Virtual</italic> survival probability after hypothetical revascularization in 283 pre- and post-revascularization positron emission tomography pairs</title>", "<p>For the 283 pre- and post-revascularization PET pairs, the <italic toggle=\"yes\">Observed</italic> survival probability correlated with CFC severity before and after actual revascularization that was worse with myocardial scar (<italic toggle=\"yes\">##FIG##5##Figure 5##</italic>). The <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability after actual revascularization was compared to the <italic toggle=\"yes\">Optimal and Realistic Virtual</italic> survival probabilities after hypothetical complete or incomplete revascularization or residual diffuse CAD, respectively (<italic toggle=\"yes\">##FIG##6##Figure 6##</italic>). The <italic toggle=\"yes\">Optimal Virtual</italic> survival probability averaged 0.81 ± 0.88, i.e. significantly higher than 0.71 ± 0.21 (<italic toggle=\"yes\">P</italic> &lt; .001) of the <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability after actual revascularization due to residual diffuse or multi-stenosis CAD or incomplete revascularization (<italic toggle=\"yes\">##FIG##6##Figure 6##</italic>) as illustrated by the residual CFC abnormalities in the individual PET pairs of <italic toggle=\"yes\">##FIG##3##Figure 3##</italic> and KS plots of <italic toggle=\"yes\">##FIG##4##Figure 4##</italic>. The <italic toggle=\"yes\">Realistic Virtual</italic> survival probability of 0.74 ± 0.15 approximated the 0.71 ± 0.21 of the <italic toggle=\"yes\">Observed</italic> survival probability after actual revascularization. Bland-Altman plot showed a small mean bias of +0.03 for the <italic toggle=\"yes\">Realistic</italic> over <italic toggle=\"yes\">Observed</italic> survival probability (<italic toggle=\"yes\">P</italic> = .001) due to residual CAD (see ##SUPPL##0##Supplementary data online##, <italic toggle=\"yes\">##SUPPL##0##Figure S2##</italic>).</p>", "<title>Post-revascularization coronary flow capacity in 283 positron emission tomography pairs compared with 2552 post-revascularization positron emission tomography</title>", "<p>The average post-revascularization CFC in the 283 PET pairs (<italic toggle=\"yes\">##FIG##7##Figure 7##</italic>, dash-dot lines) was comparable by KS test to the post-revascularization CFC of the 2552 PET images from patients with prior revascularization (<italic toggle=\"yes\">##FIG##7##Figure 7##</italic>, solid lines). Both had CFC that was significantly more severe than with no prior revascularization (<italic toggle=\"yes\">##FIG##7##Figure 7##</italic>, light dashed lines) (<italic toggle=\"yes\">P</italic> &lt; .00001). Both were much worse than 125 healthy young volunteers without risk factors as a reference (<italic toggle=\"yes\">##FIG##7##Figure 7##</italic>, heavy dashed line with solid squares).</p>", "<title>Repeat revascularization and <italic toggle=\"yes\">Observed</italic> survival probability</title>", "<p>For the 2552 patients with prior revascularization, multi-variable Cox regression modelling shows CFC severe associated with severely reduced survival probability (<italic toggle=\"yes\">##FIG##8##Figure 8##</italic>, solid blue line) compared to no severely reduced CFC (<italic toggle=\"yes\">##FIG##8##Figure 8##</italic>, black line). Additional revascularization is associated with improved survival probability (<italic toggle=\"yes\">##FIG##8##Figure 8##</italic>, dashed blue line). However, after revascularization, substantial mortality risk remains due to residual CFC reflecting residual diffuse or segmental disease or incomplete revascularization.</p>" ]
[ "<title>Discussion</title>", "<p>The data confirm our hypothesis. Severely reduced CFC associates with or predicts low 10-year survival probability that is significantly improved after revascularization compared to no revascularization for comparable CFC severity. However, effects of revascularization on CFC and survival probability are heterogeneous with residual CFC abnormalities and associated limited survival due to residual diffuse CAD, multiple stenosis, incomplete or failed revascularization procedures, or disease progression. Only 5.7% of post-revascularization PETs normalize the CFC map. Survival probability is not improved or worse after revascularization in non-severe CFC (<italic toggle=\"yes\">##FIG##0##Structured Graphical Abstract##</italic>).</p>", "<p>In 283 pre- and post-revascularization PET pairs, the change in CFC maps confirms this heterogeneous response with significant residual CFC abnormalities by KS tests comparing CFC severity histogram plots. The associated <italic toggle=\"yes\">Observed</italic> CFC-associated survival probability as a fraction of 1.0 was on average better (higher) after than before revascularization in the same subject. However, individual changes in the pre- and post-revascularization CFC pair and associated changes in survival probability were heterogeneously better or worse after revascularization. Gender, ethnicity, or microvascular dysfunction played no role in these heterogeneous outcomes likely related to the comprehensive CFC maps that integrate and account for cumulative risk factors, stenosis, and diffuse CAD.</p>", "<p>Finally, in 2552 post-revascularization PET, substantial residual CFC abnormalities were common in association with <italic toggle=\"yes\">Observed</italic> CFC-associated lower survival probability. Both were significantly improved after repeat revascularization compared with no-repeat revascularization for comparable CFC severity.</p>", "<p>Importantly, for pre-revascularization PET with primarily normal CFC and good survival probability, revascularization is associated with worsening CFC and significantly reduced survival probability due to stent-jailed branches, procedure failures, or complications of procedures performed despite normal or adequate CFC.</p>", "<title>Implications for randomized trials</title>", "<p>Our data suggest that survival probability might not have improved in reported revascularization trials for chronic coronary syndromes due to several quantifiable pathophysiologic reasons. Patient selection by angiogram without quantitative perfusion may not select sufficient physiologic severity associated with sufficient mortality to be reduced by revascularization comparably to severely reduced CFC by PET. Second, residual diffuse or multi-stenosis CAD and incomplete or failed revascularization may cause substantial remaining severely reduced CFC incurring high residual mortality risk that is reduced by repeat revascularization that can be <italic toggle=\"yes\">virtually</italic> predicted before intervention. These results parallel and offer a mechanistic explanation for only 43% and 58% of revascularized patients achieving anatomic or functional complete revascularization respectively reported for the ISCHEMIA trial.<sup>##REF##37468185##17##</sup></p>", "<title>Coronary flow capacity as gatekeeper to interventions</title>", "<p>Of the large referral cohort of 6979 patients, 77% had known or suspected CAD, coronary calcium, high-risk factors, or non-severe quantitative PET abnormalities. However, only 21% had severely reduced CFC and only 12% had revascularization based on clinical judgement, comorbidities, size, and distal vs. proximal severe stress defects. Moreover, of 283 pre- and post-revascularization paired PETs, only 5.7% completely normalized the CFC after revascularization with associated normalized survival probability. Adding up to 23 additional clinical covariates did not change the mortality benefit after revascularization (see ##SUPPL##0##Supplementary data online##, <italic toggle=\"yes\">##SUPPL##0##Table S1##</italic>). Therefore, the cumulative data suggest that CFC is the final common cumulative physiologic expression of risk factors and CAD severity for predicting mortality risk that is modifiable by revascularization.</p>", "<title>Contributions of coronary pathophysiology to clinical decision making in chronic CAD</title>", "<p>This study presents a comprehensive extension of the established concepts of experimental pathophysiology of coronary stenosis,<sup>##REF##4808557##18##</sup> quantitative angiographic anatomic-pressure-flow relations,<sup>##REF##4808557##18–23##</sup> fractional flow reserve (FFR),<sup>##REF##8462157##24##</sup> anatomic simulation of relative CFR (FFR<sub>PET</sub>),<sup>##REF##668056##19–23##</sup> subendocardial perfusion,<sup>##REF##36599572##6##,##UREF##1##15##,##REF##30466522##16##,##REF##707289##23##</sup> and severity of diffuse or focal CAD with associated individual survival probability before and after actual and virtual hypothetical revascularization by quantitative CFC<sup>##REF##30115688##4–12##</sup> for clinical decision making in chronic coronary syndromes.</p>", "<p>As the next step in this evolution, the randomized CENTURY trial of a personalized, comprehensive, integrated strategy of lifestyle and medical treatment to goals with interventions for only severely reduced CFC documents significant survival benefit, reduced MI, reduced revascularizations, and angina relief in chronic CAD vs. standard community care (NCT00756379, <ext-link xlink:href=\"https://www.ClinicalTrials.gov\" ext-link-type=\"uri\">ClinicalTrials.gov</ext-link>).<sup>##UREF##2##25##</sup> The considered ESC Guidelines emphasize functional testing before revascularization, its limited survival benefit, and completeness of revascularization.<sup>##REF##31504439##26##,##REF##30165437##27##</sup> The current study and the survival benefit of the randomized CENTURY trial<sup>##UREF##2##25##</sup> suggest that CFC combining stress mL/min/g and CFR per regional LV pixel provide a precise metric of coronary artery-specific size-severity pathophysiology for optimal medical-lifestyle management with PET-guided interventions reserved for severe perfusion abnormalities at high mortality risk that is reduced by revascularization or for refractory angina.</p>", "<title>Limitations of the study</title>", "<p>This report on physiologic CAD severity quantified by CFC is a natural history study of survival probability at a tertiary care referral centre in a large cohort unrestricted by assumed CAD severity thresholds required by randomization, with unknown bias for PET imaging intervention or medical treatment, paralleling potential selection bias for randomized trials. Quantitative invasive or non-invasive physiologic metrics by PET are not widely used, employ diverse protocols, methodology, and criteria for intervention. The study has the limitations of a single tertiary care cardiovascular centre with high prevalence of CAD and quantitative PET perfusion imaging with highly developed, validated analytical software that is not widely available.</p>", "<p>The results are conceptually relevant to all invasive and non-invasive physiologic measurement technologies—quantitative magnetic resonance imaging, CT flow, attenuation corrected single photon emission CT, echo flow, invasive Doppler or thermodilution or FFR, and of course quantitative PET used as the gold standard to prove the clinical concepts. However, implementation requires accurate myocardial perfusion in mL/min/g for quantifying CFC per regional pixel that currently only PET is provided.</p>", "<p>On the other hand, the data have unique strengths that include a precision of ±10% for quantitative perfusion, all cases undergoing the same protocol, on the same scanner, carried out by the same physicians, technical team, using the same software, relational database, and same prospective systematic follow-up with entry-signed consent over 14-year follow-up.</p>" ]
[ "<title>Conclusions</title>", "<p>Severely reduced baseline CFC as a comprehensive integrated physiologic measure of CAD severity and associated <italic toggle=\"yes\">observed</italic> low survival probability is significantly but heterogeneously improved after revascularization compared to no revascularization for comparable severity. For non-severe CFC, survival probability was high with revascularization having no added survival benefit or causing harm. CFC derived <italic toggle=\"yes\">Virtual</italic> survival probability after conceptual hypothetical revascularization was higher on average than CFC-associated <italic toggle=\"yes\">Observed</italic> survival probability after actual revascularization due to residual CAD or failed revascularization. Substantial residual severe CFC abnormalities were common after revascularization with associated reduced survival probability both of which were significantly but heterogeneously improved after <italic toggle=\"yes\">repeat</italic> revascularization compared with no repeat revascularization for comparable CFC severity.</p>" ]
[ "<title>Abstract</title>", "<title>Background and Aims</title>", "<p>Coronary flow capacity (CFC) is associated with an observed 10-year survival probability for individual patients before and after actual revascularization for comparison to virtual hypothetical ideal complete revascularization.</p>", "<title>Methods</title>", "<p>Stress myocardial perfusion (mL/min/g) and coronary flow reserve (CFR) per pixel were quantified in 6979 coronary artery disease (CAD) subjects using Rb-82 positron emission tomography (PET) for CFC maps of artery-specific size-severity abnormalities expressed as percent left ventricle with prospective follow-up to define survival probability per-decade as fraction of 1.0.</p>", "<title>Results</title>", "<p>Severely reduced CFC in 6979 subjects predicted low survival probability that improved by 42% after revascularization compared with no revascularization for comparable severity (<italic toggle=\"yes\">P</italic> = .0015). For 283 pre-and-post-procedure PET pairs, severely reduced regional CFC-associated survival probability improved heterogeneously after revascularization (<italic toggle=\"yes\">P</italic> &lt; .001), more so after bypass surgery than percutaneous coronary interventions (<italic toggle=\"yes\">P</italic> &lt; .001) but normalized in only 5.7%; non-severe baseline CFC or survival probability did not improve compared with severe CFC (<italic toggle=\"yes\">P</italic> = .00001). Observed CFC-associated survival probability after actual revascularization was lower than virtual ideal hypothetical complete post-revascularization survival probability due to residual CAD or failed revascularization (<italic toggle=\"yes\">P</italic> &lt; .001) unrelated to gender or microvascular dysfunction. Severely reduced CFC in 2552 post-revascularization subjects associated with low survival probability also improved after repeat revascularization compared with no repeat procedures (<italic toggle=\"yes\">P</italic> = .025).</p>", "<title>Conclusions</title>", "<p>Severely reduced CFC and associated observed survival probability improved after first and repeat revascularization compared with no revascularization for comparable CFC severity. Non-severe CFC showed no benefit. Discordance between observed actual and virtual hypothetical post-revascularization survival probability revealed residual CAD or failed revascularization.</p>", "<title>Structured Graphical Abstract</title>", "<p>\n\n</p>" ]
[ "<p>\n<bold>See the editorial comment for this article ‘Leveraging global coronary flow assessments to inform revascularization benefit in chronic coronary disease: time to test total heart flow’, by V.R. Taqueti, <ext-link xlink:href=\"https://doi.org/10.1093/eurheartj/ehad812\" ext-link-type=\"uri\">https://doi.org/10.1093/eurheartj/ehad812</ext-link>.</bold>\n</p>", "<title>Supplementary Data</title>", "<p>\n##SUPPL##0##Supplementary data## are available at <italic toggle=\"yes\">European Heart Journal</italic> online.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Declarations</title>", "<title>Disclosure of Interest</title>", "<p>Nothing to declare.</p>", "<title>Data Availability</title>", "<p>If accepted for publication, de-identified data and statistical analysis will be made available on request.</p>", "<title>Funding</title>", "<p>Research supported by internal funds of the Weatherhead PET Center endowment at the University of Texas – Houston.</p>", "<title>Ethical Approval</title>", "<p>All research for this study is approved by the Committee for Protection of Human Subjects (CPHS) at the University of Texas Medical School—Houston, Texas.</p>", "<title>Pre-registered Clinical Trial Number</title>", "<p>Not applicable.</p>" ]
[ "<fig position=\"anchor\" id=\"ehad579_ga1\"><label>Structured Graphical Abstract</label><caption><p>Coronary flow capacity (CFC) and survival prediction after revascularization: physiological basis and clinical implications. CFC maps in one view before (upper row) and after revascularization (lower row) show residual diffuse coronary artery disease (CAD), stenosis, and incomplete or inappropriate revascularization as examples from 283 pre-post-revascularization positron emission tomography (PET) pairs. The 10-year survival probability is determined as a fraction of one for normal CFC (all red) by the proportional distribution of regional size-severity CFC abnormalities. The individual observed survival probabilities for each CFC map before and after actual coronary revascularization are listed below each CFC map. The virtual survival probability predicted for the baseline CFC map after virtual theoretical ideal complete revascularization is listed in the lowest (3rd) row for each case. The plots show the observed survival probability of 6979 PET cases with and without severely reduced CFC with and without non-randomized revascularization followed over 12 years as the database from which individual survival probability is determined by multi-variable CFC components by Cox regression modelling. LAD, left anterior descending coronary artery; LCx, left circumflex artery; RCA, right coronary artery.</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F1\" fig-type=\"figure\"><label>Figure 1</label><caption><p>Regional pixel values of coronary flow reserve and stress (cc/min/g) are colour coded in prespecified ranges and mapped into their left ventricle location to produce a coronary flow capacity map for % of the left ventricle as excellent (red), adequate (orange), mildly reduced (yellow), moderately reduced (green), severely reduced (blue), or myocardial steal (dark blue) in artery specific distributions down to tertiary branches as further defined in the text</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F2\" fig-type=\"figure\"><label>Figure 2</label><caption><p>Survival probability for severely reduced (blue lines) and non-severely reduced coronary flow reserve (black lines) with (dashed lines) and without (solid lines) revascularization by multi-variable Cox regression analysis of 6979 coronary flow reserve maps over 12 years follow-up. While survival probability is significantly improved after revascularization for severely reduced coronary flow reserve, residual reduced survival probability persists. CI, confidence intervals</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F3\" fig-type=\"figure\"><label>Figure 3</label><caption><p>Coronary flow capacity maps show the range of quantitative regional severity-size of perfusion abnormalities in a set of 283 cases with coronary flow capacity maps before and after revascularization with coronary flow capacity derived individual <italic toggle=\"yes\">Observed</italic> coronary flow capacity associated survival probability after actual revascularization beside each image. The <italic toggle=\"yes\">Optimal Virtual</italic> survival probability after optimal, ideal, complete hypothetical revascularization for each individual is listed in italics of the beige column</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F4\" fig-type=\"figure\"><label>Figure 4</label><caption><p>Comparisons of coronary flow reserve size-severity cumulative histograms of PET before and after revascularization by the Kolmogorov–Smirnov test for the 283 pre-post-revascularization paired PET’s, and pre-revascularization PET 1 having (<italic toggle=\"yes\">A</italic>) severely reduced coronary flow reserve; (<italic toggle=\"yes\">B</italic>) no severely reduced coronary flow reserve in the pre-revascularization PET 1; and normal CFC for ≥90% of left ventricle in the pre-revascularization PET 1 (<italic toggle=\"yes\">C</italic>), showing improved (<italic toggle=\"yes\">D</italic>) or worsened survival probability for baseline severely reduced coronary flow reserve (<italic toggle=\"yes\">E</italic>), and markedly worse survival probability after revascularization of no severely reduced coronary flow reserve (<italic toggle=\"yes\">F</italic>)</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F5\" fig-type=\"figure\"><label>Figure 5</label><caption><p>Observed survival probability associated with abnormal coronary flow reserve as % of left ventricle. For 283 paired PET before and after revascularization in the same patient, the observed survival probability is lower (worse) with increasing size of abnormal coronary flow reserve defined as mild, moderate, or severe as % of left ventricle, that is worse with myocardial scar</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F6\" fig-type=\"figure\"><label>Figure 6</label><caption><p>\n<italic toggle=\"yes\">Virtual</italic> survival probabilities after hypothetical revascularization vs. <italic toggle=\"yes\">Observed</italic> survival probability after actual revascularization</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F7\" fig-type=\"figure\"><label>Figure 7</label><caption><p>Kolmogorov—Smirnov statistic for cumulative histogram distribution of coronary flow capacity severity 2552 cases with revascularization before PET (solid line) vs. 4427 without revascularization before PET (fine dashed line) vs. the post revascularization PET of the 283 pre- post-revascularization PET pairs (dash-dot line) vs. healthy young volunteers without risk factors (heavy dashed line). Positron emission tomography after prior revascularization has substantial residual diffuse (mild) and focal (moderate or severe) coronary flow capacity abnormalities that are significantly more severe than PET with no prior revascularization or healthy volunteers</p></caption></fig>", "<fig position=\"float\" id=\"ehad579-F8\" fig-type=\"figure\"><label>Figure 8</label><caption><p>Multi-variable Cox regression analysis of 2552 coronary flow reserve maps after prior revascularization with 12 years follow-up for, <italic toggle=\"yes\">Observed</italic> coronary flow reserve associated survival probability for severely reduced (blue lines) and non-severely reduced coronary flow reserve (black lines) with (dashed lines) and without (solid lines) revascularization. While survival probability is significantly improved for severely reduced coronary flow reserve after another revascularization, substantially reduced survival probability persists (dashed blue line) that is comparable to the post-revascularization survival probability with no severely reduced coronary flow reserve (solid black lines) after the first revascularization. The numbers with no severely reduced coronary flow reserve after the first revascularization having another revascularization are too small for statistical analysis. CI, confidence intervals</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"ehad579-T1\"><label>Table 1</label><caption><p>Analysis schema</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col valign=\"top\" align=\"left\" span=\"1\"/></colgroup><tbody><tr><td rowspan=\"1\" colspan=\"1\">\n\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"ehad579-T2\"><label>Table 2</label><caption><p>Characteristics of 283 pre- and post-revascularization positron emission tomography pairs</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">N</italic> = 283 PET pairs</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic toggle=\"yes\">N</italic> = 283 pre-procedure</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic toggle=\"yes\">N</italic> = 283 post procedure</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Pre vs. post</th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristic</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Avg or #</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">SD or %</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Avg or #</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">SD or %</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Test</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">P</italic>-value</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">65.2</td><td rowspan=\"1\" colspan=\"1\">9.9</td><td rowspan=\"1\" colspan=\"1\">66.7</td><td rowspan=\"1\" colspan=\"1\">9.9</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">.076</td></tr><tr><td rowspan=\"1\" colspan=\"1\">BMI</td><td rowspan=\"1\" colspan=\"1\">29.0</td><td rowspan=\"1\" colspan=\"1\">4.7</td><td rowspan=\"1\" colspan=\"1\">28.7</td><td rowspan=\"1\" colspan=\"1\">4.7</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">.484</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">234</td><td rowspan=\"1\" colspan=\"1\">83%</td><td rowspan=\"1\" colspan=\"1\">234</td><td rowspan=\"1\" colspan=\"1\">83%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of PCI</td><td rowspan=\"1\" colspan=\"1\">182</td><td rowspan=\"1\" colspan=\"1\">64%</td><td rowspan=\"1\" colspan=\"1\">261</td><td rowspan=\"1\" colspan=\"1\">92%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">&lt;.00001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of CABG</td><td rowspan=\"1\" colspan=\"1\">94</td><td rowspan=\"1\" colspan=\"1\">33%</td><td rowspan=\"1\" colspan=\"1\">134</td><td rowspan=\"1\" colspan=\"1\">47%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of MI recent</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">4%</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">4%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of MI distant</td><td rowspan=\"1\" colspan=\"1\">80</td><td rowspan=\"1\" colspan=\"1\">28%</td><td rowspan=\"1\" colspan=\"1\">109</td><td rowspan=\"1\" colspan=\"1\">39%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.010</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of hypertension</td><td rowspan=\"1\" colspan=\"1\">256</td><td rowspan=\"1\" colspan=\"1\">91%</td><td rowspan=\"1\" colspan=\"1\">259</td><td rowspan=\"1\" colspan=\"1\">92%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.660</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of dyslipidemia</td><td rowspan=\"1\" colspan=\"1\">281</td><td rowspan=\"1\" colspan=\"1\">99%</td><td rowspan=\"1\" colspan=\"1\">281</td><td rowspan=\"1\" colspan=\"1\">99%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of diabetes</td><td rowspan=\"1\" colspan=\"1\">163</td><td rowspan=\"1\" colspan=\"1\">58%</td><td rowspan=\"1\" colspan=\"1\">167</td><td rowspan=\"1\" colspan=\"1\">59%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.733</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx of smoking</td><td rowspan=\"1\" colspan=\"1\">186</td><td rowspan=\"1\" colspan=\"1\">66%</td><td rowspan=\"1\" colspan=\"1\">182</td><td rowspan=\"1\" colspan=\"1\">64%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.724</td></tr><tr><td rowspan=\"1\" colspan=\"1\">medication_statin</td><td rowspan=\"1\" colspan=\"1\">242</td><td rowspan=\"1\" colspan=\"1\">86%</td><td rowspan=\"1\" colspan=\"1\">264</td><td rowspan=\"1\" colspan=\"1\">93%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.003</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">medication_antiplatelet</td><td rowspan=\"1\" colspan=\"1\">252</td><td rowspan=\"1\" colspan=\"1\">89.%</td><td rowspan=\"1\" colspan=\"1\">278</td><td rowspan=\"1\" colspan=\"1\">98%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">&lt;.00001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">medication_betablocker</td><td rowspan=\"1\" colspan=\"1\">190</td><td rowspan=\"1\" colspan=\"1\">67%</td><td rowspan=\"1\" colspan=\"1\">210</td><td rowspan=\"1\" colspan=\"1\">74%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.065</td></tr><tr><td rowspan=\"1\" colspan=\"1\">medication_ACEIorARB</td><td rowspan=\"1\" colspan=\"1\">194</td><td rowspan=\"1\" colspan=\"1\">69%</td><td rowspan=\"1\" colspan=\"1\">193</td><td rowspan=\"1\" colspan=\"1\">68%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.928</td></tr><tr><td rowspan=\"1\" colspan=\"1\">medication_ccb</td><td rowspan=\"1\" colspan=\"1\">54</td><td rowspan=\"1\" colspan=\"1\">19%</td><td rowspan=\"1\" colspan=\"1\">54</td><td rowspan=\"1\" colspan=\"1\">19%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">medication_diuretic</td><td rowspan=\"1\" colspan=\"1\">99</td><td rowspan=\"1\" colspan=\"1\">35%</td><td rowspan=\"1\" colspan=\"1\">102</td><td rowspan=\"1\" colspan=\"1\">36%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.792</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Stress EF</td><td rowspan=\"1\" colspan=\"1\">59%</td><td rowspan=\"1\" colspan=\"1\">13%</td><td rowspan=\"1\" colspan=\"1\">60%</td><td rowspan=\"1\" colspan=\"1\">13%</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">.358</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Abnor relative stress image</td><td rowspan=\"1\" colspan=\"1\">246</td><td rowspan=\"1\" colspan=\"1\">87%</td><td rowspan=\"1\" colspan=\"1\">228</td><td rowspan=\"1\" colspan=\"1\">81%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.040</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Coronary calcium &gt; 120 HU</td><td rowspan=\"1\" colspan=\"1\">276</td><td rowspan=\"1\" colspan=\"1\">98%</td><td rowspan=\"1\" colspan=\"1\">283</td><td rowspan=\"1\" colspan=\"1\">100%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.003</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx_CAD</td><td rowspan=\"1\" colspan=\"1\">230</td><td rowspan=\"1\" colspan=\"1\">81%</td><td rowspan=\"1\" colspan=\"1\">283</td><td rowspan=\"1\" colspan=\"1\">100%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.0001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx current angina typical</td><td rowspan=\"1\" colspan=\"1\">85</td><td rowspan=\"1\" colspan=\"1\">30%</td><td rowspan=\"1\" colspan=\"1\">35</td><td rowspan=\"1\" colspan=\"1\">12%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">&lt;.00001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx current angina atypical</td><td rowspan=\"1\" colspan=\"1\">23</td><td rowspan=\"1\" colspan=\"1\">8%</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">10%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.383</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx typical/atypical angina</td><td rowspan=\"1\" colspan=\"1\">108</td><td rowspan=\"1\" colspan=\"1\">38%</td><td rowspan=\"1\" colspan=\"1\">64</td><td rowspan=\"1\" colspan=\"1\">23%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">&lt;.00001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx current dyspnoea</td><td rowspan=\"1\" colspan=\"1\">97</td><td rowspan=\"1\" colspan=\"1\">34%</td><td rowspan=\"1\" colspan=\"1\">70</td><td rowspan=\"1\" colspan=\"1\">25%</td><td rowspan=\"1\" colspan=\"1\">chi3</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.013</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hx past dyspnoea</td><td rowspan=\"1\" colspan=\"1\">58</td><td rowspan=\"1\" colspan=\"1\">21%</td><td rowspan=\"1\" colspan=\"1\">69</td><td rowspan=\"1\" colspan=\"1\">24%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">.268</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PET stress angina #</td><td rowspan=\"1\" colspan=\"1\">241</td><td rowspan=\"1\" colspan=\"1\">85%</td><td rowspan=\"1\" colspan=\"1\">132</td><td rowspan=\"1\" colspan=\"1\">47%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">&lt;.00001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PET stress ST Δ #</td><td rowspan=\"1\" colspan=\"1\">241</td><td rowspan=\"1\" colspan=\"1\">85%</td><td rowspan=\"1\" colspan=\"1\">153</td><td rowspan=\"1\" colspan=\"1\">54%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">&lt;.00001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rest relat defect ≤ 60% of LV</td><td rowspan=\"1\" colspan=\"1\">6.9%</td><td rowspan=\"1\" colspan=\"1\">9.2%</td><td rowspan=\"1\" colspan=\"1\">7.1%</td><td rowspan=\"1\" colspan=\"1\">9.7%</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">.792</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Strs rel defect ≤ 60% of LV</td><td rowspan=\"1\" colspan=\"1\">21.4%</td><td rowspan=\"1\" colspan=\"1\">17.2%</td><td rowspan=\"1\" colspan=\"1\">14.9%</td><td rowspan=\"1\" colspan=\"1\">15.6%</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">&lt;.00001</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CFR minimum quad avg</td><td rowspan=\"1\" colspan=\"1\">1.6</td><td rowspan=\"1\" colspan=\"1\">0.7</td><td rowspan=\"1\" colspan=\"1\">1.8</td><td rowspan=\"1\" colspan=\"1\">0.6</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.0001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CFR minimum quad avg &lt; 2</td><td rowspan=\"1\" colspan=\"1\">207</td><td rowspan=\"1\" colspan=\"1\">73%</td><td rowspan=\"1\" colspan=\"1\">167</td><td rowspan=\"1\" colspan=\"1\">59%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.0001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CFR global avg</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">0.7</td><td rowspan=\"1\" colspan=\"1\">2.1</td><td rowspan=\"1\" colspan=\"1\">0.6</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.017</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CFR maximum</td><td rowspan=\"1\" colspan=\"1\">3.1</td><td rowspan=\"1\" colspan=\"1\">0.97</td><td rowspan=\"1\" colspan=\"1\">3.1</td><td rowspan=\"1\" colspan=\"1\">0.9</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">.918</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Severe CFC fraction of LV</td><td rowspan=\"1\" colspan=\"1\">11.2%</td><td rowspan=\"1\" colspan=\"1\">15.9%</td><td rowspan=\"1\" colspan=\"1\">6%</td><td rowspan=\"1\" colspan=\"1\">12%</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">&lt;.00001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mild CFC &gt; 15% of LV</td><td rowspan=\"1\" colspan=\"1\">226</td><td rowspan=\"1\" colspan=\"1\">80%</td><td rowspan=\"1\" colspan=\"1\">198</td><td rowspan=\"1\" colspan=\"1\">70%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.007</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mod CFC &gt; 15% of LV</td><td rowspan=\"1\" colspan=\"1\">73</td><td rowspan=\"1\" colspan=\"1\">26%</td><td rowspan=\"1\" colspan=\"1\">47</td><td rowspan=\"1\" colspan=\"1\">17%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.008</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Severe CFC &gt; 0% of LV</td><td rowspan=\"1\" colspan=\"1\">184</td><td rowspan=\"1\" colspan=\"1\">65%</td><td rowspan=\"1\" colspan=\"1\">142</td><td rowspan=\"1\" colspan=\"1\">50%</td><td rowspan=\"1\" colspan=\"1\">chi2</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.0001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Strs cc/min/g MQA</td><td rowspan=\"1\" colspan=\"1\">1.24</td><td rowspan=\"1\" colspan=\"1\">0.57</td><td rowspan=\"1\" colspan=\"1\">1.42</td><td rowspan=\"1\" colspan=\"1\">0.58</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.0001</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Strs mL/mim/g global avg</td><td rowspan=\"1\" colspan=\"1\">1.59</td><td rowspan=\"1\" colspan=\"1\">0.57</td><td rowspan=\"1\" colspan=\"1\">1.70</td><td rowspan=\"1\" colspan=\"1\">0.57</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">.014</italic>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Strs mL/min/g maximum</td><td rowspan=\"1\" colspan=\"1\">2.48</td><td rowspan=\"1\" colspan=\"1\">0.69</td><td rowspan=\"1\" colspan=\"1\">2.52</td><td rowspan=\"1\" colspan=\"1\">0.68</td><td rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">t</italic>-test</td><td rowspan=\"1\" colspan=\"1\">.469</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"sup1\" position=\"float\" content-type=\"local-data\"><label>ehad579_Supplementary_Data</label></supplementary-material>" ]
[ "<table-wrap-foot><fn id=\"tblfn10\"><p>The italics of &lt;.00001 indicate a highly significant difference in the clinical history of having additional revascularization procedures between the two paired PET scans.</p></fn><fn id=\"tblfn1\"><p>BMI, body mass index; CFR, coronary flow reserve; CFC, coronary flow capacity; HU, Hounsfield units; MI, myocardial infarction; MQA, minimum quadrant average; Rest relat, % of LV with rest relative defect ≤ 60% of maximum activity; Strs rel, % of LV with stress relative defect ≤ 60% of maximum activity; quad avg, quadrant average.</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"ehad579_ga1\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f1\" position=\"float\"/>", "<inline-graphic xlink:href=\"ehad579il1.jpg\"/>", "<graphic xlink:href=\"ehad579f2\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f3\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f4\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f5\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f6\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f7\" position=\"float\"/>", "<graphic xlink:href=\"ehad579f8\" position=\"float\"/>" ]
[ "<media xlink:href=\"ehad579_supplementary_data.docx\"><caption><p>Click here for additional data file.</p></caption></media>" ]
[{"label": ["14"], "mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Lai", "Shiao"], "given-names": ["D", "SP"], "article-title": ["Comparing two clinical measurements: a linear mixed model approach"], "source": ["J Appl Stat"], "year": ["2005"], "volume": ["32"], "fpage": ["855"], "lpage": ["60"], "pub-id": ["10.1080/02664760500080157"]}, {"label": ["15"], "mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Gould", "Johnson", "Narula"], "given-names": ["KL", "NP", "J"], "article-title": ["Alternative viewpoint\u2014microvascular dysfunction or diffuse epicardial CAD with normal stress vasodilation"], "source": ["JACC Cardiovasc Imaging"], "year": ["2023"], "volume": ["14"], "fpage": ["549"], "lpage": ["55"], "pub-id": ["10.1016/j.jcmg.2022.10.029"]}, {"label": ["25"], "mixed-citation": ["\n"], "person-group": ["\n"], "string-name": ["\n"], "surname": ["Gould", "Johnson", "Kitkungvan", "Kirkeeide", "Haynie", "Nguyen"], "given-names": ["KL", "NP", "D", "R", "M", "T"], "etal": ["et al"], "comment": ["The randomized CENTURY trial in chronic CAD of comprehensive integrated lifestyle modification, maximal pharmacological treatment and using coronary flow capacity by quantitative PET to guide interventions. Reported at the ESC 2023 Congress, Late Breaking Trials, Amsterdam, August 27"], "year": ["2023"]}]
{ "acronym": [], "definition": [] }
27
CC BY
no
2024-01-14 23:43:50
Eur Heart J. 2023 Aug 27; 45(3):181-194
oa_package/03/bc/PMC10787661.tar.gz
PMC10787662
0
[ "<title>Introduction</title>", "<p><italic>Dirofilaria immitis</italic> is a mosquito-borne parasitic nematode that primarily infects canines, causing heartworm disease. Although human infections are rare, they have been reported in regions endemic to <italic>Dirofilaria</italic>. Human dirofilariasis is caused mainly by <italic>Dirofilaria</italic> belonging to two species: <italic>Dirofilaria repens</italic> and <italic>Dirofilaria immitis</italic> [##REF##22763636##1##], and it usually presents as subcutaneous nodules and pulmonary lesions [##REF##8758146##2##]. <italic>Dirofilaria</italic> presenting as a breast lump is rare and occurs as solid or mostly cystic lesions [##REF##19705438##3##]. Given the potential for misdiagnosis, clinicians need to consider parasitic infections when evaluating patients with unusual breast lumps.</p>" ]
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[ "<title>Discussion</title>", "<p>Dirofilariasis is a zoonotic infection transmitted to humans through mosquito bites. Human dirofilariasis is an infrequent helminthic infection caused by filarial worms of the <italic>Dirofilaria</italic> species, with occasional zoonotic transmission. These worms naturally parasitize dogs, cats, foxes, and various wild mammals [##REF##15937374##4##]. Transmission to humans occurs through mosquitoes. While most cases of dirofilariasis are asymptomatic, symptomatic individuals typically present with subcutaneous nodules or lung parenchymal disease.</p>", "<p>In India, where most documented cases of human dirofilariasis occur, ocular infections are prevalent [##REF##11556571##5##,##REF##12082307##6##]. <italic>Dirofilaria repens</italic> is the primary causative agent in subcutaneous human dirofilariasis in India and the Asian subcontinent. Coastal Kerala is particularly endemic to dirofilariasis due to the presence of suitable vectors and climatic conditions [##REF##15937374##4##]. While <italic>Dirofilaria repens</italic> is the predominant cause in the northern and western parts of India [##REF##15949193##7##], there have been a few reported cases of <italic>Dirofilaria immitis</italic> [##REF##2607577##8##]. The mosquitoes <italic>Culex</italic>, <italic>Aedes</italic>, and even <italic>Anopheles</italic> species form the vector for this nematode [##REF##23776842##9##].</p>", "<p>Clinically, human dirofilariasis poses challenges as subcutaneous lesions may be initially misidentified as malignant tumors. This misidentification often leads to invasive investigations and surgeries before an accurate diagnosis is established. The pathology of the condition arises from the abnormal localization of immature worms intended for nonhuman hosts.</p>", "<p>The incidence of zoonotic filariasis is increasing worldwide, possibly attributed to global warming and climate change [##REF##22763636##1##,##REF##32959768##10##, ####REF##33027710##11##, ##REF##23961434##12####23961434##12##]. Studies have demonstrated that elevated temperatures can accelerate larval stage 3 development in mosquitoes, shorten their developmental period, modify seasonal transmission patterns, influence mosquito feeding behavior, and expand breeding areas [##REF##22763636##1##,##REF##28642878##13##]. Although the lungs and subcutaneous tissues are the most common sites of involvement [##REF##27579206##14##], breast involvement is extremely rare. The clinical presentation of dirofilariasis can mimic various subcutaneous conditions, including breast masses, leading to potential misdiagnosis. The subcutaneous nodules, which may be located anywhere in the body, often evoke suspicion of tumor growth, necessitating an excisional biopsy to exclude malignancy [##REF##1575206##15##,##REF##11976852##16##].</p>", "<p>In this case, the patient presented with a breast lump, which was initially considered to be a benign fibroepithelial lesion based on FNAC. However, further investigations revealed the presence of <italic>Dirofilaria immitis</italic>, highlighting the importance of considering parasitic infections in the differential diagnosis of breast masses, particularly in regions where the parasite is endemic.</p>", "<p>Histopathological examination remains crucial in establishing the diagnosis of dirofilariasis. However, serological testing can help confirm the infection. Treatment options for human dirofilariasis include surgical excision, antiparasitic medications, or a combination of both.</p>" ]
[ "<title>Conclusions</title>", "<p>This case report emphasizes the importance of considering parasitic infections, such as <italic>Dirofilaria immitis</italic>, in the differential diagnosis of breast masses. Clinicians should maintain a high index of suspicion, especially in areas where the parasite is endemic. Histopathological examination and serological testing are essential for confirming the diagnosis. Prompt diagnosis and appropriate management can lead to favorable outcomes for patients presenting with this rare manifestation of dirofilariasis.</p>" ]
[ "<p><italic>Dirofilaria</italic>, commonly known as heartworm, is a parasitic nematode that primarily infects canines. However, human infections have been reported and can present as subcutaneous nodules in different parts of the body. We present a case of a 43-year-old female who presented with a breast lump that was ultimately diagnosed as a <italic>Dirofilaria</italic> infection, a rare occurrence in humans. This case report shows that considering parasites in unusual presentations is of utmost importance, especially in regions known to have a high prevalence of such infections.</p>" ]
[ "<title>Case presentation</title>", "<p>A 43-year-old female from mid-Kerala, South India, presented to the clinic with a painless lump in her left breast that had been present for three weeks. She had no significant medical history. She is of low socioeconomic status, and there are many mosquito breeding places in and around her house, and there are dogs in her neighbor's home. Her mother died of carcinoma lung, and her mother's sister was diagnosed with carcinoma breast. Physical examination revealed a firm, mobile, non-tender lump measuring 2 x 1 cm located in the upper inner quadrant of the left breast with a benign-looking lymph node in the left axilla. Considering the patient's age, family history, and the presence of a palpable breast mass, a mammogram and fine-needle aspiration cytology (FNAC) were performed. The mammogram (Figure ##FIG##0##1##) with ultrasonogram correlation showed a well-defined hypoechoic lesion with multiple internal septations and echogenic areas - breast imaging-reporting and data system (BI-RADS 4A).</p>", "<p>FNAC revealed a suppurative inflammatory lesion, and the sample revealed no malignant cells, indicating a benign nature. Her blood count was normal. Given the patient's persistent concern and the atypical appearance of the mass, a wide local excision was performed. Histopathological examination revealed greyish-white firm soft tissue measuring 2 x 2 cm and oval in shape. The cut section surprisingly showed a thread-like worm 1 cm long. On microscopy, the lesion demonstrated a chronic inflammatory lesion (Figure ##FIG##1##2##) with suppuration and a foreign body giant cell reaction (Figure ##FIG##2##3##). A cross-section of the worm is seen (Figure ##FIG##3##4##), morphologically consistent with <italic>Dirofilaria</italic>. Subsequent evaluation of the patient's serology confirmed the diagnosis of <italic>Dirofilaria immitis</italic> infection. No complications or recurrences were observed during the follow-up period.</p>" ]
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[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Mammogram</title><p>Dense breast tissue with benign axillary lymph node</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Histopathology</title><p>H&amp;E stain with 400x magnification showing numerous eosinophils and plasma cells in the surrounding fibrous tissue close to the parasite</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>Histopathology</title><p>100x magnification, H &amp;E stain showing multinucleated foreign body giant cells close to the parasite</p></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG4\"><label>Figure 4</label><caption><title>Microscopy</title><p>400x magnification, H&amp;E stain showing a cross-section of the worm</p></caption></fig>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Ravindran Chirukandath, Sumin V. Sulaiman, Sharath K. Krishnan</p><p><bold>Drafting of the manuscript:</bold>  Ravindran Chirukandath, Sumin V. Sulaiman, Sharath K. Krishnan, Rajesh M. Subramanian, Shahina Salim Aysha</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Ravindran Chirukandath, Sumin V. Sulaiman, Sharath K. Krishnan</p><p><bold>Supervision:</bold>  Ravindran Chirukandath</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Sumin V. Sulaiman, Sharath K. Krishnan, Rajesh M. Subramanian, Shahina Salim Aysha</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050535-i01\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050535-i02\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050535-i03\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050535-i04\" position=\"float\"/>" ]
[]
[]
{ "acronym": [], "definition": [] }
16
CC BY
no
2024-01-14 23:43:50
Cureus.; 15(12):e50535
oa_package/ec/a1/PMC10787662.tar.gz
PMC10787663
0
[ "<title>Introduction</title>", "<p>Similar to all subtypes of lymphoma, mantle cell lymphoma (MCL) most commonly presents with constitutional “B-symptoms” (fevers, night sweats, weight loss), lymphadenopathy (LAD), and blood count abnormalities [##UREF##0##1##]. While it is less common, extranodal involvement of MCL can be the initial presentation and most commonly involves the spleen, bone marrow, and gastrointestinal (GI) tract [##UREF##0##1##]. When presenting with extranodal involvement, it is determined to be stage IV, consistent with the frequently advanced stage of MCL at initial diagnosis [##REF##24857076##2##]. </p>", "<p>The frequency of GI involvement of MCL has previously been reported to be 15-30%, with the lower GI tract being the most common site of GI involvement (53.6% lower GI tract involvement vs. 14.3% upper GI tract involvement) [##REF##12548600##3##,##UREF##1##4##]. Most commonly, GI involvement is characterized by nodules or polyps present on endoscopic evaluation. Other studies have demonstrated even higher rates, reporting the presence of GI tract involvement in up to 90% of patients with MCL who underwent routine endoscopic evaluation [##REF##12548600##3##, ####UREF##1##4##, ##REF##33414416##5##, ##REF##17001159##6####17001159##6##]. While the stomach and intestines are the most common sites of GI involvement in MCL, involvement of the gallbladder is exceedingly rare in any type of lymphoma, including MCL [##REF##20679881##7##]. In a retrospective review of lymphoma cases at the National Institute of Health, the largest case series of gallbladder lymphomas to date found only 19 total cases of gallbladder involvement [##REF##20679881##7##]. Of the 19 cases identified, only one was found to be MCL, with extranodal marginal zone lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and lymphoblastic lymphoma being more commonly associated with gallbladder involvement [##REF##20679881##7##]. Of these 19 cases, 92% presented with symptoms of cholecystitis, cholelithiasis, and/or jaundice [##REF##20679881##7##]. A literature review was performed and found 36 reported cases of lymphoma involving the gallbladder, though none of these 36 cases were found to be MCL [##REF##20679881##7##].</p>", "<p>Similar to gallbladder involvement of lymphoma, direct kidney involvement of lymphoma is extremely rare. A retrospective review over an 18-year time period at two tertiary hospitals identified 20 total cases of patients with renal parenchymal infiltration of lymphoma [##REF##33729651##8##]. Of the 20 cases identified, only one was found to be MCL, with diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma being more commonly associated with parenchymal involvement of the kidneys [##REF##33729651##8##]. Of the 20 cases, 14 patients (70%) presented with acute kidney injury and one patient (5%) presented with bilateral nephromegaly [##REF##33729651##8##]. In patients presenting with lymphomatous involvement of the kidneys, an elevated serum creatinine has been reported in up to 56% of patients [##UREF##2##9##]. Literature review has shown that with lymphomatous involvement of the kidneys, the most common finding on imaging is the presence of multiple parenchymal nodular masses, occurring in approximately 50-60% of cases [##UREF##2##9##]. Less commonly noted on imaging is the presence of nephromegaly, which almost always occurs bilaterally, and is present in only 6-19% of cases [##UREF##2##9##].</p>", "<p>Overall, gallbladder involvement of MCL is exceptionally rare and has only been reported in case studies [##UREF##3##10##, ####REF##30676368##11##, ##UREF##4##12####4##12##]. Similarly, renal involvement of MCL is also exceptionally uncommon and typically only reported in case studies [##REF##22098707##13##, ####UREF##5##14##, ##REF##35768844##15####35768844##15##]. Here, we present a rare case of a 53-year-old man presenting with both cholecystitis and bilateral nephromegaly secondary to newly diagnosed MCL.</p>" ]
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[ "<title>Discussion</title>", "<p>MCL is a rare subtype of B-cell non-Hodgkin’s lymphoma, comprising between 3-10% of all non-Hodgkin’s lymphoma and having an annual incidence of one per 200,000 people [##UREF##0##1##,##REF##33074750##19##]. Due to overall advancements in medical care, the average life expectancy in the general population continues to increase. Given that the median age at diagnosis of MCL ranges from 60-70 years of age, the incidence of MCL continues to rise due to the prolonged life expectancy [##UREF##0##1##,##REF##28444739##20##]. MCL typically presents as advanced staged disease with widespread disease involvement at initial diagnosis [##REF##24857076##2##]. The overall five-year relative survival rate of patients with MCL is around 50%, with the rate decreasing to approximately 36% in patients &gt;75 years old [##REF##28444739##20##]. Given the frequently advanced stage of MCL at the time of presentation, prompt diagnosis and treatment are crucial to improve patient outcomes [##REF##24857076##2##]. While recognizing the more common presenting symptoms of lymphoma (B-symptoms, LAD, blood count abnormalities) is vital, awareness of the less common presenting symptoms is necessary to diagnose and manage patients with rare presentations in a timely manner [##UREF##0##1##].</p>", "<p>Two of the uncommon extranodal sites of MCL involvement are the gallbladder and kidneys, as seen in our case. While our patient did not have biopsy-proven renal involvement, it was strongly suspected as there was no alternative clinical explanation, and renal function improved with dexamethasone. Given the rarity of these sites of involvement, only case reports detailing these presentations have been described in literature, leading to lack of physician awareness necessary to make a prompt diagnosis [##UREF##3##10##, ####REF##30676368##11##, ##UREF##4##12##, ##REF##22098707##13##, ##UREF##5##14##, ##REF##35768844##15####35768844##15##]. In this case report, the patient initially presented with cholecystitis and bilateral nephromegaly. While cholecystitis isn’t an uncommon presenting symptom for GI involvement of lymphoma, it is rare for MCL to be the underlying lymphoma subtype [##REF##20679881##7##]. Additionally, renal lymphomatous involvement presenting as nephromegaly is rare in all subtypes of lymphoma, including MCL [##REF##33729651##8##,##UREF##2##9##].</p>", "<p>With a new diagnosis of MCL that is symptomatic on presentation, single-agent therapy with corticosteroids is commonly utilized to produce a temporary response [##UREF##9##21##]. However, it is paramount to obtain histologic evidence of lymphoma prior to the initiation of steroids to avoid reductions in the diagnostic yield of biopsy whenever possible. In this case report, the patient had symptomatic involvement of the gallbladder and kidneys which was initially treated with a five-day course of dexamethasone, resulting in symptomatic improvement and improvement in renal function. Once patients are clinically stabilized and a final diagnosis of MCL has been made, decisions regarding treatment with chemoimmunotherapy and/or HSCT are determined. Generally, fit patients with advanced staged, symptomatic MCL initially undergo chemoimmunotherapy [##UREF##0##1##,##REF##24857076##2##]. Some commonly utilized chemoimmunotherapy regimens include R-CHOP (rituximab + cyclophosphamide, doxorubicin, vincristine, prednisone), R-DHAP (rituximab + dexamethasone, cytarabine, cisplatin), BR, and R-BAC [##UREF##0##1##,##REF##24857076##2##]. Patients with advanced, fast-growing MCL commonly undergo an aggressive, intensified treatment regimen. Some commonly utilized induction treatment regimens in these scenarios include the LYMA regimen (four cycles of R-DHAP followed by R-CHOP), NORDIC regimen (R-CHOP alternating with rituximab + high-dose cytarabine), BR followed by rituximab + high-dose cytarabine, TRIANGLE regimen (R-CHOP + ibrutinib alternating with R-DHAP), and HyperCVAD + R (cyclophosphamide, vincristine, doxorubicin, dexamethasone + rituximab) [##UREF##10##22##]. The patient in this case report was initially treated with BR, followed by R-BAC, which has been shown to be an effective approach in patients with previously untreated MCL [##REF##23401442##23##]. In eligible patients who achieve remission after initial induction therapy, utilization of HSCT consolidation is a commonly utilized approach in the management of MCL [##UREF##0##1##,##UREF##10##22##,##REF##30154113##24##].</p>" ]
[ "<title>Conclusions</title>", "<p>In conclusion, we report the case of a patient who presented with cholecystitis and bilateral nephromegaly found to be secondary to newly diagnosed MCL. Given the patient had associated LAD noted on imaging and physical examination, cervical lymph node biopsy and bone marrow biopsy were completed, confirming the diagnosis of MCL. A laparoscopic cholecystectomy was performed, and gallbladder pathology was consistent with MCL involvement. Initial presentation of MCL with extranodal involvement of the gallbladder and/or kidneys is extraordinarily rare and is infrequently documented in literature. We have reported the present case study to assist in the prompt diagnosis and management of patients with atypical presentations of MCL.</p>" ]
[ "<p>Mantle cell lymphoma (MCL) most commonly presents as lymphadenopathy (LAD), fevers, night sweats, weight loss, splenomegaly, and blood count abnormalities. While extranodal involvement as an initial presentation can occur, it is uncommon. At initial diagnosis, MCL most commonly presents as widespread, advanced stage III or IV lymphoma. Given advanced stage MCL at presentation, it is important for medical practitioners to recognize unusual extranodal presentations of MCL for earlier diagnosis and treatment planning. Here, we present a case of MCL initially presenting as cholecystitis and bilateral nephromegaly in a 53-year-old male patient.</p>" ]
[ "<title>Case presentation</title>", "<p>A 53-year-old male with a past medical history of type 2 diabetes mellitus, hypertension, and hyperlipidemia presented to the emergency department with a three-week history of progressive right upper quadrant abdominal pain with associated nausea and vomiting. Of note, he did not have constitutional symptoms at the presentation. Upon arrival, he was found to have acute kidney injury with a creatinine of 3.73 milligrams per deciliter (mg/dl) (reference range: 0.4 - 1.24). He had no known history of renal disease and had a normal creatinine at baseline. He was oliguric upon initial evaluation. Additionally, the patient’s hemoglobin on admission was 8.8 grams per deciliter (reference range: 13.5 - 16.5) without any known history of anemia. The remainder of the initial laboratory evaluation was unremarkable. Physical examination demonstrated right upper quadrant abdominal tenderness to palpation with a positive Murphy’s sign. Additionally, bilateral palpable supraclavicular lymph nodes were noted.</p>", "<p>While in the emergency department, a point-of-care right upper quadrant abdominal ultrasound was performed and showed cholelithiasis with gallbladder wall thickening and a positive sonographic Murphy’s sign indicating cholecystitis. Subsequently, computed tomography (CT) of the abdomen and pelvis was performed and confirmed gallbladder wall thickening. There is a wide range of underlying medical conditions that can lead to gallbladder wall thickening, including cholecystitis, liver disease (hepatitis, cirrhosis, portal hypertension), extra cholecystic inflammation (pancreatitis, colitis, pyelonephritis), systemic diseases (congestive heart failure, renal failure, sepsis), and malignancy (primary gallbladder carcinoma, lymphoma) [##UREF##6##16##]. In addition to these findings, CT of the abdomen and pelvis also showed severe bilateral nephromegaly measuring up to 20 cm (Figures ##FIG##0##1##, ##FIG##1##2##), multifocal LAD (abdominal, pelvic, lower thoracic), and mild splenomegaly. Bilateral nephromegaly can be secondary to a variety of medical conditions, including diabetic nephropathy, pyelonephritis, vasculitis, renal involvement of lymphoma, polycystic kidney disease, bilateral renal cell carcinoma, renal metastases, and many more [##UREF##7##17##]. A CT scan of the chest and neck were performed and demonstrated prominent thoracic LAD and the CT neck demonstrated diffuse cervical LAD and symmetric prominence of the submandibular and parotid glands. Some of the more common underlying causes of cervical lymphadenopathy include malignancy (lymphoma, leukemia, multiple myeloma, metastatic malignancy), infection (viral, bacterial, fungal), autoimmune disease, hyperthyroidism, sarcoidosis, medication-induced, and many more [##UREF##8##18##]. Cholecystectomy was deferred pending further evaluation of LAD.</p>", "<p>Review of a peripheral blood smear showed atypical lymphocytes with small- to medium-sized nuclei. Peripheral blood flow cytometry showed CD5+ (dim) B-cells comprising 27% of total events. B-cells were also positive for CD1d, CD20, CD38, FMC7, and monoclonal surface kappa, while they were negative for CD10, CD23, CD34, and CD200. A bone marrow biopsy was obtained, and pathology showed 13% atypical lymphocytes consistent with CD5+ B-cell lymphoma. Bone marrow flow cytometry showed 20% CD5+ (dim) lymphocytes consistent with dim CD5+ B-cell lymphoma. Additionally, fluorescence in situ hybridization (FISH) was performed on the bone marrow specimen and was positive for t(11;14). A right cervical lymph node core needle biopsy was performed, and immunohistochemical stains were positive for CD5, CD20, Cyclin D1, and BCL-2, while they were negative for CD3 and BCL-6. Ki-67 of the cervical lymph node biopsy showed a proliferation index of 20-30%. Similar to the bone marrow, FISH of the lymph node specimen was positive for t(11;14). These findings were consistent with a diagnosis of MCL. Of note, the patient’s LDH level on presentation was 198 units per liter (reference range: 100 - 210), his mantle cell lymphoma international prognostic index (MIPI) score was 6.1 indicating intermediate risk, and he was determined to have stage IV disease given his extranodal organ involvement.</p>", "<p>After cervical lymph node and bone marrow biopsies had been completed, the patient was started on oral dexamethasone 20 mg daily for a three-day course. His kidney function improved with a decline in creatinine from 4.00 mg/dl to 2.34 mg/dl upon completion of the three-day course of steroids. He subsequently underwent laparoscopic cholecystectomy with the gallbladder specimen being sent for pathology.</p>", "<p>Hematoxylin and Eosin (H&amp;E) sections show extensive lymphomatous involvement of the gallbladder with transmural infiltration and denudation of the mucosal surface (Figure ##FIG##2##3A##, indicated by arrow). In some areas of the gallbladder with remaining intact mucosal surface, lymphoma cells obliterate underlying normal anatomic structure with one intact gland as indicated by an arrow (Figure ##FIG##2##3B##, ##FIG##2##3C##). The lymphoma cells are medium in size and show irregular nuclear contour, vesicular chromatin, occasional indistinct nucleoli and scant amount of cytoplasm. Brisk mitotic figures and necrosis are not evident. CD20 and Cyclin D1 immunohistochemical stains (Figure ##FIG##2##3D##, ##FIG##2##3E## respectively) highlight lymphoma cells and confirm the diagnosis of MCL.</p>", "<p>Following cholecystectomy, the patient completed two additional doses of oral dexamethasone 20 mg daily prior to discharge from the hospital. At the time of discharge, his creatinine had improved to 1.65 mg/dl.</p>", "<p>After discharge, the patient established care with an outpatient hematologist for further management of his newly diagnosed MCL. Of note, his next generation sequencing testing had returned positive for MLH1 A681T variant mutation, but negative for TP53 mutation. Chemoimmunotherapy with BR (bendamustine, rituximab) was initiated, and he completed three cycles. Following this, he was transitioned to treatment with R-BAC (rituximab + bendamustine, cytarabine) to complete three additional cycles. He had been referred for hematopoietic stem cell transplantation (HSCT) evaluation in preparation for potential bone marrow transplant in the future.</p>" ]
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[ "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>CT abdomen and pelvis without contrast (axial orientation) demonstrating severe bilateral nephromegaly measuring up to 20 cm indicated by arrows</title></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>CT abdomen and pelvis without contrast (coronal orientation) demonstrating severe bilateral nephromegaly measuring up to 20 cm indicated by arrows</title></caption></fig>", "<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>Mantle cell lymphoma involving gallbladder. H&amp;E of mantle cell lymphoma (A, x20; B, x200; C, x400). CD20 immunohistochemical stain (D, x200). Cyclin D1 immunohistochemical stain (E, x200). </title></caption></fig>" ]
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[ "<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold>  Kamen W. Kossow, Joseph G. Bennett, Robert J. Kribs, Aung M. Tun, Wei Cui</p><p><bold>Acquisition, analysis, or interpretation of data:</bold>  Kamen W. Kossow</p><p><bold>Drafting of the manuscript:</bold>  Kamen W. Kossow</p><p><bold>Critical review of the manuscript for important intellectual content:</bold>  Kamen W. Kossow, Joseph G. Bennett, Robert J. Kribs, Aung M. Tun, Wei Cui</p><p><bold>Supervision:</bold>  Joseph G. Bennett, Robert J. Kribs, Aung M. Tun</p></fn></fn-group>", "<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>", "<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"cureus-0015-00000050536-i01\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050536-i02\" position=\"float\"/>", "<graphic xlink:href=\"cureus-0015-00000050536-i03\" position=\"float\"/>" ]
[]
[{"label": ["1"], "article-title": ["Mantle cell lymphoma"], "source": ["StatPearls"], "person-group": ["\n"], "surname": ["Lynch", "Koya", "Acharya"], "given-names": ["D", "S", "U"], "publisher-loc": ["Treasure Island"], "publisher-name": ["StatPearls Publishing"], "year": ["2023"], "uri": ["https://www.ncbi.nlm.nih.gov/books/NBK536985/"]}, {"label": ["4"], "article-title": ["Mantle cell lymphoma with gastrointestinal involvement and the role of endoscopic examinations"], "source": ["PLoS One"], "person-group": ["\n"], "surname": ["Lee", "Cho", "Lee"], "given-names": ["HH", "SG", "IS"], "fpage": ["0"], "volume": ["15"], "year": ["2020"]}, {"label": ["9"], "article-title": ["Renal lymphoma"], "source": ["StatPearls"], "person-group": ["\n"], "surname": ["Bokhari", "Rana", "Bokhari"], "given-names": ["M", "U", "S"], "publisher-loc": ["Treasure Island"], "publisher-name": ["StatPearls Publishing"], "year": ["2023"], "uri": ["https://www.ncbi.nlm.nih.gov/books/NBK526034/"]}, {"label": ["10"], "article-title": ["Mantle cell lymphoma presented as enteric intussusception and involved gallbladder"], "source": ["J Cancer Res Pract"], "person-group": ["\n"], "surname": ["Chin", "Shiau", "Wu"], "given-names": ["C", "J", "W"], "fpage": ["105"], "lpage": ["108"], "volume": ["4"], "year": ["2017"]}, {"label": ["12"], "article-title": ["Primary gastrointestinal mantle cell lymphoma involving colon and gallbladder: a case report"], "source": ["Am J Gastroenterol"], "person-group": ["\n"], "surname": ["Prakash", "Siddiqui", "Ashangari"], "given-names": ["S", "S", "C"], "fpage": ["839"], "lpage": ["841"], "volume": ["113"], "year": ["2018"], "uri": ["https://journals.lww.com/ajg/Fulltext/2018/10001/Primary_Gastrointestinal_Mantle_Cell_Lymphoma.1465.aspx"]}, {"label": ["14"], "article-title": ["Renal lymphomatous infiltration by mantle cell lymphoma: treatment with chemoradiation and initiation of angiotensin converting enzyme (ACE) inhibitor for renal protection"], "source": ["J Onconephrol"], "person-group": ["\n"], "surname": ["Damron", "Gunther", "Kala"], "given-names": ["E", "J", "J"], "fpage": ["183"], "lpage": ["187"], "volume": ["5"], "year": ["2021"]}, {"label": ["16"], "article-title": ["Gallbladder wall thickening"], "source": ["AJR Am J Roentgenol"], "person-group": ["\n"], "surname": ["Runner", "Corwin", "Siewert", "Eisenberg"], "given-names": ["GJ", "MT", "B", "RL"], "fpage": ["0"], "volume": ["202"], "year": ["2014"]}, {"label": ["17"], "article-title": ["Bilateral renal enlargement"], "source": ["Radiopaedia"], "person-group": ["\n"], "surname": ["Jha", "Weerakkody", "Bell"], "given-names": ["P", "Y", "D"], "year": ["2023"]}, {"label": ["18"], "article-title": ["Cervical: lymphadenopathy"], "source": ["Head and Neck and Endocrine Surgery"], "person-group": ["\n"], "surname": ["Sakr"], "given-names": ["M"], "fpage": ["163"], "lpage": ["190"], "publisher-loc": ["Cham"], "publisher-name": ["Springer"], "year": ["2016"]}, {"label": ["21"], "article-title": ["Corticosteroids in the treatment of neoplasm"], "source": ["Holland-Frei Cancer Medicine. 6th edition"], "person-group": ["\n"], "surname": ["McKay", "Cidlowski"], "given-names": ["L", "J"], "fpage": ["163"], "lpage": ["190"], "publisher-loc": ["Hamilton"], "publisher-name": ["BC Decker"], "year": ["2003"], "uri": ["https://www.ncbi.nlm.nih.gov/books/NBK13383/"]}, {"label": ["22"], "article-title": ["Mantle Cell Lymphoma. NCCN Guidelines Recommendations (version 4.2023)"], "person-group": ["\n"], "surname": ["National Comprehensive Cancer"], "given-names": ["Network"], "publisher-loc": ["Plymouth Meeting, Pennsylvania"], "publisher-name": ["NCCN"], "year": ["2023"], "uri": ["https://www.nccn.org/patients/guidelines/content/PDF/Mantle-patient-guideline.pdf"]}]
{ "acronym": [], "definition": [] }
24
CC BY
no
2024-01-14 23:43:50
Cureus.; 15(12):e50536
oa_package/8b/0b/PMC10787663.tar.gz
PMC3634987
23456481
[ "<title>Introduction</title>", "<p>Reperfusion strategies have substantially contributed to the effective treatment of ischemic heart diseases despite the potential negative impact of ischemia/reperfusion (I/R) injury[##REF##12181231##1##]. I/R injury is characterized by robust local and systemic inflammatory responses which may aggravate tissue injury and adversely affect left ventricular (LV) recovery [##REF##11744011##2##]. A variety of studies in the last decades has shown that the extent of postischemic tissue damage strongly correlates with the number of leukocytes recruited to the reperfused tissue [##REF##8997297##3##–##REF##6831665##5##]. It is known that increased chemokine expression post I/R could promote the adhesion of neutrophils and increase leukocyte infiltration in the cardiac tissue [##REF##15320517##6##], which could serve as one of the important mechanisms mediating the ischemic myocardial damage. Over the past years, chemokines and their receptors have become the subject of intensive investigations and there is a growing body of evidence that chemokines and their receptors are also critically involved in the pathogenesis of I/R. Chemokines are small, secreted proteins that are produced constitutively or in an inducible manner by most cell types and that induce directed cell migration [##REF##16467548##7##]. CC-chemokine receptor 5 (CCR5) is expressed on T-lymphocytes with memory/effector phenotype, macrophages, monocytes, as well as the immature dendritic cells [##REF##15337520##8##]. The expression levels of CCR5 are very low in the mononuclear cells and T cells of human peripheral blood under normal conditions, but could increase significantly after the inflammatory stimulation both in vivo and in vitro [##REF##2566624##9##]. Previous study showed that TAK-779, a small-molecule, nonpeptide compound that selectively binds to a certain subtype of the CC-chemokine receptor, CCR5, with high affinity [##REF##10318947##10##], could effectively reduce leukocyte infiltration of the reperfused tissue and attenuate subsequent postischemic organ failure in mouse models of focal cerebral ischemia [##REF##16275892##11##]. In this study, we tested the hypothesis that myocardial I/R injury could be attenuated by CCR5 antibody or aggravated by CCR5 agonist RANTES in rats.</p>" ]
[ "<title>Materials and methods</title>", "<title>Animals and reagents</title>", "<p>Healthy adult male Wistar rats (200–250 g) were purchased from Vital River Laboratories, Beijing, China. All experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University. CCR5 antibody and CCR5 agonist RANTES were purchased from Sigma (USA); creatine kinase (CK) and myeloperoxidase (MPO) kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). TNF-α ELISA kit was purchased from Wuhan Boster Bioengineering Co. Ltd. (China). Electrophoretic mobility shift assay (EMSA) kit was purchased from Promega Corp. (Madison, WI, USA).</p>", "<title>Surgical preparation</title>", "<p>The surgical protocol was performed as described previously [##REF##15023554##12##]. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg). After endotracheal intubation with a 14 gauge tube, the rats were then connected to a rodent respirator (TKR-400H, Jiangxi, China, 70 breaths per minute, tidal volume was set to 1.0 ml/100 mg body weight). Body temperature was measured by a rectal thermometer and maintained between 36 and 37 °C by infrared heating lamp. Hemodynamics (left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); and heart rate (HR), +d<italic>P</italic>/d<italic>t</italic>\n<sub>max</sub> and −dP/d<italic>t</italic>\n<sub>max</sub>) were measured through a short segment of saline-filled PE50 tubing which was advanced to left ventricle through right carotid artery and connected to a multi-channel physiological monitoring system (LEAD 2000, Sichuan, China) before left anterior descending coronary artery (LAD) ligation or sham operation. Hemodynamic parameters were obtained immediately after 2-h reperfusion. Electrocardiograph (ECG) leads were connected to the chest and limbs for continuous ECG monitoring throughout the experiment (LEAD 2000, Sichuan, China). Then, the chest was opened via left thoracotomy through the fourth or fifth intercostal space, and the ribs were gently retracted to expose the heart. A 7-0 prolene suture was placed under left anterior descending coronary artery (LAD) after pericardiotomy.</p>", "<title>Experimental protocol</title>", "<p>Sixty rats were randomly divided into five groups: sham operation group (SHAM, <italic>n</italic> = 12), I/R group (I/R, <italic>n</italic> = 12), ischemic preconditioning group (I/R + Pre, <italic>n</italic> = 12), CCR5 antibody group (I/R + CCR5Ab, <italic>n</italic> = 12), and CCR5 agonist (RANTES) group (I/R + CCR5Ago, <italic>n</italic> = 12). Each group was subjected to 30 min of coronary artery occlusion followed by 2 h of reperfusion except sham group. (1) SHAM group: 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and LAD was not ligated; (2) I/R group: LAD ligation for 30 min followed by 2-h reperfusion, 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and after 20-min ischemia; (3) I/R + Pre group: two cycles of 5-min ischemia followed by 5-min reperfusion and one cycle of 10-min ischemia followed by 10-min reperfusion, 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein before the 3rd circle reperfusion; (4) I/R + CCR5Ab group [##REF##19577762##13##]: LAD ligation for 30 min followed by 2-h reperfusion, 0.2 mg/kg CCR5 antibody diluted in 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and after 20-min ischemia; (5) I/R + CCR5Ag group [##REF##19665464##14##]: LAD ligation for 30 min followed by 2-h reperfusion, 0.1 mg/kg RANTES diluted in 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and after 20-min ischemia. For each individual group, six rats were assigned for myocardial MPO activity determination and measurement of infarct zone and risk area and another 6 rats were assigned for myocardial inflammatory cells counting in HE-stained slices, myocardial CCR5 and ICAM-1 expression, and NF-κB activity determination.</p>", "<title>Measurement of infarct zone and risk area</title>", "<p>Immediately after hemodynamic measurements, LAD was re-occluded with a 7-0 prolene suture which was used previously at the same place for rats assigned for myocardial MPO activity determination and measurement of infarct zone and risk area, and Evans blue dye (2 ml of a 1 % solution) was injected via the external jugular vein to delineate the area at risk (AAR). The rats were sacrificed under deep pentobarbital anesthesia (60 mg/kg, i.p.) after blood sampling. The heart was then rapidly excised and washed in 0.9 % saline. After removal of the atrium, the ventricle was cut into transverse slices of equal thickness (3 mm) from the apex to the base. The slices were then incubated for 20 min in phosphate-buffered 1 % 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C, and then fixed in 10 % formalin solution. The AAR was defined as the area not stained with Evans blue dye. The area not stained by TTC was defined as the infarcted zone (AI). The border zones (Evans blue stained area neighboring Evans blue-unstained area), infarcted zones [TTC and The border zones (TTC-stained), infarcted zones (TTC and Evans blue-unstained)], and the nonischemic zones (Evens blue-unstained area remote from Evans blue-unstained area) were photographed and analyzed by the software program Image J 1.36. The AAR, AI, and ventricle size (VS) were assessed by a technician who was blinded to the experimental protocol using computer-assisted planimetry (NIH Image 1.57 software).</p>", "<p>The infarct zone, border zones, and risk area were digital recorded. The percentage of the ischemic region (AR) in the whole LV (AR/LV) represents the severity of myocardial ischemia. The percentage of the infarcted region (IS) in the whole ischemic region (AR) (IS/AR) represents the extent of myocardial infarction. The three parts of LV samples (nonischemic zone, border zone, and infract zone) were stored in −80 °C refrigerator for determining MPO activity.</p>", "<title>Blood collection of tissue sampling</title>", "<p>After hemodynamic measurements, 4-ml blood was obtained from the carotid artery of all rats. Blood samples were placed static for 30 min at room temperature, and then centrifuged at 4,000 r/min for 10 min at 4 °C. The upper serum was removed into new EP tubes and stored in −80 °C refrigerator for detection of CK and TNF-α.</p>", "<p>For rats assigned myocardial inflammatory cells counting in HE-stained slices, myocardial CCR5 and ICAM-1 expression, and NF-κB binding activity determination, rats were sacrificed under deep pentobarbital anesthesia (60 mg/kg, i.p.) after blood sampling, hearts were excised and washed with ice-cold saline solution. Two transversal sections (3-mm thick) from the middle part of each heart were prepared and stained with hematoxylin–eosin (HE) for evaluation of the inflammatory response in the cardiac tissues. Severity of inflammatory cell infiltration on HE staining was scored using the following scale [##REF##10580801##15##]: 0 = no inflammation; 1 = cellular infiltrates only around blood vessel and meninges; 2 = mild cellular infiltrates in parenchyma (1–10/section); 3 = moderate cellular infiltrates in parenchyma (11–100/section); 4 = serious cellular infiltrates in parenchyma (100/section).</p>", "<p>The remaining LV free wall was divided into three parts. One portion of LV free wall was used for the determination of myocardial ICAM-1 and fixed in 4 % paraformaldehyde. The second portion of LV free wall was stored in −80 °C refrigerator until use for NF-κB binding activity determination. The third portion was stored in −80 °C refrigerator until use for myocardial CCR5 protein expression determination.</p>", "<title>Determination of Serum CK, TNF-α, and myocardial MPO activity</title>", "<p>Serum CK level was determined by chemical colorimetric method. The serum level TNF-α was determined by rat TNF-α ELISA kit, referring to the manual. Tetramethyl benzidine method was applied for the determination of myocardial MPO activity.</p>", "<title>Immunohistochemistry</title>", "<p>Immunohistochemical staining of ICAM-1 was performed by the Strept Avidin Biotin Complex (SABC) method. Mouse monoclonal ICAM-1 antibody sc-107 (Santa Cruz Biotechnology, CA) was diluted at 1:100 as primary antibodies. The streptavidin–biotin complex kit was purchased from Wuhan Boster Biological Technology, Ltd. Wuhan, China. All the procedures were carried out according to the manufacturer’s manual. The data of the extent and intensity of staining were obtained using Image Pro Plus Version 6.0 (Media Cybernetics, Bethesda, MD). Five fields of each slice were photographed and analyzed by mean optical density.</p>", "<title>Electrophoretic mobility shift assay</title>", "<p>EMSA method was used to detect the DNA-binding activities of NF-κB in nuclear extracts. NF-κB oligonucleotide’s sequence was 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and 5′-GCCTGGGAAAGTCCCCTCAACT-3′. Protein-DNA binding assays were performed with 20 μg of nuclear protein. In order to block the unspecific binding, 1 μg of poly (dI-dC) • poly (dI-dC) was added to the samples; then apply the binding medium containing 5 % glycerol, 1 % NP40, 1 mM MgCl<sub>2</sub>, 50 mM NaCl, 0.5 mM EDTA, 2 mM DTT, and 10 mM Tris/HCl, and with its pH around 7.5. In each reaction, 20,000 cpm of a radiolabeled probe was included. Samples were incubated at room temperature for 20 min. In order to separate the nuclear protein oligonucleotide complex labeled with 32P from free 32P-labeled oligonucleotide, the samples were subjected to electrophoresis through a 5 % native polyacrylamide gel for 2 h in a running buffer containing 50 mM Tris, pH 8.0, 45 mM borate, and 0.5 mM EDTA. After the separation was achieved, the gel was vacuum-dried for autoradiography and exposed to Fuji X-ray film for 24–48 h at −80 °C. The results were analyzed by medical image analysis system, with its gray-scale value representing the activity of NF-κB [##REF##12470896##16##].</p>", "<title>Western blotting</title>", "<p>Western blotting was conducted to determine protein levels of CCR5 from myocardial tissues. Total cellular membrane proteins were extracted by Plasma Membrane Protein Extraction Kit (Catalog #K268-50) according to Membrane Protein Extraction Protocol. Supernatants were boiled for 10 min in loading buffer, and then separated by SDSPAGE and transferred onto nitrocellulose membranes. After blockage with 5 % skim milk in Tris-buffered saline (TBS) for 1 h at room temperature, the membranes were incubated with primary antibody at 4 °C overnight. After three washings with TBST, (HRP)-labeled secondary antibodies were added and incubated for another 1.5 h on the shaker at room temperature; the blots were then washed two times with TBST. The developed signal was detected using ECL as per the manufacturer’s instructions and exposed to Hyperfilm.</p>", "<p>Antibodies against CCR5 were obtained from Cell Signaling Technology (Boston, MA, USA). The antibody against β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit and anti-mouse HRP-labeled antibodies and the ECL detection reagents were from Santa Cruz Biotechnology. The X-ray film used for Western-blot analysis was from Kodak (Rochester, NY, USA). Other chemicals and reagents were of analytical grade.</p>", "<title>Statistical analysis</title>", "<p>Data are presented as mean ± standard deviation (SD). Normality of distribution of all continuous variables was explored by examining skewness, kurtosis, and Q–Q plots. Differences on continuous data among groups were compared using one-way analysis of variance (ANOVA) followed by either Tukey’s or Games-Howell multiple comparison post-hoc tests as appropriate. Variables with non-normal distribution were compared using the non-parametric Mann–Whitney <italic>U</italic>-statistic test. A <italic>P</italic> value &lt;0.05 was considered statistically significant. Statistical analysis was performed using IBM SPSS, version 20 for Windows.</p>" ]
[ "<title>Results</title>", "<title>Mortality</title>", "<p>Five rats died post LAD ligation due to malignant arrhythmias and the remaining 55 rats were used in the study and 5–6 rats were examined in each individual group.</p>", "<title>Cardiac function</title>", "<p>The cardiac function indexes after 2-h reperfusion or sham operation are shown in Table ##TAB##0##1##. LVSP, +dP/d<italic>t</italic>\n<sub>max</sub>, and −d<italic>P</italic>/d<italic>t</italic>\n<sub>max</sub> were all significantly lower in I/R group than those in sham group and were significantly higher in I/R + Pre group and I/R + CCR5Ab group while significantly lower in I/R + CCR5Ago group as compared to I/R group. LVEDP was increased in I/R group than those in sham group and were significantly reduced in I/R + Pre group and I/R + CCR5Ab group while significantly higher in I/R + CCR5Ago group as compared to I/R group.\n</p>", "<title>Serum levels of CK and TNF-α</title>", "<p>Similarly, serum levels of CK and TNF-α were both significantly higher in I/R group compared to sham group and were significantly reduced in I/R + Pre group and I/R + CCR5Ab groups while significantly increased in I/R + CCR5Ago group compared to I/R group (Fig. ##FIG##0##1##).\n</p>", "<title>Myocardial infarct size</title>", "<p>As shown in Fig. ##FIG##1##2##, the ischemia region (AR/LV) was similar among group. The myocardial infarct size was significantly smaller in I/R + Pre group and I/R + CCR5Ab group while was significantly larger in I/R + CCR5Ago group than those in I/R group, and was significantly smaller in I/R + CCR5Ab group as compared to I/R + Pre group (<italic>P</italic> &lt; 0.05).\n</p>", "<title>MPO activity in myocardial tissues</title>", "<p>MPO activities were significantly increased in I/R group compared to sham group in normal zone; MPO activities were significantly lower in I/R + Pre group and I/R + CCR5Ab group while was significantly higher in I/R + CCR5Ago group in normal, risk and infract zone than in I/R group (Fig. ##FIG##2##3##).\n</p>", "<title>Inflammatory levels in myocardial tissues</title>", "<p>The extent of inflammation of myocardial tissues from LV free wall in different groups was examined in HE-stained transversal myocardial slides. Histopathological images and histological score are listed in Fig. ##FIG##3##4##. Myocardial tissue in I/R and I/R + CCR5Ago groups presented massive inflammatory cell infiltration as compared to sham group indicating that I/R injury could trigger the inflammatory response which was reduced in I/R + Pre and I/R + CCR5Ab group. Accordingly, histological score was significantly lower in I/R + Pre and I/R + CCR5Ab groups than in I/R group and higher in I/R + CCR5Ago group than in I/R + Pre and I/R + CCR5Ab groups.\n</p>", "<title>Myocardial ICAM-1 expression</title>", "<p>Brown particles indicated the expression levels of ICAM-1 in myocardial tissues from LV free wall (Fig. ##FIG##4##5##). Myocardial ICAM-1 expression was significantly upregulated in I/R group compared to sham group and was significantly attenuated in I/R + Pre group and I/R + CCR5Ab group while was significantly increased in I/R + CCR5Ago group compared to that in I/R group.\n</p>", "<title>Myocardial NF-κB binding activity</title>", "<p>The myocardial DNA-binding activity of NF-κB was significantly higher in I/R group than in sham group, and was significantly lower in I/R + Pre and I/R + CCR5Ab groups while higher in I/R + CCR5Ago group as compared to I/R group (Fig. ##FIG##5##6##).\n</p>", "<title>CCR5 protein level of myocardial tissues</title>", "<p>We also determined the membrane protein levels of CCR5 in myocardial tissue from LV free wall (Fig. ##FIG##6##7##). The specific protein expression levels of CCR5 were normalized to β-actin. There is no difference in expression levels of membrane protein CCR5 between sham and I/R groups which was downregulated in I/R + Pre and I/R + CCR5Ab and I/R + CCR5Ago groups.\n</p>" ]
[ "<title>Discussion</title>", "<p>Our study showed that blocking CCR5 attenuates while enhancing CCR5 aggravates myocardial I/R injury through modulating inflammatory responses in rat heart. Thus, strategies modulating CCR5 might serve as potential therapeutic modalities to reducing I/R injury.</p>", "<p>Although the precise mechanism of I/R injury has not been fully revealed, a series of studies have demonstrated that I–R could activate the intrinsic inflammatory network. The adhesion and aggregation of neutrophils in the cardiac tissue might be one of the important factors mediating myocardial I/R injury [##REF##16202368##17##]. CCR5 is a receptor for various proinflammatory chemokines. Blocking CCR5 has been proposed as a novel therapeutic approach for cardiovascular conditions by interfering with systemic inflammation. This concept is supported by an animal study by Veillard et al. [##REF##14656931##18##] in which treatment of hypercholesterolemic mice with the CCR5 antagonist Met-RANTES reduced progression of atherosclerosis and CCL5/RANTES inhibition attenuated myocardial reperfusion injury in atherosclerotic mice [##REF##19665464##14##]. Moreover, treatment of apoE-deficient mice with Met-RANTES reduced neointimal plaque area and macrophage infiltration [##REF##12234959##19##] and treatment with TAK-799, a CCR5 chemokine receptor antagonist, reduced lesion development in a collar-induced carotid artery atherosclerosis model [##REF##16239591##20##]. Finally, TAK-779 treatment also reduced leukocyte infiltration and ischemic injury in a mouse model of focal cerebral ischemia [##REF##16275892##11##]. In line with the above findings, we demonstrated that CCR5 antibody effectively reduced myocardial inflammatory cell infiltration and myocardial infarct size in this rat I/R model. It is to note that CCR5 activation was not evidenced in this I/R model; however, our results showed that CCR5 antibody treatment reduced myocardial injury in this model by reducing inflammatory responses. The exact mechanism responsible for the CCR5 antibody treatment effects in this model warrants further studies. Previous studies found that stimulation of increased TNF-α activity could upregulate ICAM-1 expression which then could function as an adhesion molecule promoting neutrophils infiltration [##REF##2566624##9##]. Treatment with specific antibody of ICAM-1 resulted in coronary vascular and myocardial protection as shown by the decrease of myocardial infarct size [##REF##8997301##21##]. Similarly, we showed that treatment with CCR5 antibody significantly reduced the myocardial expression of ICAM-1. In addition, MPO activity (an indicator of neutrophil accumulation in tissue) decreased significantly in both the risk area and infarcted area in I/R + Pre and I/R + CCR5Ab groups while increased in I/R + CCR5Ago group compared with I/R group. Thus, CCR5 antibody reduced while CCR5 agonist enhanced the inflammation in ischemic hearts by down- or upregulating the expression of TNF-α and ICAM-1. Taken together, treatment with CCR5 antibody that reduced infiltration of neutrophils in the ischemic myocardium might contribute to the reduced infarcted size and serum level of CK in this rat I/R model.</p>" ]
[ "<title>Conclusion</title>", "<p>In conclusion, our study provides the first evidence that CCR5 antibody could reduce cardiac inflammation and protect the heart from I/R injury via inhibition of the activity of NF-κB, ICAM-1 expression, and MPO activities in this rat I/R model. We propose that targeting CCR5 might serve as a potential novel promising strategy for the treatment of ischemic myocardial disease.</p>" ]
[ "<p>The expression level of CC-chemokine receptor 5 (CCR5) is enhanced post inflammatory stimulations and might play a crucial role on inflammatory cells infiltration post myocardial ischemia. The purpose of this study was to evaluate the role of CCR5 on myocardial ischemia–reperfusion (I/R) injury in rats. Adult male rats were randomized to sham group, I/R group (I/R, 30 min coronary artery occlusion followed by 2-h reperfusion), ischemic preconditioning (I/R + Pre), CCR5 antibody group [I/R + CCR5Ab (0.2 mg/kg)], and CCR5 agonist group [I/R + CCR5Ago, RNATES (0.1 mg/kg)], <italic>n</italic> = 12 each group. The serum level of creatine kinase (CK) and tumor necrosis factor α (TNF-α) were measured by ELISA. Myocardial infarction size and myeloperoxidase (MPO) activity were determined. Myocardial protein expression of CCR5 and intercellular adhesion molecule-1 (ICAM-1) were evaluated by Western blotting and immunohistochemistry staining, respectively. Myocardial nuclear factor-kappa B (NF-κB) activity was assayed by electrophoretic mobility shift assay. Myocardial CCR5 protein expression was significantly reduced in I/R + Pre group (<italic>P</italic> &lt; 0.05 vs. I/R) and further reduced in I/R + CCR5Ab group (<italic>P</italic> &lt; 0.05 vs. I/R + Pre). LVSP and ±d<italic>P</italic>/d<italic>t</italic>\n<sub>max</sub> were significantly lower while serum CK and TNF-α as well as myocardial MPO activity, ICAM-1 expression, and NF-κB activity were significantly higher in I/R group than in sham group (all <italic>P</italic> &lt; 0.05), which were significantly reversed by I/R + Pre (all <italic>P</italic> &lt; 0.05 vs. I/R) and I/R + CCR5Ab (all <italic>P</italic> &lt; 0.05 vs. I/R + Pre) while aggravated by I/R + CCR5Ago (all <italic>P</italic> &lt; 0.05 vs. I/R). Our results suggest that blocking CCR5 attenuates while enhancing CCR5 aggravates myocardial I/R injury through modulating inflammatory responses in rat heart.</p>", "<title>Keywords</title>" ]
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[ "<title>Conflict of interest</title>", "<p>None.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Serum levels of CK and TNF-α. Serum levels of CK and TNF-α were both significantly higher in I/R group compared to sham group and were significantly reduced in I/R + Pre group and I/R + CCR5Ab groups while significantly increased in I/R + CCR5Ago group compared to I/R group</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Myocardial infarct size. The ischemia region (AR/LV) was similar among group. The myocardial infarct size was significantly smaller in I/R + Pre group and I/R + CCR5Ab group while was significantly larger in I/R + CCR5Ago group than those in I/R group, and was significantly smaller in I/R + CCR5Ab group as compared to I/R + Pre group (<italic>P</italic> &lt; 0.05)</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>MPO activity in myocardial tissues. MPO activities were significantly increased in I/R group compared to sham group in normal zone, MPO activities were significantly lower in I/R + Pre group and I/R + CCR5Ab group while was significantly higher in I/R + CCR5Ago group in normal, risk, and infract zone than in I/R group</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Inflammatory levels in myocardial tissues staining by HE (magnification ×400). Representative sections from rats in I/R and I/R + CCR5Ago groups presented massive inflammatory cell infiltration (<italic>arrows</italic>) as compared to sham group indicating that I/R injury could trigger the inflammatory response which was reduced in I/R + Pre and I/R + CCR5Ab group. Accordingly, histological score was significantly lower in I/R + Pre and I/R + CCR5Ab groups than in I/R group and higher in I/R + CCR5Ago group than in I/R + Pre and I/R + CCR5Ab groups</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Myocardial ICAM-1 expression. <italic>Brown</italic> particles indicated the expression levels of ICAM-1 in myocardial tissues from LV free wall. Myocardial ICAM-1 expression was significantly upregulated in I/R group compared to sham group and was significantly attenuated in I/R + Pre group and I/R + CCR5Ab group while was significantly increased in I/R + CCR5Ago group than in I/R group. (Color figure online)</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Myocardial NF-κB binding activities. The myocardial DNA-binding activity of NF-κB was significantly higher in I/R group than in sham group, and was significantly lower in I/R + Pre and I/R + CCR5Ab groups while higher I/R + CCR5Ago group as compared to I/R group</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>CCR5 protein level of myocardial tissues. The specific protein expression levels of CCR5 were normalized to β-actin. There is no difference in expression levels of membrane protein CCR5 between sham and I/R groups which was downregulated in I/R + Pre and I/R + CCR5Ab and I/R + CCR5Ago groups</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Cardiac function in vivo</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">\n<italic>n</italic>\n</th><th align=\"left\">HR (beats/min)</th><th align=\"left\">LVSP (mmHg)</th><th align=\"left\">LVEDP (mmHg)</th><th align=\"left\">+d<italic>P</italic>/d<italic>t</italic>\n<sub>max</sub> (mmHg/s)</th><th align=\"left\">−d<italic>P</italic>/d<italic>t</italic>\n<sub>max</sub> (mmHg/s)</th></tr></thead><tbody><tr><td align=\"left\">SHAM</td><td char=\".\" align=\"char\">12</td><td char=\"±\" align=\"char\">322 ± 17</td><td char=\"±\" align=\"char\">139 ± 14</td><td char=\"±\" align=\"char\">2.6 ± 0.2</td><td char=\"±\" align=\"char\">7999 ± 772</td><td char=\"±\" align=\"char\">−5005 ± 711</td></tr><tr><td align=\"left\">I/R</td><td char=\".\" align=\"char\">11</td><td char=\"±\" align=\"char\">367 ± 14*</td><td char=\"±\" align=\"char\">93 ± 6*</td><td char=\"±\" align=\"char\">7.4 ± 0.5*</td><td char=\"±\" align=\"char\">4868 ± 525*</td><td char=\"±\" align=\"char\">−2556 ± 444*</td></tr><tr><td align=\"left\">I/R + Pre</td><td char=\".\" align=\"char\">10</td><td char=\"±\" align=\"char\">358 ± 9*</td><td char=\"±\" align=\"char\">116 ± 6*<sup>,†</sup>\n</td><td char=\"±\" align=\"char\">4.1 ± 0.3*<sup>,†</sup>\n</td><td char=\"±\" align=\"char\">6316 ± 603*<sup>,†</sup>\n</td><td char=\"±\" align=\"char\">−3512 ± 551*<sup>,†</sup>\n</td></tr><tr><td align=\"left\">I/R + CCR5Ab</td><td char=\".\" align=\"char\">12</td><td char=\"±\" align=\"char\">352 ± 10*<sup>,†</sup>\n</td><td char=\"±\" align=\"char\">126 ± 5<sup>†,‡</sup>\n</td><td char=\"±\" align=\"char\">4.3 ± 0.38*<sup>,†</sup>\n</td><td char=\"±\" align=\"char\">7077 ± 445*<sup>,†,‡</sup>\n</td><td char=\"±\" align=\"char\">−4253 ± 666*<sup>,†,‡</sup>\n</td></tr><tr><td align=\"left\">I/R + CCR5Ago</td><td char=\".\" align=\"char\">10</td><td char=\"±\" align=\"char\">370 ± 21*<sup>,§</sup>\n</td><td char=\"±\" align=\"char\">77 ± 6*<sup>,†,‡,§</sup>\n</td><td char=\"±\" align=\"char\">7.5 ± 0.5*<sup>,‡,§</sup>\n</td><td char=\"±\" align=\"char\">2987 ± 685*<sup>,†‡,§</sup>\n</td><td char=\"±\" align=\"char\">−1705 ± 515*<sup>,†‡,§</sup>\n</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>* <italic>P</italic> &lt; 0.05 vs. SHAM; <sup>†</sup> <italic>P</italic> &lt; 0.05 vs. I/R; <sup>‡</sup> <italic>P</italic> &lt; 0.05 vs. I/R + Pre; <sup>§</sup> <italic>P</italic> &lt; 0.05 vs. I/R + CCR5Ab</p></table-wrap-foot>" ]
[ "<graphic xlink:href=\"11010_2013_1604_Fig1_HTML\" id=\"MO1\"/>", "<graphic xlink:href=\"11010_2013_1604_Fig2_HTML\" id=\"MO2\"/>", "<graphic xlink:href=\"11010_2013_1604_Fig3_HTML\" id=\"MO3\"/>", "<graphic xlink:href=\"11010_2013_1604_Fig4_HTML\" id=\"MO4\"/>", "<graphic xlink:href=\"11010_2013_1604_Fig5_HTML\" id=\"MO5\"/>", "<graphic xlink:href=\"11010_2013_1604_Fig6_HTML\" id=\"MO6\"/>", "<graphic xlink:href=\"11010_2013_1604_Fig7_HTML\" id=\"MO7\"/>" ]
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{ "acronym": [], "definition": [] }
21
CC BY
no
2024-01-13 23:35:10
Mol Cell Biochem. 2013 Mar 3; 378(1-2):137-144
oa_package/a5/ee/PMC3634987.tar.gz
PMC7289538
32528043
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[ "<title>Subject terms</title>" ]
[ "<p id=\"Par1\">We read with interest the article titled “COVID-19 in persons with haematological cancers” by Wenjuan He et al. published recently [##REF##32332856##1##]. The authors conducted a retrospective cohort study of 13 hospitalized patients with hematological malignancies (PHM) from Wuhan, China, who developed coronavirus infectious disease 2019 (COVID-19) and compared their clinical characteristics and outcomes with eleven hospitalized health care providers (HCP). They showed that COVID-19 led to more severe disease and significantly higher case fatality rate (CFR) of 62% in PHM compared to zero in HCP (<italic>P</italic> = 0.002). We acknowledge these findings since data on COVID-19 infections in PHM have been scarce so far, but we see important limitations that were not explicitly addressed in the article. Furthermore, we present our own retrospective cohort data from Graz, Austria, which differs in terms of demographics and outcome.</p>", "<p id=\"Par2\">First, the cohort of PHM with COVID-19 exhibiting a median age of 35 years and selected by lung CT scan is likely not representative for most PHM whose median age reportedly is around 60 years [##REF##23752112##2##, ##UREF##0##3##]. In Wuhan, PHM with COVID-19 were a median of 14 years younger compared with PHM without COVID-19 (<italic>P</italic> = 0.082). Moreover, 62% (8/13) of PHM with COVID-19 had already been in the intensive care unit (ICU) before the COVID-19 pandemic began. Depending on the reason for ICU admission, which is not specified in the article, mortality rates of up to 60% are to be expected [##REF##23752112##2##]. Taken together, younger but obviously already critically ill patients prior to COVID-19 infection were reported by He et al.</p>", "<p id=\"Par3\">Second, predominantly female HCP with COVID-19 having a median age of 32 years are not an ideal comparator group since in these subjects CFR is expected to be below 2.5% [##REF##32320003##4##]. Only 18% (2/11) of HCP were male compared with 54% (7/13) of PHM with COVID-19 and 57% (65/115) of PHM without COVID-19, respectively (Fisher’s exact test, <italic>P</italic> = 0.0492). It has been previously shown that male gender is strongly associated with worse outcome of COVID-19 [##REF##32320003##4##].</p>", "<p id=\"Par4\">In summary, the expectedly very low CFR in young female HCP and the high mortality in presumably preselected PHM likely overestimated the difference in CFR between these two groups.</p>", "<p id=\"Par5\">We similarly analyzed all consecutive patients infected with SARS-CoV-2 (<italic>N</italic> = 78; Supplementary Table ##SUPPL##0##1##) diagnosed at the University Hospital in Graz, Austria, until May 1, 2020. COVID-19 and acute respiratory distress syndrome (ARDS) were diagnosed as previously described [##REF##32109013##5##]. Importantly, all SARS-CoV-2 infections were confirmed by nucleic acid test (NAT) and were not preselected by lung CT scan.</p>", "<p id=\"Par6\">There were eight PHM with a median age of 57 years showing equal sex distribution (Table ##TAB##0##1##). Surprisingly, there was no difference in overall survival between PHM and subjects without hematologic cancers given the limitation that the latter were 16 years older (Supplementary Fig. ##SUPPL##0##1##; Supplementary Table ##SUPPL##0##1##). Despite thorough precautions, four PHM (UPN5–8) got infected most likely by asymptomatic HCP demonstrating the danger of nosocomial transmission as has been noted [##REF##32332856##1##]. Two female patients had undergone allogeneic hematopoietic cell transplantation (HCT) 6 months (UPN7) and 5 weeks (UPN8) prior to SARS-CoV-2 infection and had ongoing immunosuppressive therapy including cyclosporine. Overall, six patients developed bilateral pneumonia diagnosed by chest X-ray and of these, three male PHM (UPN3, UPN5–6) progressed to severe ARDS requiring mechanical ventilation in the ICU. Importantly, these three PHM had recently received cytotoxic therapy, two including anti-CD20 antibodies and granulocyte-colony stimulating factors (G-CSF), confirming antitumor treatment &lt;14 days previously as risk factor for severe COVID-19 course [##UREF##1##6##].</p>", "<p id=\"Par7\">Several recurrent laboratory findings have been previously described in patients with COVID-19 [##REF##32109013##5##]. In accordance with prior reports, lymphopenia was observed in seven PHM including five prior to infection. Systemic hyperinflammation was documented in all but one PHM (UPN4), but compared to patients without hematologic cancers, PHM showed significantly higher peak levels of IL-6 (median of 1207 vs. 36.4 pg/mL, <italic>P</italic> = 0.033) and serum ferritin (3756 vs. 558 ng/mL, <italic>P</italic> &lt; 0.001) but not CRP (Fig. ##FIG##0##1a##). The latter is in contrast to He et al. reporting significantly higher CRP and procalcitonin levels possibly associated with high bacterial and fungal coinfection rates that resulted in the high CFR [##REF##32332856##1##].</p>", "<p id=\"Par8\">The experimental pharmacological therapy of COVID-19 and treatment outcomes are summarized in Table ##TAB##0##1##. The two PHM after HCT had a surprisingly unremarkable clinical course. Patients with ARDS also developed cytokine release syndrome and were treated with tocilizumab but no significant improvement of the respiratory situation could be achieved [##REF##32192578##7##]. Therefore, we decided to administer SARS2-CoV-2 convalescent plasma (CP) on a compassionate use basis to patients UPN3 and UPN6, 6 and 11 days after start of mechanical ventilation, respectively [##REF##32219428##8##]. As shown in Fig. ##FIG##0##1b##, in both cases, IL-6 and serum ferritin decreased dramatically, and patients were off the ventilator 5 and 4 days after CP therapy, respectively. UPN3 and UPN6 achieved a negative NAT whereas another three PHM remained positive after a median time of 55 days. Not surprisingly, compared with subjects without hematologic cancers, PHM needed significantly longer to achieve negative NAT (Table ##TAB##0##1##; Fig. ##FIG##0##1c##). Production of anti-SARS-CoV-2 antibodies, which reportedly occur in all COVID-19 patients 19 days after symptom onset, did not always accompany negative NAT in PHM [##UREF##2##9##]. After a median follow-up of 57 days, one patient (UPN5) died of severe ARDS, two patients (UPN3, UPN6) are in rehabilitation centers without symptoms of COVID-19, and five have been discharged.</p>", "<p id=\"Par9\">Previous studies displayed cancer patients as vulnerable population with high risk of morbidity due to COVID-19 [##REF##32332856##1##, ##UREF##1##6##, ##REF##32066541##10##]. In our observation, CFR of COVID-19 in PHM is lower than reported by He and co-workers (13% vs. 62%). Nevertheless, we must consider patient heterogeneity and admit that empirical knowledge about the experimental treatment of COVID-19 has evolved since the first outbreak in Wuhan, China. Whether additional administration of anti-CD20 therapy and G-CSF had impact on hyperinflammation and development of ARDS must be assessed in studies with larger patient numbers. As shown in two PHM with severe ARDS due to COVID-19, treatment with CP seems to be promising but requires further evaluation in randomized controlled trials.</p>", "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0914-x) contains supplementary material, which is available to authorized users.</p>", "<title>Author contributions</title>", "<p>All authors provided care for the patients. SH and ES conceived the study. SH, FE, PK, and ES collected clinical data. SH and ES analyzed data. PS provided convalescent plasma. MHS provided immunologic assays. HG revised the paper. SH, FE, and ES wrote the paper, which was reviewed and approved by all authors.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par10\">The authors declare that they have no conflict of interest.</p>", "<title>Ethical approval</title>", "<p id=\"Par11\">This study was approved by the ethics committee of the Medical University of Graz, Graz, Austria.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Inflammatory markers, clinical courses after SARS-CoV-2 convalescent plasma administration and time to negative nucleic acid test in patients with hematologic malignancies and COVID-19.</title><p><bold>a</bold> Comparison of inflammatory markers in patients with and without hematologic malignancies. Box plots display serum ferritin (left), interleukin-6 (IL-6; middle), and C-reactive protein (CRP; right) in eight hematologic versus 70 non-hematologic patients on log-transformed <italic>y</italic>-axis. The maximum values were used in every subject, except in patients who received tocilizumab. Here, the maximum values before tocilizumab infusion were selected because serum ferritin and IL-6 would regularly increase, and CRP would decrease after tocilizumab administration. <italic>P</italic>-values were calculated with the Kruskal–Wallis test (see Supplementary information). <bold>b</bold> Clinical courses of patients UPN3 and UPN6 receiving SARS-CoV-2 convalescent plasma. Serum ferritin (ng/mL), CRP (mg/L), IL-6 (pg/mL) and the ratio of partial pressure of oxygen in blood (PaO<sub>2</sub> in millimeters of mercury) and the fraction of oxygen in the inhaled air (FiO<sub>2</sub>) are depicted as a measure of inflammation and respiratory function, respectively. Tocilizumab was administered at a dose of 8 mg/kg body weight. Patients received 200 mL of ABO compatible SARS-CoV-2 convalescent plasma (CP) every other day for three times. CP was collected by standard apheresis and further pathogen-inactivated by INTERCEPT Blood System (Cerus, B.V. Europe). IV invasive mechanical ventilation, NIV non-invasive ventilation, P convalescent plasma, T tocilizumab, UPN unique patient number. <bold>c</bold> Time to SARS-CoV-2 qRT-PCR negativity in hematologic and non-hematologic patients. Analysis was performed with competing risk cumulative incidence estimators and Gray’s tests (see Supplementary information). Data cut off was May 12, 2020. The black line indicates non-hematologic patients, the red line patients with hematologic diseases.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Demographics, baseline characteristics, and clinical outcomes of hematological patients with SARS-CoV-2 infection.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>UPN1</th><th>UPN2</th><th>UPN3</th><th>UPN4</th><th>UPN5</th><th>UPN6</th><th>UPN7</th><th>UPN8</th></tr></thead><tbody><tr><td>Sex</td><td>M</td><td>W</td><td>M</td><td>W</td><td>M</td><td>M</td><td>W</td><td>W</td></tr><tr><td>Age</td><td>61</td><td>63</td><td>52</td><td>64</td><td>54</td><td>55</td><td>58</td><td>56</td></tr><tr><td>Days from positive NAT to last seen or death</td><td>68</td><td>66</td><td>59</td><td>59</td><td>23</td><td>55</td><td>55</td><td>55</td></tr><tr><td>Days from positive NAT to first symptoms<sup><bold>a</bold></sup></td><td>−5</td><td>−8</td><td>−1</td><td>N/A</td><td>−2</td><td>2</td><td>7</td><td>10</td></tr><tr><td>First symptoms</td><td>Fever, cough</td><td>Cough, diarrhea</td><td>Fever, malaise</td><td>None</td><td>Fever</td><td>Fever</td><td>Dyspnea</td><td>Dysgeusia, nausea</td></tr><tr><td>Hematological disease<sup><bold>b</bold></sup></td><td>DLBCL (12 months continuous complete remission)</td><td>Coombs positive Evans syndrome, Hodgkin lymphoma (10 year continuous complete remission)</td><td>DLBCL</td><td>Multiple myeloma</td><td>AML-MRC, CRi (severe pancytopenia)</td><td>Follicular lymphoma</td><td>Lymphoid blast crisis of CML, HLA-identical unrelated donor alloHCT, molecular remission, chronic GvHD</td><td>AML-MRC, HLA-identical sibling donor alloHCT, acute skin GvHD</td></tr><tr><td>Specific hematologic therapy in the last 12 months before COVID-19 diagnosis</td><td>EPOCH-R, high-dose MTX</td><td>Eltrombopag<sup><bold>c</bold></sup>, prednisolone<sup><bold>c</bold></sup>, intravenous immunoglobulins<sup><bold>c</bold></sup></td><td>R-CHOP and pegfilgrastim<sup><bold>c</bold></sup></td><td>RVD induction; high dose cyclophosphamide (priming therapy) and filgrastim<sup><bold>c</bold></sup></td><td>daunorubicin, cytarabine (7 + 3 induction)</td><td>G-CHOP and lipegfilgrastim<sup><bold>c</bold></sup></td><td>TBI 8 Gy, fludarabine, rabbit ATG, methotrexate (reduced-intensity myeloablative conditioning); dasatinib; methylprednisolone and cyclosporine<sup><bold>c</bold></sup></td><td>High-dose cytarabine (consolidation); busulfan, fludarabine (myeloablative conditioning); mycophenolate mofetil and cyclosporine<sup><bold>c</bold></sup></td></tr><tr><td>Relevant coexisting disorders</td><td>Secondary immunoglobulin deficiency</td><td>Iatrogenic Cushing’s syndrome, Parkinson’s disease, severe osteoporosis, recurrent deep vein thrombosis, splenectomy</td><td>Obesity</td><td>None</td><td>Diabetes mellitus, peptic ulcer disease</td><td>Chronic obstructive pulmonary disease, arterial hypertension, clear cell renal cell carcinoma (in remission), obesity</td><td>Arterial hypertension, QTc-prolongation, extrapulmonary tuberculosis</td><td>Arterial hypertension, paroxysmal atrial fibrillation, hyperlipidemia</td></tr><tr><td>Smoking history</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes, 122 pack years</td><td>No</td><td>No</td></tr><tr><td>All symptoms</td><td>Fever, cough, sore throat, respiratory distress</td><td>Fever, cough, dyspnea, diarrhea</td><td>Fever, mailase, cough, respiratory distress</td><td>None</td><td>Fever, cough, respiratory distress</td><td>Fever, cough, dysgeusia, respiratory distress</td><td>Dyspnea, cough, malaise, fever, respiratory distress</td><td>Dysgeusia, nausea, cough, fever, respiratory distress</td></tr><tr><td>Chest X-ray</td><td>Bilateral pneumonia</td><td>None</td><td>Bilateral pneumonia</td><td>None</td><td>Bilateral pneumonia</td><td>Bilateral pneumonia</td><td>Bilateral pneumonia</td><td>Bilateral pneumonia</td></tr><tr><td>Abnormal blood count<sup><bold>d</bold></sup> before SARS-CoV-2 infection</td><td>Lymphopenia</td><td>Leukocytosis</td><td>Lymphopenia</td><td>Severe neutropenia, mild anemia, severe thrombocytopenia, lymphopenia</td><td>Severe pancytopenia, lymphopenia</td><td>No</td><td>Anemia</td><td>Anemia, lymphopenia, thrombocytopenia</td></tr><tr><td>Laboratory changes since SARS-CoV-2 infection</td><td>Increased CRP, d-dimers, ferritin, LDH, PCT (IL-6, sIL-2R not measured)</td><td>Lymphocytosis, thrombocytopenia; increased IL-6 (ferritin, sIL-2R not measured)</td><td>Neutropenia, thrombocytopenia; increased AST, ALT, CRP, d-dimers, ferritin, HSTT, IL-6, LDH, PCT, sIL-2R</td><td>Increased CRP (ferritin, IL-6 and sIL-2r not measured)</td><td>Increased CRP, d-dimers, ferritin, HSTT, IL-6, LDH, sIL-2R; decreased fibrinogen</td><td>Neutropenia, thrombocytopenia, lymphocytopenia; increased AST, ALT, CRP, d-dimers, ferritin, fibrinogen, HSTT, IL-6, LDH (sIL-2R not measured)</td><td>Lymphocytopenia; increased CRP, ferritin, IL-6, sIL-2R</td><td>Increased CRP, ferritin, IL-6 (sIL-2R not measured)</td></tr><tr><td>Complications</td><td>Bacterial pneumonia</td><td>None</td><td>Severe ARDS, cytokine release syndrome, ventilator associated pneumonia</td><td>None</td><td>Severe ARDS, extubation failure, cytokine release syndrome, multi organ failure, death</td><td>Severe ARDS, cytokine release syndrome</td><td>None</td><td>CMV reactivation, bacterial enterocolitis</td></tr><tr><td>Days to ARDS<sup><bold>e</bold></sup></td><td>N/A</td><td>N/A</td><td>7</td><td>N/A</td><td>7</td><td>6</td><td>N/A</td><td>N/A</td></tr><tr><td>Treatment of COVID-19</td><td>Hydroxychloroquine, clarithromycin, zinc</td><td>None</td><td>Hydroxychloroquine, azithromycin, zinc, tocilizumab (three doses), prednisolone, convalescent plasma with prednisolone</td><td>None</td><td>Hydroxychloroquine, azithromycin, zinc, tocilizumab (three doses), dexamethasone</td><td>Hydroxychloroquine, azithromycin, zinc, tocilizumab (two doses), convalescent plasma with prednisolone</td><td>Hydroxychloroquine, zinc</td><td>Hydroxychloroquine, azithromycin, zinc</td></tr><tr><td>SARS-CoV-2 S1/S2 IgG (EIA) at time of last NAT</td><td>N/A</td><td>Positive</td><td>Weakly positive</td><td>N/A</td><td>N/A</td><td>Negative</td><td>Negative</td><td>Positive</td></tr><tr><td>Days to negative NAT<sup><bold>f</bold></sup></td><td>11</td><td>N/R</td><td>34</td><td>7</td><td>N/R</td><td>48</td><td>N/R</td><td>N/R</td></tr><tr><td>Outcome</td><td>Cured, well, discharged</td><td>Alive, well, outpatient</td><td>Cured, well, rehabilitation</td><td>Cured, well, outpatient</td><td>Dead</td><td>Cured, well, rehabilitation</td><td>Alive, well, discharged</td><td>Alive, well, discharged</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p><italic>AlloHCT</italic> allogeneic hematopoietic cell transplantation, <italic>ALT</italic> alanine aminotransferase, <italic>AML-MRC</italic> acute myeloid leukemia with myelodysplasia-related changes, <italic>ARDS</italic> acute respiratory distress syndrome, <italic>AST</italic> aspartate aminotransferase, <italic>ATG</italic> anti-thymocyte globulin, <italic>CRi</italic> complete remission with incomplete hematologic recovery, <italic>CRP</italic> C-reactive protein, <italic>CML</italic> chronic myeloid leukemia, <italic>DLBCL</italic> diffuse large B-cell lymphoma, <italic>EIA</italic> enzyme immunoassay, <italic>EPOCH-R</italic> etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin, rituximab, <italic>G-CHOP</italic> obinutuzumab, cyclophosphamide, doxorubicin, vincristine, prednisolone, <italic>GvHD</italic> graft versus host disease, <italic>HLA</italic> human leukocyte antigen, <italic>HSTT</italic> highly sensitive troponin t, <italic>IgG</italic> immunoglobulin G, <italic>IL-6</italic> interleukin-6, <italic>LDH</italic> lactate dehydrogenase, <italic>NAT</italic> nucleic acid test, <italic>N/A</italic> not applicable, <italic>N/R</italic> not reached, <italic>PBSC</italic> peripheral blood stem cell, <italic>PCT</italic> procalcitonin, <italic>R-CHOP</italic> rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone, <italic>RVD</italic> lenalidomide, bortezomib, dexamethasone, <italic>sIL-2R</italic> soluble interleukin-2 receptor, <italic>TBI</italic> total body irradiation, <italic>UPN</italic> unique patient number.</p><p><sup>a</sup>Some patients were identified by NAT screening after contact with infected patient before development of symptoms.</p><p><sup>b</sup>Hematologic disease were classified according to WHO classification.</p><p><sup>c</sup>Administered in the 14 days prior to COVID-19 onset.</p><p><sup>d</sup>Lymphocytopenia was defined as a lymphocyte count of &lt;1000 per cubic millimeter. Thrombocytopenia was defined as a platelet count of &lt;150,000 per cubic millimeter.</p><p><sup>e</sup>Days to ARDS were counted from onset of clinical symptoms.</p><p><sup>f</sup>Days to negative NAT were counted from first positive NAT until the first of two consecutive negative NAT 24 h apart.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Stefan Hatzl, Florian Eisner.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_914_Fig1_HTML\" id=\"d32e945\"/>" ]
[ "<media xlink:href=\"41375_2020_914_MOESM1_ESM.pdf\"><caption><p>Supplementary information</p></caption></media>" ]
[{"label": ["3."], "mixed-citation": ["Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, et al. (eds.) SEER Cancer Statistics Review, 1975\u20132017, National Cancer Institute. Bethesda, MD. 2020. "], "ext-link": ["https://seer.cancer.gov/csr/1975_2017/"]}, {"label": ["6."], "mixed-citation": ["Zhang L, Zhu F, Xie L, Wang C, Wang J, Chen R, et al. Clinical characteristics of COVID-19-infected cancer patients: A retrospective case study in three hospitals within Wuhan, China. Ann Oncol. 2020. 10.1016/j.annonc.2020.03.296."]}, {"label": ["9."], "mixed-citation": ["Long QX, Liu BZ, Deng HJ, Wu GC, Deng K, Chen YK, et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. 2020. 10.1038/s41591-020-0897-1."]}]
{ "acronym": [], "definition": [] }
10
CC BY
no
2024-01-13 23:35:05
Leukemia. 2020 Jun 11; 34(8):2265-2270
oa_package/cc/75/PMC7289538.tar.gz
PMC7298698
32555296
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[ "<title>Subject terms</title>" ]
[ "<p id=\"Par1\">Since its outbreak in December 2019 in Wuhan, China [##REF##31978945##1##], the novel coronavirus, SARS-CoV-2, has created a dramatic global health and economic crisis. COVID-19 is the disease caused by SARS-CoV-2. In most cases, COVID-19 is associated with mild respiratory symptoms. However, in ~15% of the patients, hospitalization is required, and about 5% of patients develop severe lung injury that can result in acute respiratory distress syndrome (ARDS). ARDS may be accompanied by sepsis and septic shock, and multiorgan failure, including acute kidney injury and cardiac injury. Older age, obesity, pulmonary and other comorbidities are risk factors for higher mortality [##UREF##0##2##, ##UREF##1##3##]. It has also been reported that the extent of inflammation—mirrored by peripheral blood cytokine levels—is associated with a worse outcome [##REF##32217835##4##].</p>", "<p id=\"Par2\">LaRosée et al. describe a very interesting series of 14 COVID-19 patients with heavy inflammatory syndrome treated successfully with ruxolitinib [##UREF##2##5##]. As COVID-19 induces significant burden to societies worldwide, appropriate treatment also for COVID-19 associated ARDS would be beneficial. However, in the 14 patients described in this series, only one patient was mechanically ventilated before start of ruxolitinib.</p>", "<p id=\"Par3\">We were interested whether inhibition of Janus kinases could also revert overt ARDS in COVID-19.</p>", "<p id=\"Par4\">We report on our first case of a 65-year old Asian woman with COVID-19-induced ARDS that was successfully treated with ruxolitinib. The patient presented with progressive dyspnea (respiratory rate &gt;40/min, SpO2 of 60% on ambient air) and a history of fever for 3 days. Although there was no pre-existing disease in this patient, her respiratory distress deteriorated rapidly and she had to be intubated 3 hours after first contact in the emergency room. Computed tomography of the chest revealed extensive bilateral ground glass opacities and consolidations (Fig. ##FIG##0##1##). The patient was transferred to the ICU and intubated immediately. SARS-CoV-2 infection was confirmed by combined E- and S-specific PCR (RealStar<sup>®</sup> SARS-CoV-2 RT-PCR Kit, Altona Diagnostics, Hamburg, Germany) from a nasopharyngeal swab. The initial laboratory investigations revealed no abnormalities except for a lymphopenia of 5,4% (reference value 20–44%), and elevated levels for LDH (928 U/l, reference value &lt; 247 U/l), ferritin (1756 μg/l, reference value 15–400 μg/l) and IL-6 (120 pg/ml, reference value &lt;7 pg/ml) and a high sensitive troponin I (39,8 ng/l, reference value &lt;15,8 ng/l), parameters suggesting an adverse clinical course [##REF##32217835##4##]. Twelve hours later, the respiratory status deteriorated to a Horowitz index (paO2/FiO2) of 110 under pressure controlled ventilation (PCV) with a positive end expiratory pressure (PEEP) of 16mBar, a plateau pressure (P Peak) of 31mBar and a set respiratory rate of 30/min. To achieve an adequate perfusion pressure, norepinephrine was commenced at 4–16 µg/min. Prone positioning was performed on the sedated patient for intervals of 24 h. As a bacterial superinfection was suspected due to worsening of gas exchange, leukocytosis and substantial increase of CRP and procalcitonin, antibiotic treatment with meropenem was started (with dose adjustments based on therapeutic drug monitoring). Within a few hours NT-proBNP and TNI values increased substantially and a severe left ventricular dysfunction was diagnosed. The overall prognosis of this patient was considered to be very poor [##UREF##3##6##]. After counseling the ethics committee at our institution, an experimental treatment with 10 mg ruxolitinib BID was started. The drug was administered via a nasogastral tube. In parallel, standard of care treatment was continued. During the following days, the Horowitz index improved continuously under assisted spontaneous ventilation with a PEEP of 12mBar and P<sub>ASB</sub> of 9mBar with 24 h intervals of prone and supine positioning facilitated by sedation with dexmedetomidine, propofol and sufentanil (Fig. ##FIG##1##2a##). At day 8 after ICU admission, percutaneous dilatational tracheotomy was performed and the patient was intermittently weaned from the respirator starting at day 10. RT-PCR from serum samples before ruxolitinib (Ct = 35.84) and 5 days after start of ruxolitinib (Ct = 35.86) suggested that SARS-CoV-2 viremia did not increase. IL6 and ferritin levels returned to normal (Fig. ##FIG##1##2b##).</p>", "<p id=\"Par5\">To the best of our knowledge, we here describe the first successful treatment of COVID-19-associated ARDS using ruxolitinib. COVID-19 is characterized by an exuberant inflammatory response [##REF##31986264##7##] that can result in massive lung injury (ARDS), multiorgan failure and death. COVID-19-associated ARDS requiring invasive ventilation is characterized by low survival rates—especially in older patients [##REF##31986264##7##].</p>", "<p id=\"Par6\">The decision to treat our patient with ruxolitinib to inhibit JAKs was based on the current knowledge of COVID-19 pathophysiology, which is thought to be mediated by an overwhelming inflammatory cytokine response and thus is very likely to involve JAK-signaling. We reasoned that by blocking JAK1/2-kinases the devastating consequences especially of inflammatory lung tissue damage could be diminished. The use of ruxolitinib as an immunosuppressive agent is not without precedence: in steroid–refractory graft versus host disease (GvHD)—an aggressive form of organ-damaging, cytokine-mediated hyperinflammation after allogeneic hematopoietic stem cell transplantation, ruxolitinib resulted in impressive clinical improvements [##REF##26228813##8##].</p>", "<p id=\"Par7\">In our patient, ruxolitinib not only potently reduced ARDS-associated inflammatory blood cytokine levels such as IL-6 and the acute phase protein ferritin, but was also associated with a rapid respiratory and cardiac improvement and clinical stabilization. This course was remarkable when compared to other patients [##UREF##3##6##]. Based on the close temporal association between ruxolitinib start and clinical response, it is tempting to speculate that JAK1/2-inhibition effectively contributed to the favorable clinical course. Importantly, the virus load as determined by PCR did not increase in our patient during ruxolitinib treatment.</p>", "<p id=\"Par8\">Artificial intelligence and pre-clinical studies have recently suggested that baricitinib, a numb associated kinase (NAK) and JAK1/2-inhibitor, inhibits clathrin-mediated endocytosis and thereby antagonizes SARS-CoV-2 infection of cells [##REF##32113509##9##]. Thus, besides ruxolitinib, baricitinib, which is approved in the treatment of rheumatoid arthritis, is another promising candidate with significant potential in the treatment of COVID-19 disease. However, since baricitinib may not penetrate into lung tissue [##REF##28358994##10##], we hypothesized that it may be less effective in COVID-19-associated ARDS.</p>", "<p id=\"Par9\">The finding in this patient may have important implications for the ongoing search for optimal therapy for patients suffering from severe COVID-19. Clinical trials are under way to study both ruxolitinib and baricitinib in a prospective manner (e.g. NCT04359290).</p>" ]
[ "<title>Acknowledgements</title>", "<p>Supported in part by the German José Carreras Leukemia foundation (AH 06-01; to A.N.). Open access funding provided by Projekt DEAL.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par10\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Ruxolitinib clears lung infiltration in SARS-CoV-2 induced ARDS.</title><p><bold>a</bold> Chest CT scans of the patient before transfer to the ICU reveals diffuse bilateral ground glass opacities, consolidations, and motion artifacts due to tachypnea. <bold>b</bold> After 11 days of treatment with ruxolitinib, markedly decreased ground glass opacities and motion artifacts, less pronounced regression of consolidations, and atelectasis.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Ruxolitinib improves functional and inflammatory parameters in SARS-CoV-2 induced ARDS.</title><p>Functional lung parameters (<bold>a</bold>) and laboratory parameters (<bold>b</bold>; ferritin and IL6) during treatment with ruxolitinib in a case of COVID-19 related ARDS.</p></caption></fig>" ]
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[ "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_907_Fig1_HTML\" id=\"d32e335\"/>", "<graphic xlink:href=\"41375_2020_907_Fig2_HTML\" id=\"d32e350\"/>" ]
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[{"label": ["2."], "mixed-citation": ["Lippi G, Mattiuzzi C, Sanchis-Gomar F, Henry BM. Clinical and demographic characteristics of patients dying from COVID-19 in Italy versus China. J Med Virol. 2020."]}, {"label": ["3."], "mixed-citation": ["Lighter J, Phillips M, Hochman S, Sterling S, Johnson D, Francois F, et al. Obesity in patients younger than 60 years is a risk factor for Covid-19 hospital admission. Clin Infect Dis. 2020."]}, {"label": ["5."], "mixed-citation": ["La Ros\u00e9e F, Bremer HC, Gehrke I, Kehr A, Hochhaus A, Birndt S, et al. The Janus kinase 1/2 inhibitor Ruxolitinib in COVID-19 with severe systemic hyperinflammation. Leukemia. 2020."]}, {"label": ["6."], "surname": ["Yang", "Yu", "Xu", "Shu", "Xia", "Liu"], "given-names": ["X", "Y", "J", "H", "J", "H"], "article-title": ["Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study"], "source": ["Lancet Resp Med"], "year": ["2020"], "volume": ["8"], "fpage": ["475"], "lpage": ["81"], "pub-id": ["10.1016/S2213-2600(20)30079-5"]}]
{ "acronym": [], "definition": [] }
10
CC BY
no
2024-01-13 23:35:06
Leukemia. 2020 Jun 17; 34(8):2276-2278
oa_package/55/96/PMC7298698.tar.gz
PMC7301049
32555367
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[ "<title>Subject terms</title>" ]
[ "<title>To the Editor:</title>", "<p id=\"Par1\">We read with great interest the recent thorough study from Miller et al. [##UREF##0##1##], who reported a beneficial impact of early Bacillus Calmette–Guérin (BCG) vaccination, and linked morbidity and mortality due to SARS-CoV-2 with BCG vaccination policy. They found that countries without policies of universal BCG vaccination (like Italy, the Netherlands, or the United States) have been more severely affected compared with countries with universal and long-standing BCG policies. Since senior citizens are particularly sensitive to COVID-19, newborn vaccination programs implemented in the 1940s and 1950s should be the most beneficial. Thus, we were impressed by the linear correlation between the year of the establishment of universal BCG vaccination and the mortality rate presented by Miller et al. [##UREF##0##1##]. In our view, this is a convincing argument for the hypothesis that the earlier the vaccination policy was established, the larger the segment of the elderly population being protected.</p>", "<p id=\"Par2\">We would like to make the scientific community aware of a unique historical circumstance in Germany, where divergent BCG vaccination policies existed in the politically divided country (1949–1989) before German reunification in 1990. In East Germany, BCG vaccination programs were established by the communist government in 1951, and soon became compulsory in 1953, leading to near-universal (99.8%) BCG vaccination of newborns by day 3. By contrast, voluntary BCG vaccination (recommended since 1955) was far less common in West Germany, due to low incidence of the disease after the Second World War. In early years, only 7–20% of all newborns became BCG-vaccinated in Western Germany, with almost complete cessation of vaccination between 1975 and 1977 (Fig. ##FIG##0##1a##). Thus, we believe that the comparison of morbidity and mortality of SARS-CoV-2 would be particular informative in the light of the rather uniform genetic, social, and cultural background. Here, we record those data in formerly East and West German federal states (excluding Berlin, Fig. ##FIG##0##1b##). Our observations strongly support the analysis from Miller et al., and point toward BCG vaccination having a protective effect. We did not observe a significant difference in the lethality of SARS-CoV-2; once people became infected, the course of disease is not significantly different between formerly East and West German parts of the reunified country. It is fair to conclude that, 30 years after German reunification, the standard of intensive care has been completely harmonized throughout the country, and at the time of our analysis, the health care system in Germany still had large capacities. Besides BCG vaccination, we cannot exclude other factors influencing morbidity and mortality, e.g., the speed of the pandemic spread, differences in the social behavior, or the potentially greater likelihood of being infected with SARS-CoV-2 in the larger cities of Western Germany. We also acknowledge the fact that statistical correlation does not prove causation, but SARS-CoV-2 testing capacities, a frequent confounding factor when comparing different populations across countries, were early on expanded to far more than 100,000/day (end of March 2020) all over Germany. Furthermore, demographic characteristics show that relatively more elderly people live in East Germany than in West Germany. The age distribution shows that the proportion of people above 64 years of age is generally higher in Eastern federal states formerly belonging to the German Democratic Republic (GDR) [##UREF##1##2##]. Thus, age-wise, the population with a greater risk to succumb due to COVID-19 is even more abundant in East, compared with West Germany.</p>", "<p id=\"Par3\">We would like to remind readers that a correlation between BCG vaccination and reduced disease risk beyond tuberculosis has long been suspected. In particular, many older studies link BCG vaccination to a lower cancer risk, and there was even an international symposium held in 1982 entitled “BCG Vaccination against Cancer and Leukemia” (Chicago). Mortality related to childhood leukemia was reported to be significantly reduced in a retrospective study of 54 414 BCG-vaccinated versus 172 986 non-vaccinated infants in Chicago [##UREF##2##3##]. The question was brought up again in 1978 in a meta-analysis of Rosenthal’s data, and two other cohorts confirmed the initial observation [##UREF##3##4##]. With respect to childhood leukemia, the potentially beneficial effects of the East German BCG vaccination program were also reported by Spix et al. [##REF##18076067##5##]. The authors compared the rate of childhood leukemia before and after German reunification. Somewhat counterintuitively, the rate of childhood leukemia was clearly lower in the former GDR (East Germany) with 3/100,000 children compared with 3.7/100,000 in former West Germany, but reached West German levels ~8 years after reunification with four cases of acute lymphoblastic leukemia (ALL)/100,000 children below 15 years of age. It should be emphasized that cancer registries were exemplary in the former GDR, and thus this “catch-up effect” cannot be attributed to poor registration. In addition, meta-analysis of several epidemiological studies has shown that protection from childhood ALL, the most common malignancy in children, is observed only when children are vaccinated with BCG very early in life (&lt;3 months) [##REF##29167460##6##].</p>", "<p id=\"Par4\">In recent years, a new immunological concept—trained immunity—has emerged, improving our understanding of the role of BCG vaccination in shaping the innate immune memory response. Innate immune cells, such as macrophages, monocytes, or NK cells, can change their epigenome after exposure to infection, vaccination, or other stressors, which modifies their expression profile and cell physiology [##REF##27102489##7##]. BCG vaccination in particular, educates hematopoietic stem cells and generates trained monocytes and macrophages [##REF##29328912##8##]. In a double-blinded, placebo-controlled study with healthy male subjects who were challenged by attenuated Yellow Fever Virus after having received a BCG vaccine or placebo, the viremia was significantly lower in the vaccinated group [##REF##29324233##9##]. Besides infection with SARS-CoV-2 and childhood ALL, there may be many other malignant and nonmalignant, e.g., infectious diseases for which measures that augment our innate immune responses are beneficial [##REF##27903713##10##, ##REF##30967658##11##]. However, it remains unclear to date whether BCG vaccination across different age groups, e.g., newborns, adolescents, young, and elderly people, has uniformly the same beneficial effect.</p>", "<p id=\"Par5\">With regard to the current SARS-CoV-2 pandemic situation, we fully support the idea of implementing controlled clinical trials for BCG vaccination, as already started by Curtis et al. [##REF##32359402##12##]. Only well-controlled clinical trials may prove a benefit of this old vaccine that was developed by the two French immunologists almost 100 years ago. Low- and medium-income regions like Africa or India may benefit mostly if such trials prove a clinical benefit, at least until a specific and effective SARS-CoV-2 vaccine becomes available.</p>" ]
[ "<title>Acknowledgements</title>", "<p>JH was supported by DJCLS 02R/2016, ERAPERMED 2018-2019 “GEPARD” and ERCStg 852222 “PreventALL”. AB and UF were supported by DJCLS 02R/2016 and DJCLS 21R/2019, respectively. Open access funding provided by Projekt DEAL.</p>", "<title>Author contributions</title>", "<p>JH designed the research, analyzed the data, and wrote the paper. UF wrote the paper. FA analyzed the data, performed the research, and wrote the paper. AB designed research, analyzed the data, and wrote the paper.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par6\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Morbidity and mortality due to SARS-CoV-2.</title><p><bold>a</bold> Vaccination policy in East and West Germany. When BCG vaccination was first introduced against tuberculosis in Germany (in the city of Lübeck) in 1930, tragically 131 newborns fell ill, and 77 died due to a contamination of the vaccine with virulent tubercle bacilli. This was the greatest vaccination disaster of the twentieth century, and delayed introduction of BCG vaccination in Germany till after World War II. <bold>b</bold> Morbidity and mortality due to SARS-CoV-2 in federal states formerly belonging to East and West Germany, excluding Berlin. Data were extracted from Johns Hopkins University (as per 28.4.2020). Population numbers were extracted for the federal states as per December 2018 (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.statista.com\">www.statista.com</ext-link>). <italic>p</italic>-values were calculated using the Studentʼs <italic>t</italic>-test.</p></caption></fig>" ]
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[ "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[{"label": ["1."], "mixed-citation": ["Miller A, Reandelar MJ, Fasciglione K, Roumenova V, Li Y, Otazu GH. Correlation between universal BCG vaccination policy and reduced morbidity and mortality for COVID-19: an epidemiological study. medRxiv. 2020; preprint at 10.1101/2020.03.24.20042937."]}, {"label": ["2."], "mixed-citation": ["Statistisches Bundesamt. Statistisches Jahrbuch, Deutschland und Internationales. 2017;32."]}, {"label": ["3."], "surname": ["Rosenthal", "Crispen", "Thorne", "Piekarski", "Raisys", "Rettig"], "given-names": ["SR", "RG", "MG", "N", "N", "PG"], "article-title": ["BCG vaccination and leukemia mortality"], "source": ["J Am Med Assoc."], "year": ["1972"], "volume": ["222"], "fpage": ["1543"], "lpage": ["4"], "pub-id": ["10.1001/jama.1972.03210120041010"]}, {"label": ["4."], "surname": ["Ambrosch", "Krepler", "Wiedermann"], "given-names": ["F", "P", "G"], "article-title": ["The effect of neonatal BCG vaccination on the incidence of leukemia (author\u2019s transl)"], "source": ["MMW, Munch medizinische Wochenschr."], "year": ["1978"], "volume": ["120"], "fpage": ["243"], "lpage": ["6"]}]
{ "acronym": [], "definition": [] }
12
CC BY
no
2024-01-13 23:35:06
Leukemia. 2020 Jun 18; 34(8):2217-2219
oa_package/47/37/PMC7301049.tar.gz
PMC7323604
32601378
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[ "<title>Subject terms</title>" ]
[ "<title>To the Editor:</title>", "<p id=\"Par1\">With more than 5 million proven infections and more than 300,000 associated deaths worldwide [##UREF##0##1##], the SARS-CoV-2 pandemic poses unprecedented challenges to health-care professionals and especially those treating and caring for patients with malignant hematological diseases. These patients often have multiple different risk factors for severe infections [##REF##32332856##2##]. Chronic lymphocytic leukemia (CLL) is the most common form of leukemia and infections are a known contributor to morbidity and mortality due to a disease-inherent immunodeficiency [##REF##29519862##3##, ##REF##26084672##4##]. Considering this multifactorial immune defect, it appears conceivable that patients with CLL are more susceptible to infections with SARS-CoV-2 and more likely to develop severe courses of the associated respiratory disease COVID-19, especially when under additional immunosuppression by chemoimmunotherapy (CIT). Few case reports on COVID-19 in CLL patients from countries with suspected different prevalence rates of COVID-19 have been published so far. The publications report a patient after first-line treatment with single-agent chlorambucil, a case series with four treatment-naive CLL patients, a case series of eight patients on Bruton tyrosine kinase (BTK) inhibitors and most recently a heterogeneously treated population of four patients from the Hospital Clinic of Barcelona [##REF##32220344##5##–##UREF##2##8##]. While it has been hypothesized that the BTK inhibitor ibrutinib might have protective effects against COVID-19 by attenuating hyperinflammatory responses, there is currently no data on COVID-19 in patients receiving venetoclax-based treatments [##UREF##1##7##, ##REF##32302379##9##]. A recent study has suggested a reduction of CLL-inherent immunosuppression after successful treatment with venetoclax-based regimens [##REF##31506282##10##]. In light of these data we sought to determine the incidence, severity, and possible risk factors of COVID-19 cases in a well-defined cohort of patients with CLL receiving venetoclax-based combination treatments as first-line therapy in a prospective clinical trial.</p>", "<p id=\"Par2\">The GAIA/CLL13 trial (NCT02950051) is a multicenter phase 3 investigator-initiated trial with sites in nine European countries plus Israel. From December 2016 to September 2019, 926 physically fit and treatment-naive patients were randomized into four treatment arms. In the standard arm, CIT with fludarabine, cyclophosphamide plus rituximab (FCR, patients ≤ 65 years) or bendamustine plus rituximab (BR, patients &gt; 65 years) is administered. In the experimental arms 12 cycles of venetoclax-containing regimes are tested: venetoclax plus rituximab (RVe), venetoclax plus obinutuzumab (GVe) and venetoclax plus ibrutinib and obinutuzumab (GIVe). Patients with del(17p) or <italic>TP53</italic> mutation were not eligible.</p>", "<p id=\"Par3\">Between March and April 2020 seven patients within the GAIA/CLL13 trial developed COVID-19, one in the CIT arm and six patients in the experimental treatment arms (Table ##TAB##0##1##). The baseline characteristics show a median age of 61 years (range 52–78) and in accordance with the inclusion criteria of the study only few comorbidities and no <italic>TP53</italic> aberrations were documented. All but one patient had completed study treatment at the time point of COVID-19 diagnosis with a median time after end of treatment of 22 (range 1–30) months. All seven patients were tested positive for SARS-CoV-2 by PCR collected from nasopharyngeal swabs. While one patient was isolated in home quarantine, six of seven patients (85.7%) had to be hospitalized and two (28.6%) required treatment on an intensive care unit (ICU), only one patient required invasive mechanical ventilation (Table ##TAB##0##1##). Two patients died as a result of their SARS-CoV-2 infection, one 58-year-old patient (patient 4) after a prolonged treatment with mechanical ventilation (52 days) on an ICU and a 78-year-old patient (patient 7) who decided against ICU treatment and was treated with best supportive care.</p>", "<p id=\"Par4\">We assessed different surrogate markers for immune function to elucidate the mechanisms of susceptibility to COVID-19 in our patient cohort (Fig. ##FIG##0##1##). The frequency of infections observed after start of study treatment differed strongly between the patients. Three patients had a history of multiple (≥2) infections per year since start of study treatment and six of seven (85.7%) patients had at least one episode of neutropenia (Fig. ##FIG##0##1a##). An analysis of immunoglobulin levels before and after study treatment revealed a substantial humoral immune deficiency with abnormal pretreatment IgG levels in six of seven patients (85.7%) (Fig. ##FIG##0##1b##). In line with previous data, we show a decrease of the initially expanded CD3+, CD4+ and CD8+ T-cell populations in the course of study treatment (Fig. ##FIG##0##1c##).</p>", "<p id=\"Par5\">Between March and April 2020, we observed seven cases of COVID-19 among 926 patients in our phase 3 GAIA/CLL13 trial. The estimated cumulative incidence of 755.9 COVID-19 cases per 100,000 persons appears high when compared to age-specific (60–79 years) incidence rates, for instance in Germany (female: 169.5; male: 209) [##UREF##3##11##]. We also observed a substantially higher hospitalization rate of 85.7% in our patients compared to a study that estimated patients requiring hospitalization at 11.8% (60–69 years) and 16.6% (70–79 years), respectively [##UREF##4##12##].</p>", "<p id=\"Par6\">This difference is likely due to the multifactorial immune suppression in our patients (Fig. ##FIG##0##1##). Besides an increased frequency of infections in some and CLL-associated hypogammaglobulinaemia in most patients we also found reduced CD4+ and CD8+ T-cell subpopulations. Adding to this quantitative cellular immune deficiency, T cells are known to be functionally impaired in CLL [##REF##23247726##13##]. In COVID-19, decreased levels of CD4+ and CD8+ T cells were associated with more severe disease courses, suggesting that pre-existing cellular defects might lead to an impaired T-cell response in infected individuals with CLL [##REF##32425950##14##]. Furthermore, in this relatively small case series, the most severe respiratory failures were observed in patients who were still under treatment (patient 7) or had stopped treatment 2 months before (patient 4), which might reflect more severe immune deficiency during ongoing combination treatment. However, the comparably high incidence and hospitalization rate could also reflect a more stringent observation and precautious hospitalization of these patients treated within a clinical trial, though none of the patients had an asymptomatic SARS-CoV-2 infection and the number of unknown cases could be even higher.</p>", "<p id=\"Par7\">Despite the high hospitalization rate, the here observed case fatality rate of 28.6% is similar to the recently published cohort of BTK inhibitor-treated patients with CLL (25%) and lower than in the case series of four treatment-naive patients, of which three had a fatal outcome (75%) [##REF##32388230##6##, ##UREF##1##7##]. The different cases fatality rates observed between the treatment-naive cohort and our study are most likely due to the different ages of the analyzed populations. All patients with fatal COVID-19 courses described in the publication by Paneesha et al. were between 79 and 81 years of age compared to a median age of 61 years in our cohort. Furthermore, our study population comprises of comparably fit CLL patients with few comorbidities (median CIRS score: 2 [range 0-5]). However, five of our patients had additional risk factors (hypertension, chronic respiratory diseases, cardiovascular disease) for severe COVID-19 as established by recent meta-analyses [##REF##32217650##15##].</p>", "<p id=\"Par8\">To our knowledge, we here report the first analysis of COVID-19 in CLL patients receiving venetoclax-based combinations and CIT as first-line treatment within a large randomized controlled trial. This analysis suggests an increased rate of COVID-19 as well as an increased hospitalization rate in fit patients with CLL. Despite their various CLL-associated immune defects, the majority of patients recovered from COVID-19. As this is an ongoing clinical trial, a benefit-risk assessment is continuously performed by an independent data and safety monitoring board (DSMB). At this time the DSMB had no objection against continuation of the trial or subjects to continue treatment as allocated.</p>" ]
[ "<title>Acknowledgements</title>", "<p>This study is supported by research funding from Hoffmann-La Roche, AbbVie and Janssen-Cilag Ltd to the German CLL Study Group. The GAIA/CLL13 trial is sponsored by the German CLL Study Group in close cooperation with HOVON, the Nordic CLL Study Group (NCLLSG), the Swiss Group for Clinical Cancer Research (SAKK), Cancer Trials Ireland and the Israeli CLL Study Group. The Data and Safety Monitoring Board (DSMB) that supervised the trial is an independent body of scientists including Carol Moreno (Barcelona, Spain), Monica Else (London, UK), Daniel Catovsky (London, UK) and Paolo Ghia (Milano, Italy). Open access funding provided by Projekt DEAL.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par9\">MF, NDS, AMF, SR, KH, JD, MvdK, TI, BS, and MG have nothing to disclose. PL reports grants, personal fees, travel support from and consultancy for Janssen-Cilag, travel support from Roche, consultancy for Abbvie and Sunesis outside the submitted work. JvT reports grants and personal fees from Hoffmann-La Roche, AbbVie, Janssen-Cilag during the conduct of the study and outside the submitted work. FS reports travel support from Gilead. EvdS. reports personal fees from Amgen outside the submitted work. KF reports travel grants from Roche and honoraria from Roche and Abbvie. CMW reports grants and personal fees from Hoffmann-La Roche, AbbVie, Janssen-Cilag during the conduct of the study and outside the submitted work. ET reports grants and personal fees (advisory board, speakers bureau) from Roche and Abbvie and personal fees and non-financial support (advisory board, speakers bureau, travel support) from Janssen. SS reports grants, personal fees, and non-financial support from AbbVie, AstraZeneca, Celgene, Gilead, GSK, Hoffmann-La Roche, Janssen, Novartis, Pharmacyclics, Sunesis and personal fees and non-financial support from Verastem during the conduct of the study and outside the submitted work. CUN reports grants and personal fees from Abbvie, Janssen, AstraZeneca, grants from Novo Nordisk Foundation and the Danish Cancer Society, all outside the submitted work. APK reports research grants from, appeared on an ad board for and received speaker’s fees from Abbvie, Janssen and Genentech. MH reports grants, non-financial support and personal fees (honoraria; speaker’s bureau and/or advisory board) from Roche, Gilead, Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie outside the submitted work and grants from Roche, Janssen and Abbvie during the conduct of the study. BE reports personal fees and research funding from Janssen-Cilag, Abbvie and Roche, research funding from Beigene and personal fees from Novartis, Celgene, ArQule, AstraZeneca, Oxford Biomedica (UK) and Adaptive Biotechnologies, all outside the submitted work.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Individual treatment courses and parameters of immune function.</title><p><bold>a</bold> The vertical axis represents absolute neutrophil counts (ANC), blue boxes show treatment regimen and duration (RVe rituximab, venetoclax, GVe obinutuzumab, venetoclax, GIVe obinutuzumab, ibrutinib, venetoclax, FCR fludarabine, cyclophosphamide, rituximab). Infections after study inclusion and onset of COVID-19 are depicted in orange boxes. The threshold for neutropenia is shown in yellow. <bold>b</bold> The vertical axis shows levels of immunoglobulins before (baseline) and after treatment (final restaging), normal ranges are indicated in green. Each cross/line represents one patient. <bold>c</bold> Changes in T-cell subpopulations in the course of first-line treatment. Patients on venetoclax combinations are depicted in black, the patient on FCR is shown in orange. Blue bars represent median values of all analyzed patients at each time point.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient and treatment characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Patient 1</th><th>Patient 2</th><th>Patient 3</th><th>Patient 4</th><th>Patient 5</th><th>Patient 6</th><th>Patient 7</th></tr></thead><tbody><tr><td colspan=\"8\">Baseline characteristics</td></tr><tr><td> Age, years</td><td>52</td><td>60</td><td>68</td><td>58</td><td>63</td><td>61</td><td>78</td></tr><tr><td> Sex</td><td>M</td><td>F</td><td>M</td><td>M</td><td>M</td><td>F</td><td>F</td></tr><tr><td> Country</td><td>GER</td><td>NL</td><td>NL</td><td>CH</td><td>GER</td><td>GER</td><td>NL</td></tr><tr><td colspan=\"8\">CLL characteristics</td></tr><tr><td> Treatment</td><td>RVe</td><td>RVe</td><td>GIVe</td><td>GIVe</td><td>GVe</td><td>FCR</td><td>GVe</td></tr><tr><td> Date of CLL diagnosis</td><td>07/2013</td><td>02/2013</td><td>03/2018</td><td>08/2018</td><td>03/2017</td><td>07/2011</td><td>09/2011</td></tr><tr><td> Time from CLL diagnosis to treatment, months</td><td>43</td><td>56</td><td>7</td><td>6</td><td>1</td><td>70</td><td>96</td></tr><tr><td> Binet stage at screening</td><td>B</td><td>A</td><td>B</td><td>B</td><td>C</td><td>B</td><td>A</td></tr><tr><td colspan=\"8\"> FISH at screening</td></tr><tr><td>  Del(11q)</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>  Trisomy 12</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>  Del(13q)</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>Y</td><td>Y</td></tr><tr><td> IGHV mutational status at screening</td><td>Unmut.</td><td>Unmut.</td><td>Unmut.</td><td>Mut.</td><td>Unmut.</td><td>Mut.</td><td>Mut.</td></tr><tr><td colspan=\"8\">Comorbidities at screening</td></tr><tr><td> Cumulative Ilness Rating Scale (CIRS) score</td><td>5</td><td>4</td><td>2</td><td>0</td><td>2</td><td>3</td><td>2</td></tr><tr><td> Hypertension</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td></tr><tr><td> Diabetes</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td> Asthma</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td> COPD</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td> Cardiovascular Diseases</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td></tr><tr><td> Obesity (BMI ≥ 30 kg/m²)</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td colspan=\"8\">COVID-19 presentation</td></tr><tr><td colspan=\"8\"> Symptoms at first presentation</td></tr><tr><td>  Fever</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Y</td></tr><tr><td>  Cough</td><td>Y</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>Y</td></tr><tr><td>  Fatigue</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td></tr><tr><td>  Dyspnea</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>  Sore throat</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>  Rhinitis</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td></tr><tr><td>  Anosmia/Ageusia</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td> Bilateral pulmonary infiltrates on CT/X-ray</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>ND</td><td>Y</td><td>Y</td></tr><tr><td> Time EOT to COVID-19, months</td><td>26</td><td>18</td><td>3</td><td>1</td><td>27</td><td>30</td><td>NA</td></tr><tr><td colspan=\"8\">COVID-19 treatment and outcome</td></tr><tr><td> Hospitalization</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>Y</td></tr><tr><td> Duration of stay, days</td><td>16</td><td>12</td><td>6</td><td>55</td><td>NA</td><td>12</td><td>14</td></tr><tr><td> Oxygen support</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>Y</td></tr><tr><td> Type of ventilation/support</td><td>HFNC</td><td>NC</td><td>NC</td><td>TI</td><td>None</td><td>None</td><td>NC</td></tr><tr><td> Outcome</td><td>Resolved</td><td>Resolved</td><td>Resolved</td><td>Death</td><td>Resolved</td><td>Resolved</td><td>Death</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p><italic>Y</italic> yes, <italic>N</italic> no, <italic>NA</italic> not applicable, <italic>ND</italic> not done. <italic>M</italic> male, <italic>F</italic> female. <italic>GER</italic> Germany, <italic>NL</italic> The Netherlands, <italic>CH</italic> Switzerland. <italic>RVe</italic> rituximab, venetoclax, <italic>GIVe</italic> obinutuzumab, ibrutinib, venetoclax, <italic>GVe</italic> obinutuzumab, venetoclax, <italic>FCR</italic> fludarabine, cyclophosphamide, rituximab. <italic>Unmut.</italic> unmutated, <italic>Mut.</italic> mutated. <italic>HFNC</italic> high flow nasal cannula, <italic>NC</italic> nasal cannula, <italic>TI</italic> tracheal intubation.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Moritz Fürstenau, Petra Langerbeins</p></fn></fn-group>" ]
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[{"label": ["1."], "mixed-citation": ["Dong E, Du H, Gardner L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis. 2020;20:533\u20134."]}, {"label": ["7."], "mixed-citation": ["Thibaud S, Tremblay D, Bhalla S, Zimmerman B, Sigel K, Gabrilove J. Protective role of BTK inhibitors in patients with chronic lymphocytic leukemia and COVID-19. Br J Haematol. 2020."]}, {"label": ["8."], "mixed-citation": ["Baumann T, Delgado J, Montserrat E. CLL and COVID-19 at the hospital clinic of Barcelona: an interim report. Leukemia. 2020."]}, {"label": ["11."], "mixed-citation": ["Robert Koch-Institut. COVID-19-Dashboard 2020. "], "ext-link": ["https://experience.arcgis.com/experience/478220a4c454480e823b17327b2bf1d4"]}, {"label": ["12."], "surname": ["Verity", "Okell", "Dorigatti", "Winskill", "Whittaker", "Imai"], "given-names": ["R", "LC", "I", "P", "C", "N"], "article-title": ["Estimates of the severity of coronavirus disease 2019: a model-based analysis"], "source": ["Lancet Infect Dis."], "year": ["2019"], "volume": ["20"], "fpage": ["669"], "lpage": ["77"], "pub-id": ["10.1016/S1473-3099(20)30243-7"]}]
{ "acronym": [], "definition": [] }
15
CC BY
no
2024-01-13 23:35:05
Leukemia. 2020 Jun 29; 34(8):2225-2229
oa_package/72/45/PMC7323604.tar.gz
PMC7387243
32572189
[ "<title>Introduction</title>", "<p id=\"Par2\">Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome (Ph) [##UREF##0##1##]. Imatinib was the first BCR-ABL1-targeting tyrosine kinase inhibitor (TKI) approved for the treatment of CML [##REF##12783372##2##, ##REF##12637609##3##]. The 2nd-generation TKIs dasatinib, nilotinib, and bosutinib can be used as first-line therapy alternatives to imatinib for chronic phase (CP) CML [##REF##20525995##4##–##REF##29091516##6##]. However, patients may become resistant or intolerant to first-line TKI treatment [##REF##29411417##7##–##REF##25814079##10##]. Therapy options in the second-line setting are dasatinib, nilotinib, and bosutinib, or the 3rd-generation BCR-ABL1 TKI ponatinib [##REF##20139391##11##–##REF##24180494##14##]. TKIs radotinib and asciminib are emerging as treatment options [##REF##31334123##9##, ##REF##32127639##15##].</p>", "<p id=\"Par3\">Approval of bosutinib for patients with Ph+ CP, accelerated phase (AP), or blast phase (BP) CML previously treated with ≥1 TKI was based on results from a phase 1/2 study [##REF##21865346##13##, ##REF##22371878##16##]. In patients with imatinib-resistant or imatinib-intolerant Ph+ CML, and in patients who had received prior imatinib plus dasatinib and/or nilotinib, bosutinib 500 mg once daily (QD) demonstrated durable efficacy and manageable toxicity after longer follow-up [##REF##26537529##17##–##REF##29773593##19##]. At year 5, 40% of patients resistant/intolerant to imatinib remained on bosutinib; cumulative major cytogenetic response (MCyR), complete cytogenetic response (CCyR), and major molecular response (MMR) rates were 60%, 50%, and 42%, respectively [##REF##29773593##19##]. In a 4-year follow-up of patients receiving bosutinib in the third- or fourth-line setting, cumulative MCyR and CCyR rates were 40% and 32%, respectively [##REF##27531525##18##]. Across all patients with Ph+ CP CML in that study, the most common (≥30%) adverse events (AEs) were diarrhea, nausea, and vomiting [##REF##27531525##18##, ##REF##29773593##19##].</p>", "<p id=\"Par4\">The purpose of the current phase 4 study was to provide further information on the treatment with bosutinib of patients with CML resistant/intolerant to prior TKIs or who were otherwise not appropriate for treatment with other TKIs. This study also aimed to fulfill a post-authorization commitment to the European Medicines Agency regarding the efficacy and safety of bosutinib in this patient population.</p>" ]
[ "<title>Methods</title>", "<title>Study design and patients</title>", "<p id=\"Par5\">BYOND (NCT02228382) is an ongoing, single-arm, open-label, non-randomized phase 4 study of bosutinib in patients with chronic or advanced Ph+ CML who have failed prior treatment with TKIs. Eligible patients were adults with a cytogenetic or qualitative polymerase chain reaction-based diagnosis of Ph+ and/or <italic>BCR-ABL1</italic>+ CML (from initial diagnosis), prior treatment with ≥1 TKI for CML and adequate hepatic/renal function. Any CML phase was permitted, as long as the patient was resistant/intolerant to prior TKIs. Patients with CP CML and treated with one or two prior TKIs were required to have Eastern Cooperative Oncology Group performance status (ECOG PS) 0 or 1; those with CP CML after three prior TKIs and with AP/BP CML could have ECOG PS 0–3. Patients with leptomeningeal leukemia or a known <italic>BCR-ABL1</italic> T315I or V299L mutation were excluded. Additional details on eligibility criteria are in Supplementary Methods.</p>", "<p id=\"Par6\">Patients received bosutinib at a starting dose of 500 mg QD. Dose escalation to a maximum of 600 mg QD was permitted due to unsatisfactory response or signs of disease progression in the absence of any grade 3/4 or persistent grade 2 AEs. Dose reduction to 400, 300, or 200 mg QD due to toxicity/tolerability was permitted (see Supplementary Methods<bold>)</bold>. Patients were to receive bosutinib for up to 4 years from the time of first dose, unless disease progression, unacceptable toxicity, withdrawal of consent, death, or study discontinuation. Patients who discontinued bosutinib prior to completing 4 years of therapy were to be followed for survival until they completed 4 years on study.</p>", "<p id=\"Par7\">The study was approved by institutional review boards and independent ethics committees at each center. The study was conducted in accordance with all local legal and regulatory requirements, as well as the general principles set forth in the International Ethical Guidelines for Biomedical Research Involving Human Patients, Guidelines for Good Clinical Practice and the Declaration of Helsinki. All patients provided written informed consent.</p>", "<title>Endpoints and analyses</title>", "<p id=\"Par8\">The primary endpoints were cumulative confirmed MCyR (in two consecutive analyses ≥28 days apart) by 1 year (52 weeks) in patients with Ph+ CP CML treated with one or two prior TKIs and three prior TKIs, and cumulative confirmed overall hematologic response (OHR; in two consecutive analyses ≥28 days apart) by 1 year (52 weeks) in patients with AP or BP CML. Cumulative confirmed MCyR was defined as CCyR (0% Ph+ from ≥20 metaphases or &lt;1% fluorescent in situ hybridization [FISH] positive cells from ≥200 interphase nuclei) or partial cytogenetic response (PCyR; &gt;0%, ≤35% Ph+). To be considered a responder, the patient must have had maintenance of baseline response for ≥52 weeks for cytogenetic response or an improvement from baseline. Patients with PCyR at baseline must have attained CCyR on-treatment to be considered a cytogenetic responder. Patients with at least MMR and a deeper molecular response (MR) than baseline were counted as confirmed CCyR. Cumulative confirmed OHR was defined as complete hematologic response (CHR) or return to CP.</p>", "<p id=\"Par9\">Key secondary and exploratory endpoints included: cumulative MCyR (unconfirmed); cumulative MMR (<italic>BCR-ABL1</italic> International Scale [IS] ≤ 0.1%), MR<sup>4</sup> (<italic>BCR-ABL1</italic> IS ≤ 0.01%), and MR<sup>4.5</sup> (<italic>BCR-ABL1</italic> IS ≤ 0.0032%); <italic>BCR-ABL1</italic> mutational analyses; on-treatment transformation to AP or BP CML; overall survival (OS); safety; and patient-reported outcome (PRO) measures.</p>", "<p id=\"Par10\">Analyses for molecular, cytogenetic, and hematologic responses are described in Supplementary Methods. CCyR was imputed from MMR on a specific date if there was no valid cytogenetic assessment. Time to response was defined as the interval from the date of first dose of bosutinib to initial response. Non-responders were censored at the last valid assessment date for the respective endpoint. OS was defined as the interval from the date of first dose of bosutinib to the date of death due to any cause. Patients not known to have died were censored at the last known alive date. Time to response was estimated using cumulative incidence, adjusting for the competing risk of treatment discontinuation without the event; OS was estimated using the Kaplan–Meier method. Two-sided 95% confidence interval (CI) for response rate was determined using the exact binomial method. For Kaplan–Meier's yearly probability estimates, two-sided 95% CI was based on Greenwood’s formula using a log(-log) transformation.</p>", "<p id=\"Par11\">Treatment-emergent AEs (TEAEs), serious AEs, and laboratory evaluations were assessed up to 28 days after last dose. Events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, v4.0. The frequency of selected adverse events of special interest was analyzed by selecting Medical Dictionary for Regulatory Activities (MedDRA) system organ class higher level group, higher level and preferred terms and standardized MedDRA queries to generate TEAE clusters (see Supplementary Methods).</p>", "<p id=\"Par12\">PROs were assessed using the Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu) quality-of-life (QoL) questionnaire (see Supplementary Methods). For each cohort at each of the timepoints, summary statistics for the observed values as well as changes from baseline were estimated. As a supplemental post hoc analysis, a repeated measures longitudinal model was used to estimate the relationship between MR (screening to month 12 represented by a log-reduction scale) as a predictor and FACT-Leu total score and each domain score as an outcome. The standardized effect sizes were calculated to determine strength of effects and allow comparisons across FACT-Leu domains.</p>", "<p id=\"Par13\">This study did not include any formal sample size determination and results are descriptive only. Approximately 150 patients with Ph+ CML were to be enrolled, including ≥45 patients with CP, AP, or BP CML treated in the fourth-line or later setting. All treated patients with Ph+ CML with a valid baseline efficacy assessment for the respective endpoint (evaluable population) were included in the molecular, cytogenetic, and hematologic efficacy analyses. All patients who received ≥1 dose of study drug (full analysis set) were included in the safety analyses and those with Ph+ CP CML were included in the PRO analyses. Ph+ CP CML patients were also analyzed by resistance or intolerance to prior TKIs as assessed by the investigator (Supplementary Methods). Data are from an unlocked trial database with a cut-off date of September 18, 2018, ≥12 months after last enrolled patient.</p>" ]
[ "<title>Results</title>", "<title>Patients and treatment</title>", "<p id=\"Par14\">A total of 163 patients were enrolled between November 20, 2014 and September 18, 2017 across 41 study centers in eight countries. Of 163 patients who received bosutinib, 156 had Ph+ CP CML, four had Ph+ AP CML, and three had Ph−/<italic>BCR-ABL1</italic>+ CP CML. Across Ph+ CP CML cohorts, 51.9% of patients were male and median age was 61.0 years; 29.5%, 39.1%, and 31.4% received bosutinib as second-, third- and fourth-line TKI therapy, respectively (Table ##TAB##0##1##). In all, 53.2% of patients with Ph+ CP CML were resistant to ≥1 prior TKI and 46.8% were intolerant to all prior TKIs. Imatinib was the most common prior TKI, received by 90.4% of patients. All patients with AP CML were male, with a median age of 40.0 years; two each received bosutinib as third- and fourth-line TKI therapy. All patients with Ph− CML were male, with a median age of 63.0 years; two received bosutinib as second-line and one as third-line TKI therapy.</p>", "<p id=\"Par15\">As of ≥1 year after last enrolled patient (~85% with ≥2-year follow-up), 56.4% of patients with Ph+ CP CML remained on bosutinib: 67.4%, 54.1%, and 49.0% in the second-, third-, and fourth-line cohorts, respectively (Fig. ##FIG##0##1##). In all, 59.0% of TKI-resistant patients and 53.4% of TKI-intolerant patients remained on bosutinib. The most common primary reasons for permanent treatment discontinuation were AEs in 39 (25.0%) and insufficient clinical response in eight (5.1%) patients. Three of four patients with AP CML discontinued bosutinib due to AE, insufficient response, or lost to follow-up (<italic>n</italic> = 1 each). All three patients with Ph− CP CML discontinued bosutinib due to AE (<italic>n</italic> = 2) or death (<italic>n</italic> = 1).</p>", "<p id=\"Par16\">Median (range) duration of bosutinib treatment was 23.7 (0.2–42.2) months in the Ph+ CP CML cohort: 25.9 (0.9–41.2), 24.2 (0.4–42.2), and 12.3 (0.2–41.9) in the second-, third-, and fourth-line cohorts, respectively. In the AP and Ph− CML cohorts, median (range) duration of bosutinib treatment was 18.0 (1.6–32.3) and 7.2 (3.4–25.8) months, respectively. Median (range) dose intensity in the Ph+ CP CML cohort was 313.1 (79.7–560.6) mg/day: 320.1 (98.4–560.6), 309.4 (79.7–500.0), and 308.0 (125.0–500.0) in the second-, third-, and fourth-line cohorts, respectively. In the AP and Ph− CML cohorts, median (range) dose intensity was 497.9 (346.6–500.0) and 296.2 (81.0–422.8) mg/day, respectively. Median (range) duration of bosutinib treatment was 23.4 (0.2–42.2) months in TKI-resistant and 25.3 (0.4–41.9) months in TKI-intolerant patients, respectively. Corresponding median (range) dose intensity was 405.9 (125.0–560.6) and 292.0 (79.7–500.0) mg/day. At all timepoints, 500 mg QD was the most commonly utilized dosage and &gt;50% of patients with Ph+ CP CML were receiving 400 or 500 mg QD (Supplementary Fig. ##SUPPL##0##S1##).</p>", "<title>Efficacy</title>", "<p id=\"Par17\">Of 144 evaluable patients with Ph+ CP CML, the primary endpoint of cumulative confirmed MCyR rate (95% CI) by 1 year was 75.8% (66.1–83.8%) in those treated with one or two prior TKIs, and 62.2% (46.5–76.2%) in those previously treated with three TKIs. In all, 112 (77.8%) patients with Ph+ CP CML had MCyR at baseline. In the overall Ph+ CP CML cohort, the cumulative confirmed MCyR rate (95% CI) by 1 year was 71.5% (63.4–78.7%); 64.6% of patients achieved a deeper response relative to baseline and 6.9% maintained their baseline response for ≥1 year. In the four patients with AP CML, the primary endpoint of cumulative confirmed OHR rate (95% CI) by 1 year was 75.0% (19.4–99.4%), as was cumulative confirmed CHR rate.</p>", "<p id=\"Par18\">Cumulative MCyR and CCyR rates, respectively, by 1 year were 83.3 and 80.6% in patients with Ph+ CP CML (TKI-resistant: 79.2 and 75.3%; TKI-intolerant: 88.1 and 86.6%); cumulative MCyR and CCyR rates by line of therapy are shown in Table ##TAB##1##2##. In patients without the respective baseline response, cumulative MCyR and CCyR rates, respectively, by 1 year were 59.4 and 63.5% (TKI-resistant: 56.5 and 58.8%; TKI-intolerant: 66.7 and 72.2%; Table ##TAB##1##2##). Cytogenetic responses were achieved within 1 year with the exception of one TKI-resistant patient who achieved a CCyR after month 12.</p>", "<p id=\"Par19\">The cumulative MMR rate by 1 year was 70.5% in the overall Ph+ CP CML cohort (TKI-resistant: 60.5%; TKI-intolerant: 80.8%); rates according to line of therapy are shown in Table ##TAB##2##3##. In patients without MMR at baseline, the cumulative MMR rate by 1 year was 58.2% (TKI-resistant: 43.8%; TKI-intolerant: 80.6%). (Table ##TAB##2##3##). By 1 year, cumulative MR<sup>4</sup> and MR<sup>4.5</sup> rates, respectively, were 51.0 and 33.6% (TKI-resistant: 39.5 and 25.0%; TKI-intolerant: 63.0 and 42.5%). In patients without the respective response at baseline, cumulative MR<sup>4</sup> and MR<sup>4.5</sup> rates were 42.0 and 26.7% (TKI-resistant: 26.7 and 20.8%; TKI-intolerant: 59.6 and 33.9%). At any time, respective cumulative MR<sup>4</sup> and MR<sup>4.5</sup> rates were 57.0% and 46.3% and 49.1% and 40.5% in patients without the respective baseline response. Cumulative MR<sup>4</sup> and MR<sup>4.5</sup> rates at any time across therapy lines and in TKI-resistant and TKI-intolerant patients are shown in Table ##TAB##2##3##. Responding patients typically achieved MMR within 1 year of bosutinib initiation and a deep MR within 2 years of bosutinib initiation, although a small proportion of patients achieved MR at later time points (Fig. ##FIG##1##2##). Of three patients with Ph−/<italic>BCR-ABL1</italic>+ CML, one each had MMR, <italic>BCR-ABL1</italic> IS ≤ 1%, and no response.</p>", "<p id=\"Par20\">Eleven patients with Ph+ CP CML had mutations at baseline; of these, two achieved MR<sup>5</sup>, one achieved MR<sup>4.5</sup>, one achieved MMR, and four achieved CHR as best response. Molecular responses were observed in patients with F359I, Y253F, A365V, and E255V mutations (Supplementary Table ##SUPPL##0##S1##). Of 20 patients with Ph+ CP CML evaluated for new <italic>BCR-ABL1</italic> point mutations, one patient in the third-line cohort with a baseline Y253F mutation had a newly detectable T315I mutation.</p>", "<p id=\"Par21\">By the cutoff date, no patient with Ph+ CP CML had progressed to AP/BP on treatment. After a median follow-up of 30.4 months (range 0.7–44.6), 1- and 2-year Kaplan–Meier OS rates, respectively, were 98.0% and 96.0%, for patients with Ph+ CP CML (second-line: 100 and 97.7%; third-line: 96.7 and 95.0%; fourth-line: 97.9 and 95.4%; Supplementary Fig. ##SUPPL##0##S2A##). The respective rates were 97.5 and 94.9% in TKI-resistant and 98.6 and 97.2% in TKI-intolerant patients (Supplementary Fig. ##SUPPL##0##S2B##). After a median follow-up of 20.6 months (range 1.6–32.3) and 26.5 months (3.5–41.4) for patients with Ph+ AP CML and Ph− CML, respectively, OS rates (95% CI) at both 1 year and 2 years were 100% (100–100%) and 66.7% (5.4–94.5%).</p>", "<title>Safety</title>", "<p id=\"Par22\">In the overall patient population (<italic>N</italic> = 163), 99.4% of patients had ≥1 any grade TEAE and 73.6% of patients had ≥1 grade 3/4 TEAE. Treatment-emergent serious AEs were reported in 35.6% of patients. TEAEs led to dose reduction and temporary discontinuation in 77.3% and 75.5% of patients, respectively, and 42 (25.8%) discontinued treatment due to AEs. The most common AEs leading to discontinuation (≥2% of patients) were increased alanine aminotransferase (4.9%) and increased aspartate aminotransferase (2.5%). In TKI-resistant and TKI-intolerant patients, respectively, rates of any grade TEAEs were 100% and 98.6%, and rates of grade 3/4 TEAEs were 69.9% and 79.5%. The rate of dose reductions due to TEAEs was 73.5% in TKI-resistant patients and 84.9% in TKI-intolerant patients; respective rate of temporary discontinuations due to TEAEs was 68.7% and 84.9%. Overall, 21.7% and 28.8% of TKI-resistant and TKI-intolerant patients, respectively, discontinued treatment due to AEs. There were no relevant differences in the overall frequency of TEAEs, grade 3/4 TEAEs, or dose reductions/temporary discontinuations due to TEAEs across lines of treatment.</p>", "<p id=\"Par23\">The most common TEAEs (&gt;30%) in the overall patient population were diarrhea (87.7%), nausea (39.9%), and vomiting (32.5%) (Table ##TAB##3##4##). However, only two (1.2%), three (1.8%), and two (1.2%) patients discontinued due to diarrhea, nausea, and vomiting, respectively. Median (range) time to first TEAE of diarrhea was 2.0 (1–304) days, and the median (range) duration of diarrhea event (any grade) was 8.0 (1–715) days. Grade 3/4 TEAEs occurring in &gt;5% of patients were diarrhea (16.0%), increased alanine aminotransferase (14.1%), thrombocytopenia (8.0%), increased lipase (6.7%), and pleural effusion (6.1%). TEAEs of special interest included cardiac (14.7%), vascular (11.7%), effusion (18.4%), metabolic (8.0%), and gastrointestinal (91.4%; Table ##TAB##4##5##).</p>", "<p id=\"Par24\">There were 12 deaths, seven of which occurred within 28 days of last dose (six due to AEs not related to bosutinib and one due to CML, as determined by the investigator) and five deaths occurred beyond 28 days of last dose (four due to AEs not related to bosutinib and one due to an unknown cause, as determined by the investigator). AEs resulting in death were: acute kidney injury, respiratory insufficiency due to aspiration, cerebral tumor, chronic briden-ileus, hemorrhagic shock, lymphoma, metastatic lung cancer, multiorgan failure, prostate adenocarcinoma, and sepsis.</p>", "<title>PROs</title>", "<p id=\"Par25\">At baseline, total FACT-Leu scores were similar (&lt;5% difference) between the second- and third-line cohorts; slightly lower scores were reported in the fourth-line cohort at baseline (Fig. ##FIG##2##3## and Supplementary Table ##SUPPL##0##S2##). Total FACT-Leu scores were maintained from baseline in all cohorts following 12 months of bosutinib treatment (Fig. ##FIG##2##3##); additionally, at month 12, no mean change in any individual FACT-Leu domain score from baseline met the MID, indicating preservation of health-related QoL (HRQoL) (Supplementary Fig. ##SUPPL##0##S3##; Fig. ##FIG##2##3##). Within these MIDs, FACT-Leu scores increased slightly from baseline to month 12 in the second-line cohort and (except for emotional well-being) decreased slightly in the third-line cohort; positive versus negative changes were less consistent in the fourth-line cohort. The effect of MR on HRQoL was variable. For patients who achieved MR<sup>5</sup>, the leukemia-specific domain showed the greatest improvement, with a large effect size, followed by the emotional well-being domain and TOI FACT-Leu, with medium effect sizes; the social well-being domain was the only domain to demonstrate a beyond-trivial reduction in HRQoL, with a medium effect size (Supplementary Fig. ##SUPPL##0##S4##).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par26\">Overall, data from the phase 4 BYOND study confirm the efficacy findings from the phase 1/2 study of bosutinib in the second-, third- and fourth-line settings [##REF##21865346##13##, ##REF##22371878##16##–##REF##24711212##20##]. High rates of cytogenetic and molecular responses were observed across patients with Ph+ CP CML treated with bosutinib, including a large proportion of patients who achieved deep MR. The majority of patients achieved deeper responses relative to baseline with bosutinib, even though most patients entered the study with at least MCyR from prior treatment. These high response rates were seen in TKI-resistant and TKI-intolerant patients and across treatment lines. Although patients with a more resistant phenotype showed lower response rates compared with TKI-intolerant patients, responses were also seen in patients with resistance to imatinib or the 2nd-generation TKIs dasatinib and nilotinib.</p>", "<p id=\"Par27\">Data in patients with CML resistant/intolerant to prior TKIs were also reported in studies of nilotinib, dasatinib, and ponatinib [##REF##20139391##11##, ##REF##24180494##14##, ##REF##21098399##21##]. In a phase 2 study of second-line nilotinib 400 mg twice daily in imatinib-resistant/intolerant patients (<italic>N</italic> = 321), MCyR, CCyR, and MMR rates at any time on treatment were 59%, 44%, and 28%, respectively, after a minimum follow-up of 24 months [##REF##21098399##21##]. In a phase 3 study of second-line dasatinib (multiple doses) in imatinib-resistant/intolerant patients (<italic>N</italic> = 670), MCyR, CCyR and MMR rates by 24 months were 61–63%, 50–54%, and 37–38%, respectively [##REF##20139391##11##]. In the present study, second-line bosutinib yielded MCyR, CCyR, and MMR rates (both by 24 months and at any time) of 80.0%, 81.3%, and 76.0%, respectively, in patients without the respective baseline response. For bosutinib, achieved rates are used for the purpose of comparisons with the nilotinib and dasatinib studies due to the higher proportion of patients with baseline MCyR enrolled in BYOND: 77.8% versus 11% and 14–20% for nilotinib and dasatinib, respectively [##REF##20139391##11##, ##REF##21098399##21##, ##REF##18541900##22##]. In the present study, response rates were higher in patients treated with fewer previous TKIs; however, response was also seen in heavily pretreated patients: 47.1% and 38.5% of patients (without the respective baseline response) treated with bosutinib fourth-line therapy achieved CCyR and MMR, respectively, by 1 year. These data are comparable to those reported in a phase 2 study of ponatinib 45 mg QD in heavily pretreated patients with CP CML without the T315I mutation (<italic>N</italic> = 203); after a median follow-up of 15 months, MCyR, CCyR, and MMR rates by 12 months were 51%, 40%, and 27%, respectively [##REF##24180494##14##]. In summary, the current data for bosutinib from BYOND, with a median follow-up of 30.4 months, showed comparable cytogenetic and molecular response rates to those reported with nilotinib, dasatinib, or ponatinib treatment.</p>", "<p id=\"Par28\">Overall, AEs with bosutinib were manageable. The reported AEs were consistent with the known safety profile of bosutinib and no new safety issues were identified [##REF##29091516##6##, ##REF##26537529##17##–##REF##24711212##20##, ##REF##24944159##23##–##REF##25196702##27##]. Patients intolerant to previous therapies had a slightly higher incidence of grade 3/4 TEAEs and required more frequent dose adjustments to manage AEs than TKI-resistant patients. Nevertheless, most patients intolerant to previous TKIs, including patients intolerant to all prior TKIs, were able to remain on treatment with bosutinib (median treatment duration, 25.3 months). The overall discontinuation rate due to AEs was consistent with the previous phase 1/2 study, despite approximately half of patients being intolerant to all prior TKI therapy, which indicates that, in general, AEs were manageable with dose reductions and temporary discontinuations. Despite a high incidence of diarrhea, which is usually transient and often improves with dietary changes and the administration of supportive care, bosutinib discontinuation due to this AE was low [##REF##29385394##28##]. In patients who have an increase in transaminases, it is advisable to avoid other hepatotoxic drugs and excess alcohol consumption, and monitor liver enzymes more frequently; dose modifications and/or discontinuation may be required in more severe cases [##REF##30587215##29##].</p>", "<p id=\"Par29\">As with imatinib, patients treated with dasatinib or nilotinib may eventually develop resistance to treatment. In addition, some patients may be unable to continue treatment with dasatinib, nilotinib, or ponatinib due to intolerance, or the safety profiles of these agents may preclude their use in patients with certain comorbidities. Safety comparisons across TKI studies are limited; however, the varying “off target” effects of bosutinib, nilotinib, dasatinib, and ponatinib are reflected in the safety of these agents, with each BCR-ABL1 TKI showing a distinct toxicity profile.</p>", "<p id=\"Par30\">Vascular AEs have been described mainly with nilotinib and ponatinib. Metabolic AEs, potentially contributing to vascular toxicity, are also frequently reported with nilotinib [##REF##24180494##14##, ##REF##21098399##21##, ##REF##29454474##30##]. The European LeukemiaNet (ELN) recommendations, therefore, state that nilotinib is contraindicated in patients with a history of coronary heart disease, cerebrovascular accidents, or peripheral arterio-occlusive disease and that previous or concomitant arteriovascular disease is a contraindication to ponatinib in second- or third-line treatment. Pulmonary toxicities, such as pleural effusion and more rarely pulmonary arterial hypertension, have been primarily associated with dasatinib treatment [##REF##20139391##11##, ##REF##22451584##31##–##REF##30093398##33##], and the ELN panel recommended to avoid the use of dasatinib in patients with respiratory failure and previous or concomitant pleuro-pulmonary or pericardial disease. While the incidence of these specific AEs with bosutinib in BYOND was higher than previously reported [##REF##21865346##13##, ##REF##22371878##16##, ##REF##26971533##34##, ##REF##27121688##35##], the heavily pretreated nature of the patients might have contributed to this. ELN recommendations state that no relevant comorbidities or contraindications have been identified for bosutinib [##REF##32127639##15##], and bosutinib is a treatment option for patients with pulmonary or cardiovascular comorbidities, diabetes mellitus, or hyperglycemia due to the lower risk of these types of events [##REF##31833784##36##]. Therefore, bosutinib is an appropriate treatment option for patients resistant or intolerant to prior TKIs, including in patients who have previously received treatment with a 2nd-generation TKI and in those who present with multiple comorbidities.</p>", "<p id=\"Par31\">HRQoL was maintained from baseline in patients with CP CML following 12 months of bosutinib treatment. Changes from baseline in patient-reported outcomes measures at month 12 were comparable to those observed in previously treated patients in the initial phase 1/2 study of bosutinib, wherein long-term efficacy and HRQoL stability were reported [##REF##29773593##19##, ##REF##22036634##37##, ##REF##27045164##38##]. In addition, FACT-G scores in the current study were consistent with those previously reported for bosutinib in newly diagnosed patients with CML, in the general population, as well as in patients with various cancers [##UREF##3##39##–##REF##32307568##43##]. Maintenance of HRQoL is important for patients with CP CML who potentially will receive lifelong TKI treatment, and PRO results from this phase 4 study suggest bosutinib is a treatment option with manageable AEs, providing further support for its use in patients with CP CML resistant/intolerant to prior TKIs. The impact of clinical improvement on different dimensions of HRQoL was variable; for the majority of domains, a deeper MR was associated with better HRQoL.</p>", "<p id=\"Par32\">In summary, high rates of cytogenetic and molecular responses, including a large proportion of patients who achieved MR<sup>4</sup> and MR<sup>4.5</sup>, were observed with bosutinib treatment. AEs that occurred with bosutinib were manageable [##REF##29385394##28##, ##REF##30587215##29##], further evidenced by maintenance of HRQoL, and the reported AEs were consistent with the known safety profile of bosutinib. The results from this phase 4 study further confirm the use of bosutinib for patients with CML resistant/intolerant to prior TKIs across all treatment lines.</p>" ]
[]
[ "<p id=\"Par1\">Bosutinib is approved for newly diagnosed Philadelphia chromosome-positive (Ph+) chronic phase (CP) chronic myeloid leukemia (CML) and for Ph+ CP, accelerated (AP), or blast (BP) phase CML after prior treatment with tyrosine kinase inhibitors (TKIs). In the ongoing phase 4 BYOND study (NCT02228382), 163 CML patients resistant/intolerant to prior TKIs (<italic>n</italic> = 156 Ph+ CP CML, <italic>n</italic> = 4 Ph+ AP CML, <italic>n</italic> = 3 Ph-negative/<italic>BCR-ABL1</italic>+ CML) received bosutinib 500 mg once daily (starting dose). As of ≥1 year after last enrolled patient (median treatment duration 23.7 months), 56.4% of Ph+ CP CML patients remained on bosutinib. Primary endpoint of cumulative confirmed major cytogenetic response (MCyR) rate by 1 year was 75.8% in Ph+ CP CML patients after one or two prior TKIs and 62.2% after three prior TKIs. Cumulative complete cytogenetic response (CCyR) and major molecular response (MMR) rates by 1 year were 80.6% and 70.5%, respectively, in Ph+ CP CML patients overall. No patient progressed to AP/BP on treatment. Across all patients, the most common treatment-emergent adverse events were diarrhea (87.7%), nausea (39.9%), and vomiting (32.5%). The majority of patients had confirmed MCyR by 1 year and MMR by 1 year, further supporting bosutinib use for Ph+ CP CML patients resistant/intolerant to prior TKIs.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0915-9) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>The study was sponsored by Pfizer Inc. Medical writing support was provided by Anne Marie McGonigal, PhD CMPP, and Gary Dever, PhD CMPP, of Engage Scientific Solutions and was funded by Pfizer Inc. Open access funding provided by Projekt DEAL.</p>", "<title>Additional BYOND Study Investigators</title>", "<p id=\"Par33\">E. Abruzzese<sup>17</sup>, L. P. Akard<sup>18</sup>, A. Bosi<sup>19</sup>, F. Cervantes<sup>20</sup>, A. Charbonnier<sup>21</sup>, F. Di Raimondo<sup>22</sup>, G. Etienne<sup>23</sup>, V. Garcia Gutierrez<sup>24</sup>, A. P. Guerci-Bresler<sup>25</sup>, H. Hjorth-Hansen<sup>26</sup>, J. M. Karsenti<sup>27</sup>, K. R. Kelly<sup>28</sup>, P. Le Coutre<sup>29</sup>, C. Martinez Chamorro<sup>30</sup>, V. G. Oehler<sup>31</sup>, G. Orti Pascual<sup>32</sup>, A. Petzer<sup>33</sup>, E. Pungolino<sup>34</sup>, G. Rege-Cambrin<sup>35</sup>, F. Rigal-Huguet<sup>36</sup>, G. J. Roboz<sup>37</sup>, P. Rousselot<sup>38</sup>, F. M. Sanchez-Guijo<sup>39</sup>, G. Sanz Santillana<sup>40</sup>, P. Schafhausen<sup>41</sup>, C. Scheid<sup>42</sup>, S. Schmidt<sup>43</sup>, G. Specchia<sup>44</sup>, J. L. Steegmann<sup>45</sup>, L. Stenke<sup>46</sup></p>", "<title>Author contributions</title>", "<p>All authors were involved in the study conception/design, or the acquisition, analysis, or interpretation of data. All authors contributed to the drafting of the manuscript and approved the final version.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par34\">Authors declare the following potential conflicts of interest: AH received honoraria from BMS, Novartis, Pfizer, and Takeda, and received research support from BMS, Incyte, Novartis, and Pfizer. CGP provides consultancy to BMS and received honoraria and research support from Pfizer. CA serves on the advisory board for Archer DX, Jazz Pharma, and Tetraphase Pharma, provides consultancy to Actinium, Agios, Bayer, Cardinal Health, Gerson Lehman Group, Incyte, Jazz Pharma, NKarta, Pfizer, Seattle Genetics, and Tetraphase Pharma, received honoraria from Agios, Cardinal Health, and Jazz Pharma, received research support from Pfizer, and serves on the speakers bureau for Jazz Pharma / provides expert testimony for Dava Oncology. BTG is a consultant for BerGenBio, Novartis, Pfizer, and Sanofi Genzyme, received research support from Pfizer, and has stock ownership in Alden Cancer Therapy AS and KinN Therapeutics AS. THB is a consultant for Janssen, Merck, Novartis, Pfizer, and Takeda, and received research support from Novartis and Pfizer. BDS received honoraria for consulting to Agios, Celgene, Jazz Pharma, Novartis and Pfizer, and received research support from Pfizer. PGC received research support from BMS and Pfizer. UOS received research support from Pfizer. SS received honoraria from BMS, Incyte, Novartis, and Pfizer, and received research support from BMS, Incyte, Novartis, and Pfizer. TE received research support from BMS, Incyte, Novartis, and Pfizer. NBB, AV, EL, ARS and JL are employees of Pfizer. GR received research support from Pfizer and served on the speaker bureau for BMS, Incyte, Novartis, and Pfizer. JW serves on the advisory board for Jazz Pharma and Takeda, received research support from Pfizer and Takeda, and serves on the data monitoring committee for Rafael Pharma. FJG is a consultant to Actuate Therapeutics Inc, provides expert testimony to Novartis, and received research support from Pfizer.</p>", "<title>Data Sharing</title>", "<p id=\"Par35\">Upon request, and subject to certain criteria, conditions and exceptions (see <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.pfizer.com/science/clinical-trials/trial-data-and-results\">https://www.pfizer.com/science/clinical-trials/trial-data-and-results</ext-link> for more information), Pfizer will provide access to individual de-identified participant data from Pfizer-sponsored global interventional clinical studies conducted for medicines, vaccines and medical devices (1) for indications that have been approved in the USA and/or Europe or (2) in programs that have been terminated (i.e., development for all indications has been discontinued). Pfizer will also consider requests for the protocol, data dictionary, and statistical analysis plan. Data may be requested from Pfizer trials 24 months after study completion. The de-identified participant data will be made available to researchers whose proposals meet the research criteria and other conditions, and for which an exception does not apply, via a secure portal. To gain access, data requestors must enter into a data access agreement with Pfizer.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Patient disposition.</title><p>Full analysis set. The 14 participants screened but not enrolled did not meet the eligibility criteria. <italic>AE</italic> adverse events, <italic>AP</italic> accelerated phase, <italic>CML</italic> chronic myeloid leukemia, <italic>CP</italic> chronic phase, <italic>FAS</italic> full analysis set, <italic>Ph</italic> Philadelphia chromosome.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Cumulative Incidence of Molecular Response in Patients with Ph+ CP CML.</title><p>(<bold>a</bold>) MMR, (<bold>b</bold>) MR<sup>4</sup>, and (<bold>c</bold>) MR<sup>4.5</sup>. <italic>CML</italic> chronic myeloid leukemia, <italic>CP</italic> chronic phase, <italic>MMR</italic> major molecular response, <italic>MR</italic> molecular response, <italic>Ph</italic> Philadelphia chromosome.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Observed mean (SE) FACT-Leu values over 12 months of bosutinib treatment.</title><p><italic>CML</italic> chronic myeloid leukemia, <italic>CP</italic> chronic phase, <italic>CP2L</italic> second-line, <italic>CP3L</italic> third-line, <italic>CP4L</italic> fourth-line, <italic>FACT-Leu</italic> functional assessment of cancer therapy–leukemia, <italic>Ph</italic> Philadelphia chromosome, <italic>SE</italic> Standard error.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Demographic and baseline characteristics across patients with Ph + CP CML.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Characteristic</th><th colspan=\"3\">Line of treatment</th><th rowspan=\"2\">Total (<italic>N</italic> = 156)</th></tr><tr><th>Second-line (<italic>n</italic> = 46)</th><th>Third-line (<italic>n</italic> = 61)</th><th>Fourth-line (<italic>n</italic> = 49)</th></tr></thead><tbody><tr><td>Male, <italic>n</italic> (%)</td><td>23 (50.0)</td><td>37 (60.7)</td><td>21 (42.9)</td><td>81 (51.9)</td></tr><tr><td>Age, median (range), years</td><td>54.0 (19.0–88.0)</td><td>65.0 (27.0–85.0)</td><td>61.0 (21.0–85.0)</td><td>61.0 (19.0–88.0)</td></tr><tr><td colspan=\"5\">Age group, <italic>n</italic> (%)</td></tr><tr><td>    &lt;65 years</td><td>34 (73.9)</td><td>30 (49.2)</td><td>32 (65.3)</td><td>96 (61.5)</td></tr><tr><td>    ≥65 years</td><td>12 (26.1)</td><td>31 (50.8)</td><td>17 (34.7)</td><td>60 (38.5)</td></tr><tr><td colspan=\"5\">ECOG PS, <italic>n</italic> (%)</td></tr><tr><td>    0</td><td>34 (73.9)</td><td>40 (65.6)</td><td>32 (65.3)</td><td>106 (67.9)</td></tr><tr><td>    1</td><td>12 (26.1)</td><td>20 (32.8)</td><td>13 (26.5)</td><td>45 (28.8)</td></tr><tr><td>    2</td><td>0</td><td>1 (1.6)</td><td>4 (8.2)</td><td>5 (3.2)</td></tr><tr><td>Median (range) duration since CML diagnosis, years</td><td>2.2 (0.2–11.4)</td><td>5.0 (0.3–18.6)</td><td>7.3 (0.7–27.7)</td><td>4.7 (0.2–27.7)</td></tr><tr><td colspan=\"5\">Prior TKI, <italic>n</italic> (%)<sup>a</sup></td></tr><tr><td>    Imatinib</td><td>35 (76.1)</td><td>57 (93.4)</td><td>49 (100)</td><td>141 (90.4)</td></tr><tr><td>    Dasatinib</td><td>5 (10.9)</td><td>41 (67.2)</td><td>49 (100)</td><td>95 (60.9)</td></tr><tr><td>    Nilotinib</td><td>6 (13.0)</td><td>24 (39.3)</td><td>49 (100)</td><td>79 (50.6)</td></tr><tr><td>Prior interferon alpha, <italic>n</italic> (%)</td><td>2 (4.3)</td><td>3 (4.9)</td><td>6 (12.2)</td><td>11 (7.1)</td></tr><tr><td>Resistant to any prior TKI, <italic>n</italic> (%)</td><td>17 (37.0)</td><td>35 (57.4)</td><td>31 (63.3)</td><td>83 (53.2)</td></tr><tr><td>Intolerant to all prior TKIs, <italic>n</italic> (%)</td><td>29 (63.0)</td><td>26 (42.6)</td><td>18 (36.7)</td><td>73 (46.8)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Cumulative cytogenetic response rates by 1 year in patients with Ph + CP CML: total cohort, by line of therapy, and by TKI resistance or intolerance (overall and excluding patients with the respective baseline response).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\"/><th rowspan=\"2\">Total <italic>N</italic> = 156</th><th colspan=\"3\">By line of therapy</th><th colspan=\"2\">By TKI resistance or intolerance</th></tr><tr><th>Second-line <italic>n</italic> = 46</th><th>Third-line <italic>n</italic> = 61</th><th>Fourth-line <italic>n</italic> = 49</th><th>Resistant <italic>n</italic> = 83</th><th>Intolerant <italic>n</italic> = 73</th></tr></thead><tbody><tr><td colspan=\"7\">Cumulative cytogenetic response, % (95% CI)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>144</td><td>43</td><td>56</td><td>45</td><td>77</td><td>67</td></tr><tr><td>   MCyR</td><td>83.3 (76.2–89.0)</td><td>88.4 (74.9–96.1)</td><td>83.9 (71.7–92.4)</td><td>77.8 (62.9–88.8)</td><td>79.2 (68.5–87.6)</td><td>88.1 (77.8–94.7)</td></tr><tr><td>   CCyR</td><td>80.6 (73.1–86.7)</td><td>83.7 (69.3–93.2)</td><td>83.9 (71.7–92.4)</td><td>73.3 (58.1–85.4)</td><td>75.3 (64.2–84.4)</td><td>86.6 (76.0–93.7)</td></tr><tr><td colspan=\"7\">Cumulative cytogenetic response in patients without the respective baseline response, % (95% CI)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>32</td><td>10</td><td>10</td><td>12</td><td>23</td><td>9</td></tr><tr><td>   MCyR</td><td>59.4 (40.6–76.3)</td><td>80.0 (44.4–97.5)</td><td>60.0 (26.2–87.8)</td><td>41.7 (15.2–72.3)</td><td>56.5 (34.5–76.8)</td><td>66.7 (29.9–92.5)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>52</td><td>16</td><td>19</td><td>17</td><td>34</td><td>18</td></tr><tr><td>   CCyR</td><td>63.5 (49.0–76.4)</td><td>75.0 (47.6–92.7)</td><td>68.4 (43.4–87.4)</td><td>47.1 (23.0–72.2)</td><td>58.8 (40.7–75.4)</td><td>72.2 (46.5–90.3)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Cumulative molecular response rates by 1 year, by 2 years, and any time on treatment in patients with Ph + CP CML: total cohort, by line of therapy and by TKI resistance or intolerance (overall and excluding patients with the respective baseline response).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\"/><th rowspan=\"2\">Total <italic>N</italic> = 156</th><th colspan=\"3\">By line of therapy</th><th colspan=\"2\">By TKI resistance or intolerance</th></tr><tr><th>Second-line <italic>n</italic> = 46</th><th>Third-line <italic>n</italic> = 61</th><th>Fourth-line <italic>n</italic> = 49</th><th>Resistant <italic>n</italic> = 83</th><th>Intolerant <italic>n</italic> = 73</th></tr></thead><tbody><tr><td colspan=\"7\">Cumulative molecular response, % (95% CI)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>149</td><td>46</td><td>55</td><td>48</td><td>76</td><td>73</td></tr><tr><td colspan=\"7\">MMR</td></tr><tr><td> By 1 year</td><td>70.5 (62.5–77.7)</td><td>80.4 (66.1–90.6)</td><td>74.5 (61.0–85.3)</td><td>56.3 (41.2–70.5)</td><td>60.5 (48.6–71.6)</td><td>80.8 (69.9–89.1)</td></tr><tr><td> By 2 years</td><td>71.1 (63.2–78.3)</td><td>82.6 (68.6–92.2)</td><td>74.5 (61.0–85.3)</td><td>56.3 (41.2–70.5)</td><td>61.8 (50.0–72.8)</td><td>80.8 (69.9–89.1)</td></tr><tr><td> Any time on treatment</td><td>71.8 (63.9–78.9)</td><td>82.6 (68.6–92.2)</td><td>76.4 (63.0–86.8)</td><td>56.3 (41.2–70.5)</td><td>61.8 (50.0–72.8)</td><td>82.2 (71.5–90.2)</td></tr><tr><td colspan=\"7\">MR<sup>4</sup></td></tr><tr><td> By 1 year</td><td>51.0 (42.7–59.3)</td><td>58.7 (43.2–73.0)</td><td>54.5 (40.6–68.0)</td><td>39.6 (25.8–54.7)</td><td>39.5 (28.4–51.4)</td><td>63.0 (50.9–74.0)</td></tr><tr><td> By 2 years</td><td>55.7 (47.3–63.8)</td><td>67.4 (52.0–80.5)</td><td>60.0 (45.9–73.0)</td><td>39.6 (25.8–54.7)</td><td>46.1 (34.5–57.9)</td><td>65.8 (53.7–76.5)</td></tr><tr><td> Any time on treatment</td><td>57.0 (48.7–65.1)</td><td>69.6 (54.2–82.3)</td><td>61.8 (47.7–74.6)</td><td>39.6 (25.8–54.7)</td><td>46.1 (34.5–57.9)</td><td>68.5 (56.6–78.9)</td></tr><tr><td colspan=\"7\">MR<sup>4.5</sup></td></tr><tr><td> By 1 year</td><td>33.6 (26.0–41.7)</td><td>32.6 (19.5–48.0)</td><td>36.4 (23.8–50.4)</td><td>31.3 (18.7–46.3)</td><td>25.0 (15.8–36.3)</td><td>42.5 (31.0–54.6)</td></tr><tr><td> By 2 years</td><td>43.0 (34.9–51.3)</td><td>47.8 (32.9–63.1)</td><td>45.5 (32.0–59.4)</td><td>35.4 (22.2–50.5)</td><td>35.5 (24.9–47.3)</td><td>50.7 (38.7–62.6)</td></tr><tr><td> Any time on treatment</td><td>46.3 (38.1–54.7)</td><td>56.5 (41.1–71.1)</td><td>47.3 (33.7–61.2)</td><td>35.4 (22.2–50.5)</td><td>36.8 (26.1–48.7)</td><td>56.2 (44.1–67.8)</td></tr><tr><td colspan=\"7\">Cumulative molecular response in patients without the respective baseline response, % (95% CI)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>79</td><td>25</td><td>28</td><td>26</td><td>48</td><td>31</td></tr><tr><td colspan=\"7\">MMR</td></tr><tr><td> By 1 year</td><td>58.2 (46.6–69.2)</td><td>72.0 (50.6–87.9)</td><td>64.3 (44.1–81.4)</td><td>38.5 (20.2–59.4)</td><td>43.8 (29.5–58.8)</td><td>80.6 (62.5–92.5)</td></tr><tr><td> By 2 years</td><td>59.5 (47.9–70.4)</td><td>76.0 (54.9–90.6)</td><td>64.3 (44.1–81.4)</td><td>38.5 (20.2–59.4)</td><td>45.8 (31.4–60.8)</td><td>80.6 (62.5–92.5)</td></tr><tr><td> Any time on treatment</td><td>59.5 (47.9–70.4)</td><td>76.0 (54.9–90.6)</td><td>64.3 (44.1–81.4)</td><td>38.5 (20.2–59.4)</td><td>45.8 (31.4–60.8)</td><td>80.6 (62.5–92.5)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>112</td><td>37</td><td>38</td><td>37</td><td>60</td><td>52</td></tr><tr><td colspan=\"7\">MR<sup>4</sup></td></tr><tr><td> By 1 year</td><td>42.0 (32.7–51.7)</td><td>51.4 (34.4–68.1)</td><td>44.7 (28.6–61.7)</td><td>29.7 (15.9–47.0)</td><td>26.7 (16.1–39.7)</td><td>59.6 (45.1–73.0)</td></tr><tr><td> By 2 years</td><td>48.2 (38.7–57.9)</td><td>62.2 (44.8–77.5)</td><td>52.6 (35.8–69.0)</td><td>29.7 (15.9–47.0)</td><td>35.0 (23.1–48.4)</td><td>63.5 (49.0–76.4)</td></tr><tr><td> Any time on treatment</td><td>49.1 (39.5–58.7)</td><td>64.9 (47.5–79.8)</td><td>52.6 (35.8–69.0)</td><td>29.7 (15.9–47.0)</td><td>35.0 (23.1–48.4)</td><td>65.4 (50.9–78.0)</td></tr><tr><td>Evaluable patients, <italic>n</italic></td><td>131</td><td>42</td><td>46</td><td>43</td><td>72</td><td>59</td></tr><tr><td colspan=\"7\">MR<sup>4.5</sup></td></tr><tr><td> By 1 year</td><td>26.7 (19.4–35.2)</td><td>26.2 (13.9–42.0)</td><td>28.3 (16.0–43.5)</td><td>25.6 (13.5–41.2)</td><td>20.8 (12.2–32.0)</td><td>33.9 (22.1–47.4)</td></tr><tr><td> By 2 years</td><td>37.4 (29.1–46.3)</td><td>42.9 (27.7–59.0)</td><td>39.1 (25.1–54.6)</td><td>30.2 (17.2–46.1)</td><td>31.9 (21.4–44.0)</td><td>44.1 (31.2–57.6)</td></tr><tr><td> Any time on treatment</td><td>40.5 (32.0–49.4)</td><td>52.4 (36.4–68.0)</td><td>39.1 (25.1–54.6)</td><td>30.2 (17.2–46.1)</td><td>33.3 (22.7–45.4)</td><td>49.2 (35.9–62.5)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Summary of TEAEs (all grade TEAEs reported in ≥10% of patients).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\"><italic>n</italic> (%)</th><th colspan=\"2\">Total (<italic>N</italic> = 163)</th></tr><tr><th>All grades</th><th>Grades 3/4</th></tr></thead><tbody><tr><td><bold>Any TEAE</bold></td><td><bold>162</bold> (<bold>99.4)</bold></td><td><bold>120 (73.6)</bold></td></tr><tr><td>Diarrhea</td><td>143 (87.7)</td><td>26 (16.0)</td></tr><tr><td>Nausea</td><td>65 (39.9)</td><td>4 (2.5)</td></tr><tr><td>Vomiting</td><td>53 (32.5)</td><td>6 (3.7)</td></tr><tr><td>Abdominal pain</td><td>46 (28.2)</td><td>7 (4.3)</td></tr><tr><td>Headache</td><td>45 (27.6)</td><td>1 (0.6)</td></tr><tr><td>ALT increased</td><td>42 (25.8)</td><td>23 (14.1)</td></tr><tr><td>Fatigue</td><td>39 (23.9)</td><td>2 (1.2)</td></tr><tr><td>Abdominal pain upper</td><td>36 (22.1)</td><td>2 (1.2)</td></tr><tr><td>Dyspnea</td><td>35 (21.5)</td><td>5 (3.1)</td></tr><tr><td>Asthenia</td><td>33 (20.2)</td><td>4 (2.5)</td></tr><tr><td>AST increased</td><td>32 (19.6)</td><td>7 (4.3)</td></tr><tr><td>Cough</td><td>30 (18.4)</td><td>1 (0.6)</td></tr><tr><td>Pyrexia</td><td>29 (17.8)</td><td>5 (3.1)</td></tr><tr><td>Constipation</td><td>28 (17.2)</td><td>2 (1.2)</td></tr><tr><td>Arthralgia</td><td>28 (17.2)</td><td>1 (0.6)</td></tr><tr><td>Pleural effusion</td><td>27 (16.6)</td><td>10 (6.1)</td></tr><tr><td>Back pain</td><td>27 (16.6)</td><td>4 (2.5)</td></tr><tr><td>Anemia</td><td>25 (15.3)</td><td>7 (4.3)</td></tr><tr><td>Rash</td><td>25 (15.3)</td><td>7 (4.3)</td></tr><tr><td>Dizziness</td><td>25 (15.3)</td><td>0</td></tr><tr><td>Blood creatinine increased</td><td>24 (14.7)</td><td>0</td></tr><tr><td>Nasopharyngitis</td><td>24 (14.7)</td><td>0</td></tr><tr><td>Lipase increased</td><td>23 (14.1)</td><td>11 (6.7)</td></tr><tr><td>Myalgia</td><td>22 (13.5)</td><td>2 (1.2)</td></tr><tr><td>Decreased appetite</td><td>22 (13.5)</td><td>1 (0.6)</td></tr><tr><td>Edema peripheral</td><td>22 (13.5)</td><td>1 (0.6)</td></tr><tr><td>Thrombocytopenia</td><td>18 (11.0)</td><td>13 (8.0)</td></tr><tr><td>Pain in extremity</td><td>17 (10.4)</td><td>2 (1.2)</td></tr><tr><td>Pruritus</td><td>17 (10.4)</td><td>2 (1.2)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>TEAEs of special interest.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th><italic>n</italic> (%)</th><th>Total (<italic>N</italic> = 163)</th></tr></thead><tbody><tr><td colspan=\"2\"><bold>Cardiac TEAEs</bold></td></tr><tr><td> Any TEAE</td><td>24 (14.7)</td></tr><tr><td> Cardiac disorders</td><td>23 (14.1)</td></tr><tr><td>   Cardiac failure</td><td>6 (3.7)</td></tr><tr><td>   Atrial fibrillation</td><td>5 (3.1)</td></tr><tr><td>   Tachycardia</td><td>3 (1.8)</td></tr><tr><td>   Arrhythmia</td><td>2 (1.2)</td></tr><tr><td>   Bradycardia</td><td>2 (1.2)</td></tr><tr><td>   Cardiac failure congestive</td><td>2 (1.2)</td></tr><tr><td>   Atrial flutter</td><td>1 (0.6)</td></tr><tr><td>   Atrioventricular block complete</td><td>1 (0.6)</td></tr><tr><td>   Bundle branch block right</td><td>1 (0.6)</td></tr><tr><td>   Cardiac failure acute</td><td>1 (0.6)</td></tr><tr><td>   Cardiac flutter</td><td>1 (0.6)</td></tr><tr><td>   Cardiogenic shock</td><td>1 (0.6)</td></tr><tr><td>   Extrasystoles</td><td>1 (0.6)</td></tr><tr><td>   Sinus bradycardia</td><td>1 (0.6)</td></tr><tr><td> Investigations</td><td>1 (0.6)</td></tr><tr><td>   Electrocardiogram QT interval prolonged</td><td>1 (0.6)</td></tr><tr><td colspan=\"2\"><bold>Vascular TEAEs</bold></td></tr><tr><td> Any TEAE</td><td>19 (11.7)</td></tr><tr><td> Cardiovascular</td><td>5 (3.1)</td></tr><tr><td>   Angina pectoris</td><td>2 (1.2)</td></tr><tr><td>   Angina unstable</td><td>1 (0.6)</td></tr><tr><td>   Coronary artery occlusion</td><td>1 (0.6)</td></tr><tr><td>   Myocardial ischemia</td><td>1 (0.6)</td></tr><tr><td> Cerebrovascular</td><td>5 (3.1)</td></tr><tr><td>   Cerebrovascular accident</td><td>2 (1.2)</td></tr><tr><td>   Transient ischemic attack</td><td>2 (1.2)</td></tr><tr><td>   Carotid artery stenosis</td><td>1 (0.6)</td></tr><tr><td> Peripheral vascular</td><td>10 (6.1)</td></tr><tr><td>   Peripheral arterial occlusive disease</td><td>3 (1.8)</td></tr><tr><td>   Peripheral ischemia</td><td>2 (1.2)</td></tr><tr><td>   Aortic stenosis</td><td>1 (0.6)</td></tr><tr><td>   Arterial rupture</td><td>1 (0.6)</td></tr><tr><td>   Intermittent claudication</td><td>1 (0.6)</td></tr><tr><td>   Peripheral coldness</td><td>1 (0.6)</td></tr><tr><td>   Vascular pain</td><td>1 (0.6)</td></tr><tr><td colspan=\"2\"><bold>Effusion TEAEs</bold></td></tr><tr><td> Any TEAE</td><td>30 (18.4)</td></tr><tr><td>   Pleural effusion</td><td>27 (16.6)</td></tr><tr><td>   Pericardial effusion</td><td>8 (4.9)</td></tr><tr><td colspan=\"2\"><bold>Metabolic TEAEs</bold></td></tr><tr><td> Any TEAE</td><td>13 (8.0)</td></tr><tr><td>   Hyperglycemia</td><td>5 (3.1)</td></tr><tr><td>   Diabetes mellitus</td><td>4 (2.5)</td></tr><tr><td>   Hypercholesterolemia</td><td>3 (1.8)</td></tr><tr><td>   Hypertriglyceridemia</td><td>3 (1.8)</td></tr><tr><td>   Hyperlipidemia</td><td>1 (0.6)</td></tr><tr><td colspan=\"2\"><bold>Gastrointestinal TEAEs</bold></td></tr><tr><td> Any TEAE</td><td>149 (91.4)</td></tr><tr><td>   Diarrhea</td><td>143 (87.7)</td></tr><tr><td>   Abdominal pain</td><td>67 (41.1)</td></tr><tr><td>   Nausea</td><td>65 (39.9)</td></tr><tr><td>   Vomiting</td><td>53 (32.5)</td></tr><tr><td>   Constipation</td><td>28 (17.2)</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p>Full analysis set.</p><p><italic>CML</italic> chronic myeloid leukemia, <italic>CP</italic> chronic phase, <italic>ECOG PS</italic> Eastern Cooperative Oncology Group performance status, <italic>Ph</italic> Philadelphia chromosome, <italic>TKI</italic> tyrosine kinase inhibitor.</p><p><sup>a</sup>In the third-line cohort, 37 (60.7%) of patients received prior imatinib and dasatinib, 20 (32.8%) of patients received prior imatinib and nilotinib and 4 (6.6%) of patients received prior dasatinib and nilotinib.</p></table-wrap-foot>", "<table-wrap-foot><p>Evaluable cytogenetic population. To be considered a responder, the patient must have maintenance of baseline response while on-treatment or an improvement from baseline. Patients with MMR or better are counted as CCyR if a valid cytogenetic assessment is not available on a specific date. Associated two-sided 95% CI based on the exact method by Clopper–Pearson.</p><p><italic>CCyR</italic> complete cytogenetic response, <italic>CI</italic> confidence interval, <italic>CML</italic> chronic myeloid leukemia, <italic>CP</italic> chronic phase, <italic>MCyR</italic> major cytogenetic response, <italic>Ph</italic> Philadelphia chromosome, <italic>TKI</italic> tyrosine kinase inhibitor.</p></table-wrap-foot>", "<table-wrap-foot><p>Evaluable molecular population. To be considered a responder, the patient must have maintenance of baseline response while on-treatment or an improvement from baseline. MMR: <italic>BCR-ABL1</italic> IS ≤ 0.1%; MR<sup>4</sup>: <italic>BCR-ABL1</italic> IS ≤ 0.01%; MR<sup>4.5</sup>: <italic>BCR-ABL1</italic> IS ≤ 0.0032%. Associated two-sided 95% CI based on the exact method by Clopper–Pearson.</p><p><italic>CI</italic> confidence interval, <italic>CML</italic> chronic myeloid leukemia, <italic>CP</italic> chronic phase, <italic>IS</italic> international scale, <italic>MMR</italic> major molecular response, <italic>MR</italic> molecular response, <italic>Ph</italic> Philadelphia chromosome, <italic>TKI</italic> tyrosine kinase inhibitor.</p></table-wrap-foot>", "<table-wrap-foot><p>Full analysis set. Classification of adverse events is based on the Medical Dictionary for Regulatory Activities (v21.1).</p><p><italic>ALT</italic> alanine aminotransferase, <italic>AST</italic> aspartate aminotransferase, <italic>CML</italic> chronic myeloid leukemia, <italic>TEAE</italic> treatment-emergent adverse event.</p></table-wrap-foot>", "<table-wrap-foot><p>Full analysis set. Classification of adverse events is based on the Medical Dictionary for Regulatory Activities (v21.1). See Supp. Methods for adverse events of special interest cluster definitions.</p><p>Totals for the number of patients at a higher level are not necessarily the sum of those at the lower levels since a patient may report two or more different TEAEs within the higher level category.</p><p><italic>TEAE</italic> treatment-emergent adverse events.</p></table-wrap-foot>", "<fn-group><fn><p>Additional BYOND Study Investigators are listed below Acknowledgements.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_915_Fig1_HTML\" id=\"d32e1251\"/>", "<graphic xlink:href=\"41375_2020_915_Fig2_HTML\" id=\"d32e2023\"/>", "<graphic xlink:href=\"41375_2020_915_Fig3_HTML\" id=\"d32e2696\"/>" ]
[ "<media xlink:href=\"41375_2020_915_MOESM1_ESM.pdf\"><caption><p>suppl. material</p></caption></media>" ]
[{"label": ["1."], "mixed-citation": ["National Cancer Institute. Chronic Myelogenous Leukemia Treatment (PDQ"], "sup": ["\u00ae"], "ext-link": ["https://www.cancer.gov/types/leukemia/hp/cml-treatment-pdq#_1"]}, {"label": ["24."], "mixed-citation": ["Cortes JE, Mauro MJ, Deininger MW, Chuah C, Kim DW, Kota V, et al. Bosutinib vs imatinib for newly diagnosed chronic myeloid leukemia in the BFORE trial: 24-month follow-up. J Clin Oncol. 2018;36(15_suppl):7002. (abstract)."]}, {"label": ["26."], "surname": ["Br\u00fcmmendorf", "Gambacorti-Passerini", "Hochhaus", "Lipton", "Kota", "Deininger"], "given-names": ["TH", "C", "A", "JH", "V", "MW"], "article-title": ["Efficacy and safety following dose reduction of bosutinib or imatinib in patients with newly diagnosed chronic myeloid leukemia: analysis of the phase 3 BFORE trial"], "source": ["Blood"], "year": ["2018"], "volume": ["132"], "fpage": ["3005"], "pub-id": ["10.1182/blood-2018-99-110543"]}, {"label": ["39."], "mixed-citation": ["Cella D, Jensen SE, Webster K, Hongyan D, Lai JS, Rosen S, et al. Measuring health-related quality of life in leukemia: the Functional Assessment of Cancer Therapy\u2013Leukemia (FACT-Leu) questionnaire. Value Health. 2012;15:1051-8."]}]
{ "acronym": [], "definition": [] }
43
CC BY
no
2024-01-13 23:35:09
Leukemia. 2020 Jun 22; 34(8):2125-2137
oa_package/9a/3f/PMC7387243.tar.gz
PMC7387244
32382082
[ "<title>Introduction</title>", "<p id=\"Par2\">Blast crisis (BC) of Philadelphia-chromosome-positive (Ph+) and/or BCR-ABL1-positive chronic myeloid leukemia (CML) is one of the remaining challenges in the management of the disease. Cytogenetic abnormalities and blast increase represent the most consistent indicators of progression to end-phase CML [##REF##11919388##1##–##REF##12560227##7##]. End-phase CML comprises early progression with emerging high-risk ACA and late progression with failing hematopoiesis and blast cell proliferation. BC represents the end stage of this evolution. Not all patients dying of CML reach the blast levels defining BC (20% or 30% blasts in blood or marrow) [##REF##369590##8##–##UREF##0##11##]. Once BC has occurred, treatment results are poor. Early allogeneic stem cell transplantation (SCT) might improve prognosis [##REF##19965667##12##, ##REF##26464170##13##]. Whether cytogenetic aberrations allow a timelier change of treatment with better outcome is unknown.</p>", "<p id=\"Par3\">Additional chromosomal abnormalities (ACAs) are thought to result from BCR-ABL1-induced genetic instability and may be causative factors of disease progression [##REF##20592475##14##, ##REF##16527898##15##]. The most frequent ACAs found in BC (+8, a second Ph-chromosome (+Ph), an isochromosome of the long arm of chromosome 17, i.e., i[17q], and +19) were termed major route by Mitelman et al. [##REF##11919388##1##–##REF##1065618##2##]. Major-route ACAs have been associated with shorter survival, if they were detected at diagnosis [##REF##22039253##16##] or if they emerged in the course of disease [##REF##27006386##17##]. A poor prognosis was also observed with 3q26.2 and 11q23 rearrangements and with −7/7q− [##REF##26243778##18##, ##REF##25888368##19##], whereas +8 and +Ph as single aberrations, but not in combination, were not equally associated with poor prognosis [##REF##25931274##20##]. Wang et al. [##REF##27006386##17##] proposed a risk stratification of the six most frequent ACAs into two groups with distinct prognoses (+8, +Ph, −Y with good prognoses and i[17q], −7/7q−, 3q26.2 rearrangements with poor prognoses). Using the same cohort of patients, a risk stratification into three groups was proposed [##REF##29296906##21##] based on BC risk associated with each ACA (high risk: 3q26.2; −7/7q−; i[17q]; complex karyotypes with high-risk ACA. Intermediate 1: +8; +Ph; other single ACA. Intermediate 2: other complex ACA).</p>", "<p id=\"Par4\">Since the prognosis with single changes (+8, +Ph) is controversial, we decided to include these in our evaluation, as well as +21 and +17, which were designated as major route later on [##REF##11919388##1##]. Other ACAs were not so clearly associated with shorter survival. This led to their tentative designation as low-risk ACA.</p>", "<p id=\"Par5\">More recently, clonal chromosomal aberrations (CCAs) found in Ph-negative cells (CCA/Ph−) were reported to also have a negative impact on survival [##REF##29122769##22##, ##REF##28835440##23##].</p>", "<p id=\"Par6\">We here made use of the data of 1551 imatinib-treated chronic phase (CP) patients recruited to CML-study IV, a randomized study comparing imatinib 400 mg with imatinib 800 mg and combinations of imatinib with interferon, simultaneously or sequentially, or low-dose cytarabine [##REF##28804124##24##]. Our aim was to analyze if defined ACA at low blast levels allow an earlier diagnosis of end-phase CML and a timelier change of treatment than current blast thresholds.</p>" ]
[ "<title>Patients and methods</title>", "<title>Patients</title>", "<p id=\"Par7\">Patient data were derived from the randomized CML-study IV (recruitment 2002–2012) with initial or predominant imatinib treatment [##REF##28804124##24##]. Documentation was done at 3–6-month intervals as previously reported [##REF##28804124##24##]. Risk assignment followed the ELTS (EUTOS Long-Term Survival) score [##REF##26416462##25##]. BC was defined by 30% blasts in blood or marrow. Patient numbers and flow are depicted in the flow chart (Fig. ##FIG##0##1a–c##).</p>", "<title>Cytogenetics</title>", "<p id=\"Par8\">By protocol, cytogenetic analysis was requested every 3–6 months during the early disease phases and every 12 months thereafter, if stable molecular remission (major molecular remission or better, BCR-ABL1 &lt;0.1% on the International Scale, IS) was achieved. In stable molecular remission, intervals between cytogenetic analyses were frequently longer due to patients’ and/or doctors’ requests. A median of four analyses per patient were documented. Cytogenetic analyses were done as described and results were reported according to the international nomenclature (ISCN 2016) [##REF##22039253##16##]. ACAs were evaluated if they were clonal according to the ISCN. Complex karyotypes were defined as three or more concurrent aberrations. High-risk ACAs were defined as the major route ACA +8, +Ph, i[17q], +19, +21, +17 (the ACA most frequently observed in BC) [##REF##11919388##1##], the minor route ACA 3q26.2, 11q23, −7/7q− (less frequently observed, but negative impact on prognosis) [##REF##27006386##17##, ##REF##25888368##19##, ##REF##26243778##18##], and complex karyotypes. Variant translocations and −Y were not considered, as they had no impact on prognosis in our and other studies [##REF##22039253##16##, ##REF##26385387##26##].</p>", "<p id=\"Par9\">CAA/Ph− have not been an objective of this study.</p>", "<title>Molecular genetics</title>", "<p id=\"Par10\">Molecular analyses followed the IS methodology and nomenclature [##REF##24297946##27##–##REF##25652737##30##].</p>", "<title>Statistics</title>", "<p id=\"Par11\">For survival analyses, patients were followed up at the start of the diagnosis, at the time of the occurrence of an ACA, or at the time of a blast increase. Patients were censored at the date of last follow-up. Mortality after the advent of blast increases was assessed by Cox models starting at the time of a blast increase. Here the presence of ACA was considered as a time-dependent covariate. <italic>P</italic> values &lt;5% were considered significant. Due to the explorative character of this work, no adjustment of <italic>p</italic> values was done and all <italic>p</italic> values have to be interpreted descriptively. All analyses were performed with SAS 9.3 or R 3.5.1.</p>" ]
[ "<title>Results</title>", "<title>Identification of ACA</title>", "<p id=\"Par12\">One thousand five hundred and thirty-six patients with Ph+ CP-CML were analyzed for blast increase and ACA, 1510 patients were cytogenetically evaluable. Patients’ characteristics are shown in Table ##TAB##0##1##. Median observation time was 8.6 years.</p>", "<p id=\"Par13\">One hundred and twenty-three patients (8.1%) displayed ACA in Ph+ metaphases (<xref rid=\"App1\" ref-type=\"app\">Appendix A1</xref>). Ninety one (6%) were high-risk ACAs (+8, +Ph, i[17q], +17, +19, +21, 11q23 and 3q26.2 rearrangements, −7/7q abnormalities, complex karyotypes) and 32 (2.1%) were low-risk ACAs (all other). Of the 91 high-risk ACAs, 25 (1.7%) were detected at baseline and 66 (4.4%) emerged in the course of disease 0.5–133 months after diagnosis. The median time to detection of high-risk ACAs was 17 months. Of the 32 low-risk ACAs, 19 (1.3%) were detected at diagnosis and 13 (0.9%) emerged in the course of disease.</p>", "<p id=\"Par14\">Frequencies of ACA are shown in Table ##TAB##1##2## grouped according to type (risk level, single, or in combination) and time of emergence (at diagnosis or in the course of disease).</p>", "<title>Impact of ACA on survival</title>", "<p id=\"Par15\">Figure ##FIG##1##2a–d## shows the impact of high- and low-risk ACAs on survival from occurrence of ACA at diagnosis or in the course of disease. Observations were synchronized for the time of emergence of ACA, and ACA detected at diagnosis and emerging in the course of disease were analyzed together. All high-risk ACAs show a negative impact on survival compared to low-risk ACAs, which serve as control. An exception is +8 as a single aberration, which carries a prognosis in between high- and low-risk ACAs (Fig. ##FIG##1##2a##). Impact of +Ph on survival was equally poor whether it occurred alone or in combination with other abnormalities (Fig. ##FIG##1##2b##). Chromosome 3, 7, 17, 19, and 21 aberrations were grouped together, as they were rare (Fig. ##FIG##1##2d##). Individual analyses of these aberrations are shown in Fig. ##FIG##1##2e–i##. Four-year survival probability after occurrence of high-risk ACA, except +8 alone, was 52.2% (95% confidence interval (CI): 41–66), after occurrence of +8 alone 77% (95% CI: 60–100), and after occurrence of low-risk ACA 87% (95% CI: 75–100).</p>", "<p id=\"Par16\">The treatment strategy after emergence of ACA including allogeneic SCT did not differ according to the type of ACA, but patients with high-risk ACA were transplanted more frequently. 42 of the 138 transplantations in CML-study IV (30%) were performed in the 6% of patients with high-risk ACA (Flow chart, Fig. ##FIG##0##1c##). Two-year survival of 26 patients transplanted in BC or accelerated phase (AP) was 46% (95% CI: 26–63%) and of 13 patients transplanted in CP 77% (95% CI: 44–92%; log-rank test: <italic>p</italic> = 0.09; phase unknown for 3 of 42 patients).</p>", "<title>Correlation of ACA with BC</title>", "<p id=\"Par17\">79 patients developed BC during the observation time. Of the 79 BC patients, 71 were cytogenetically evaluable. 44 BC patients had ACA (61%), in 27 patients no ACAs were reported. Of the 44 BC patients with ACA, 41 (93%) had high-risk and 3 low-risk ACA (Flow chart, Fig. ##FIG##0##1b##).</p>", "<title>Correlation of ACA with blast increase</title>", "<p id=\"Par18\">The close correlation of high-risk ACA with BC and the unfavorable prognosis of patients with high-risk ACA led us to ask if high-risk ACA can anticipate the diagnosis of end-phase CML. We therefore assigned patients, in whom a blast increase was observed in peripheral blood or marrow (at any time), to 6 different blast thresholds (1%, 5%, 10%, 15%, 20%, and 30%). In each of these cohorts high- and low-risk ACAs were considered as time-dependent variables. The number of patients ranged from 224 to 78 in the six cohorts with blast increases of 1% to &gt;30% in the peripheral blood, and from 1033 to 79 in the six cohorts with blast levels of 1% to &gt;30% in the marrow (Table ##TAB##2##3##). Naturally, the sets of patients who developed higher blast increases later on were subsets of the sets with lower blast increases.</p>", "<p id=\"Par19\">In the corresponding Cox proportional hazards models (Table ##TAB##2##3##, Fig. ##FIG##2##3##), we found an increased hazard to die in the presence of high-risk ACA compared to no ACA with hazard ratios of up to 3.65 (95% CI: 2.32–5.75) in the blood (Fig. ##FIG##2##3a##) and 6.12 (95% CI: 4.1–9.2) in the marrow (Fig. ##FIG##2##3b##) when only patients with low blast levels of 1–5% were considered. When restricting the cohorts to larger blast increases, the effect of high-risk ACA on the hazard to die decreased. In the last cohorts of patients with blast increases to at least 20% or 30%, no difference between patients with and without high-risk ACA was found (hazard ratio: 0.83, 95% CI: 0.50–2.89). The hazard ratios for low-risk ACA compared to no ACA were increased to much lesser extents than for high-risk ACA.</p>", "<title>Course of disease and causes of death</title>", "<p id=\"Par20\">Thirty-seven patients with high-risk ACA (41%) died (Flow chart, Fig. ##FIG##0##1a, c##). The causes of death were known for 34 patients and almost exclusively CML related. Thirty-two patients (94%) died of progression, mostly in BC, including 21 after progression-related transplantation (15 in BC, 1 in AP, 2 after loss of cytogenetic remission, 2 because of no molecular response, and 1 with unknown indication). Two patients died of CML-unrelated causes.</p>", "<p id=\"Par21\">Of 54 (59%) living patients with high-risk ACAs, 21 (23% of total) were transplanted, 8 of these in BC, 2 in AP, 9 because of no molecular response, and 2 with unknown indication. Five (5%) non-transplanted patients are alive after progression (Flow chart, Fig. ##FIG##0##1a, c##). Twenty-eight patients (31%) are alive without documented progression 0–11 years after emergence of high-risk ACA.</p>", "<p id=\"Par22\">Of the four patients with low-risk ACA who died, three died of CML-related (one after SCT) and one of CML-unrelated causes</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par23\">We here report that the combination of high-risk ACA and low-level blasts heralds progression and death by CML. This finding has implications for diagnosis and management of patients in end-phase CML. High-risk ACAs allow to identify CML end-phase at an earlier time than is possible with the current blast thresholds [##REF##22653972##9##, ##REF##16463391##10##]. These patients require a change of therapy and/or more intensive treatments, for example, with ponatinib or allogeneic SCT [##REF##22653972##9##]. A clinically relevant, but in 39 patients not statistically significant difference of 30% 2-year survival suggests that outcome of transplanted patients with high-risk ACA depends on disease stage similar to patients without ACA [##REF##19965667##12##]. Successful treatment may explain, at least in part, the plateau phase of survival curves after 4 years.</p>", "<p id=\"Par24\">High-risk ACA at low blast counts are not an indicator of AP as previously reviewed [##REF##23803709##31##, ##REF##27069254##32##], but a marker of progression and death by CML regardless whether they are present at diagnosis or emerge in the course of disease. Sixty-nine percent of patients with high-risk ACA and low blast levels progressed or died of CML during the observation period. This includes transplanted patients who as a rule were transplanted because of progression. Our findings agree with an earlier observation that patients in AP with ACA and blast increase have a worse outcome [##REF##22460758##33##].</p>", "<p id=\"Par25\">Our data suggest that the appropriate time for a change of treatment is emergence of high-risk ACA rather than waiting for an increase of blasts.</p>", "<p id=\"Par26\">High-risk ACA include, in addition to major route ACA (which were defined solely on the basis of their frequency in BC) [##REF##11919388##1##–##REF##1065618##2##], most notably −7/7q−, 3q26.2, and 11q23 rearrangements and complex karyotypes [##REF##22039253##16##–##REF##29296906##21##, ##REF##26385387##26##]. Additional high-risk ACA may surface on continued clinical and cytogenetic scrutiny.</p>", "<p id=\"Par27\">Our definition of high-risk ACA largely agrees with that proposed by others [##REF##27006386##17##, ##REF##29296906##21##], but there are differences. The prognosis with +8 alone, although clearly better than with +8 in combination, is still worse than with low-risk ACA (Fig. ##FIG##1##2a##). We thus have included +8 alone in the high-risk ACA group. +Ph has an equally unfavorable impact on survival, regardless whether it occurs as a single aberration or in combination (Fig. ##FIG##1##2b##). In agreement with earlier reports [##REF##27006386##17##, ##REF##26243778##18##], chromosome 19 aberrations exclusively occurred in combination, whereas 3q26.2 was mostly found as a single aberration and rarely at diagnosis [##REF##22039253##16##]. −Y in our analyses had a prognosis not different from no ACA.</p>", "<p id=\"Par28\">Looking at our data in context of the literature [##REF##27006386##17##, ##REF##29296906##21##], a risk stratification of ACA in two risk-groups (high risk with impact on survival and low risk with little or no impact) seems useful for clinical purposes. Due to the limited sample size and the exploratory nature of this work, we would welcome verification of these results by others.</p>", "<p id=\"Par29\">High-risk ACAs were not observed in all BC patients. We cannot rule out that other events such as genetic alterations not detectable by cytogenetics predispose to a blast increase. Gene sequencing detects such alterations [##REF##29967129##34##]. Telomere shortening [##REF##29980572##35##] or increased separase activity [##REF##26267133##36##] may contribute to progression. Likewise, low-risk ACA might include some hitherto unrecognized high-risk ACA as suggested by the three patients with BC and low-risk ACA.</p>", "<p id=\"Par30\">High-risk ACAs were observed in 6% of CP patients, but in 61% of patients who had progressed to BC, whereas low-risk ACAs were observed in 2.1% of CP patients and in 2.8% of patients with BC. This is strong support for a role of high-risk ACA in the evolution of CML and is in line with the hypothesis that BCR-ABL1 predisposes to ACA, which then promote progression. The emergence of high-risk ACA might anticipate and define the point of no return in the evolution of CML indicating non-reversibility by tyrosine kinase inhibitor (TKI). High-risk ACA could emerge as testable cause of non-mutation-related TKI resistance.</p>", "<p id=\"Par31\">A limitation of this study is the follow-up cytogenetics, which have been replaced in many instances by molecular testing increasingly done in the course of the study. Furthermore, most cytogenetic analyses were performed in the first months after diagnosis or when the patients’ conditions were worsening. This might introduce a bias, as patients doing well were usually not analyzed. Also, it is possible that analyses with low numbers of mitoses miss ACA and that the true percentage of patients with ACA is underestimated.</p>", "<p id=\"Par32\">Although the low number of follow-up cytogenetics has limited the direct correlation of ACA with blast increase, it points to the strength of the evidence for the association of high-risk ACA with end-phase CML and survival in the Cox model in spite of missing values.</p>", "<p id=\"Par33\">A correlation with BCR-ABL1 transcript numbers was not conclusive because of too few molecular measurements at early blast increase.</p>", "<p id=\"Par34\">A strength of the study is the size of the cohort as one of the largest of TKI-treated CP-CML patients on whom emergence of ACA and progression to BC were prospectively recorded in parallel over prolonged periods of time. The association of high-risk ACA with progression would not be so obvious in smaller cohorts with shorter observation.</p>", "<p id=\"Par35\">In conclusion, high-risk ACAs are an early marker of CML progression. In the presence of low blast levels, high-risk ACAs indicate death by CML earlier than is possible with standard blast thresholds. An appropriate time for a change of therapy may be emergence of high-risk ACA rather than waiting for an increase of blasts. Cytogenetic monitoring is indicated when signs of progression surface and response to therapy is unsatisfactory.</p>" ]
[]
[ "<p id=\"Par1\">Blast crisis is one of the remaining challenges in chronic myeloid leukemia (CML). Whether additional chromosomal abnormalities (ACAs) enable an earlier recognition of imminent blastic proliferation and a timelier change of treatment is unknown. One thousand five hundred and ten imatinib-treated patients with Philadelphia-chromosome-positive (Ph+) CML randomized in CML-study IV were analyzed for ACA/Ph+ and blast increase. By impact on survival, ACAs were grouped into high risk (+8, +Ph, i(17q), +17, +19, +21, 3q26.2, 11q23, −7/7q abnormalities; complex) and low risk (all other). The presence of high- and low-risk ACAs was linked to six cohorts with different blast levels (1%, 5%, 10%, 15%, 20%, and 30%) in a Cox model. One hundred and twenty-three patients displayed ACA/Ph+ (8.1%), 91 were high risk. At low blast levels (1–15%), high-risk ACA showed an increased hazard to die compared to no ACA (ratios: 3.65 in blood; 6.12 in marrow) in contrast to low-risk ACA. No effect was observed at blast levels of 20–30%. Sixty-three patients with high-risk ACA (69%) died (<italic>n</italic> = 37) or were alive after progression or progression-related transplantation (<italic>n</italic> = 26). High-risk ACA at low blast counts identify end-phase CML earlier than current diagnostic systems. Mortality was lower with earlier treatment. Cytogenetic monitoring is indicated when signs of progression surface or response to therapy is unsatisfactory.</p>", "<title>Subject terms</title>" ]
[]
[ "<title>Appendix A1. Karyotypes of patients with ACA</title>", "<p id=\"Par39\">\n\n</p>", "<title>Acknowledgements</title>", "<p>We thank E Matzat, R Pleil-Lösch, I Stalljann, G Bartsch, C Sodan-Boyer, A Elett, M Meckesheimer, U Böhm, and J Hehlmann for assistance.</p>", "<title>The SAKK and the German CML Study Group</title>", "<p id=\"Par37\">L. Fischer von Weikersthal<sup>17</sup>, M. Hahn<sup>18</sup>, G. Schlimok<sup>19</sup>, D. Reichert<sup>20</sup>, J. Janssen<sup>20</sup>, U. Martens<sup>21</sup>, P. Majunke<sup>22</sup>, Peter Reichert<sup>23</sup>, K. Neben<sup>24</sup>, S. Korsten<sup>25</sup>, Ch. Scholz<sup>26</sup>, B. Oldenkott<sup>27</sup>, J. Heßling<sup>28</sup>, D. Kingreen<sup>29</sup>, C. Sperling<sup>30</sup>, C. Schelenz<sup>30</sup>, I. Blau<sup>31</sup>, A. Urmersbach<sup>32</sup>, W. Ludwig<sup>33</sup>, P. Le Coutre<sup>34</sup>, R. Arnold<sup>34</sup>, M. de Wit<sup>35</sup>, A. Pezzutto<sup>36</sup>, E. Schäfer<sup>37</sup>, R. Schroers<sup>38</sup>, A. Lochter<sup>38</sup>, D. Behringer<sup>39</sup>, Y. Ko<sup>40</sup>, S. Weidenhöfer<sup>40</sup>, W. Verbeek<sup>41</sup>, P. Brossart<sup>42</sup>, G. Trenn<sup>43</sup>, W. Pommerien<sup>44</sup>, J. Krauter<sup>45</sup>, G. Doering<sup>46</sup>, H. Munzinger<sup>46</sup>, C. Diekmann<sup>47</sup>, B. Hertenstein<sup>48</sup>, S. Stier<sup>49</sup>, F. Möller-Faßbender<sup>50</sup>, M. Hänel<sup>51</sup>, T. Zöller<sup>52</sup>, C. Lamberti<sup>53</sup>, B. Koch<sup>54</sup>, A. Henzel<sup>55</sup>, S. Wagner<sup>56</sup>, A. Schmalenbach<sup>57</sup>, M. Hoffknecht<sup>58</sup>, G. Ehninger<sup>59</sup>, A. Kiani<sup>59</sup>, T. Illmer<sup>60</sup>, C. Aul<sup>61</sup>, M. Flaßhove<sup>62</sup>, F. Henneke<sup>62</sup>, M. Simon<sup>63</sup>, L. Müller<sup>64</sup>, H. Becker<sup>64</sup>, R. Janz<sup>65</sup>, M. J. Eckart<sup>66</sup>, R. Fuchs<sup>67</sup>, F. Schlegel<sup>67</sup>, M. Wattad<sup>68</sup>, R. Rudolph<sup>69</sup>, D. W. Beelen<sup>70</sup>, A. Lindemann<sup>71</sup>, D. Linck<sup>72</sup>, Wassman<sup>73</sup>, E. Jäger<sup>74</sup>, S. Al-Batran<sup>74</sup>, T. Reiber<sup>75</sup>, C. F. Waller<sup>76</sup>, H. Hoeffkes<sup>77</sup>, L. Schulz<sup>78</sup>, K. Tajrobehkar<sup>79</sup>, J. Mittermüller<sup>80</sup>, H. Pralle<sup>81</sup>, V. Runde<sup>82</sup>, A. Hoyer<sup>83</sup>, H. Tessen<sup>83</sup>, L. Trümper<sup>84</sup>, C. Schmidt<sup>85</sup>, M. Sieber<sup>86</sup>, H. Eschenburg<sup>87</sup>, R. Depenbusch<sup>88</sup>, S. Rösel<sup>88</sup>, H. W. Lindemann<sup>89</sup>, H. Wolf<sup>90</sup>, C. Spohn<sup>91</sup>, R. Moeller<sup>91</sup>, D. Hossfeld<sup>92</sup>, A. Zander<sup>92</sup>, P. Schafhausen<sup>92</sup>, H. Köster<sup>93</sup>, W. Hollburg<sup>94</sup>, N. Schmitz<sup>95</sup>, H. Dürk<sup>96</sup>, M. Hemeier<sup>96</sup>, A. Grote-Metke<sup>97</sup>, H. Weischer<sup>97</sup>, B. Bechtel<sup>97</sup>, L. Balleisen<sup>98</sup>, M. Sosada<sup>99</sup>, A. Ho<sup>100</sup>, V. Petersen<sup>101</sup>, J. Dengler<sup>102</sup>, S. Bildat<sup>103</sup>, L. Hahn<sup>104</sup>, H. Dietzfelbinger<sup>105</sup>, W. Gröschel<sup>106</sup>, A. Bartholomäus<sup>107</sup>, W. Freier<sup>108</sup>, B. Sievers<sup>108</sup>, I.-M. Pfreundschuh<sup>109</sup>, T. Herrmann<sup>110</sup>, A. Fauser<sup>110</sup>, J. Menzel<sup>111</sup>, M. Kemmerling<sup>112</sup>, R. Hansen<sup>113</sup>, H. Link<sup>114</sup>, M. Schatz<sup>115</sup>, M. Bentz<sup>116</sup>, O. Prümmer<sup>117</sup>, M. Kneba<sup>118</sup>, J. Heymanns<sup>119</sup>, S. Schmitz<sup>120</sup>, C. Scheid<sup>121</sup>, A. Lollert<sup>122</sup>, M. Neise<sup>122</sup>, M. Planker<sup>123</sup>, M. Stauch<sup>124</sup>, M. Schröder<sup>125</sup>, B. Kempf<sup>126</sup>, U. Vehling-Kaiser<sup>127</sup>, S. Kremers<sup>128</sup>, G. Köchling<sup>129</sup>, L. Müller<sup>130</sup>, F. Hartmann<sup>131</sup>, T. Neuhaus<sup>132</sup>, S. Fetscher<sup>133</sup>, D. Kämpfe<sup>134</sup>, G. Heil<sup>135</sup>, M. Uppenkamp<sup>136</sup>, B. Goldmann<sup>137</sup>, T. Fischer Huber<sup>138</sup>, U. Hieber<sup>139</sup>, C. Plöger<sup>140</sup>, M. Griesshammer<sup>141</sup>, C. Lange<sup>142</sup>, B. Göttler<sup>143</sup>, C. Lunscken<sup>144</sup>, X. Schiel<sup>145</sup>, C. Scheidegger<sup>146</sup>, O. Stötzer<sup>147</sup>, H. Hitz<sup>148</sup>, H. Schick<sup>149</sup>, S. Völkl<sup>150</sup>, K. Spiekermann<sup>151</sup>, W. Berdel<sup>152</sup>, H. Hebart<sup>153</sup>, E. Ladda<sup>154</sup>, P. Schmidt<sup>155</sup>, U. Burkhardt<sup>155</sup>, S. Hentschke<sup>156</sup>, C. Falge<sup>157</sup>, D. Reschke<sup>158</sup>, C. A. Köhne<sup>159</sup>, C. Müller-Naendrup<sup>160</sup>, M. Sauer<sup>161</sup>, S. Frühauf<sup>162</sup>, K. Ranft<sup>163</sup>, Y. Dencausse<sup>164</sup>, B. Sandritter<sup>165</sup>, G. Baake<sup>166</sup>, M. Hofknecht<sup>167</sup>, R. Dengler<sup>168</sup>, M. Edinger<sup>169</sup>, M. Schenk<sup>170</sup>, A. Wehmeier<sup>171</sup>, H.-P. Weidelich<sup>172</sup>, R. Pihusch<sup>173</sup>, K. Stahlhut<sup>174</sup>, M. Baldus<sup>175</sup>, A. Matzdorff<sup>176</sup>, T. Geer<sup>177</sup>, S. Schanz<sup>178</sup>, G. Käfer<sup>179</sup>, W. Gassmann<sup>180</sup>, C. Priebe-Richter<sup>181</sup>, M. Demandt<sup>182</sup>, G. Springer<sup>183</sup>, H. Fiechtner<sup>183</sup>, C. Denzlinger<sup>184</sup>, J. Schleicher<sup>185</sup>, D. Assman<sup>186</sup>, R. Gaeckler<sup>187</sup>, G. Adam<sup>188</sup>, A. Waladkhani<sup>189</sup>, B. Rendenbach<sup>190</sup>, H. Forstbauer<sup>191</sup>, L. Kanz<sup>192</sup>, S. Jacki<sup>193</sup>, F. Stegelmann<sup>193</sup>, N. Kalhori<sup>194</sup>, A. Nusch<sup>194</sup>, W. Langer<sup>194</sup>, F. Müller<sup>195</sup>, S. Brettner<sup>196</sup>, B. Uebelmesser<sup>197</sup>, T. Kamp<sup>198</sup>, C. Schadeck-Gressel<sup>199</sup>, K. Josten<sup>200</sup>, O. Klein<sup>200</sup>, R. Schwerdtfeger<sup>201</sup>, H. Baurmann<sup>201</sup>, H. Strotkötter<sup>202</sup>, W. Fett<sup>203</sup>, A. Raghavachar<sup>204</sup>, C. Maintz<sup>205</sup>, M. C. Goebler<sup>206</sup>, R. Schlag<sup>207</sup>, W. Elsel<sup>208</sup>, M. Wernli<sup>209</sup>, D. Heim<sup>210</sup>, W. Wuillemin<sup>211</sup>, U. Hess<sup>212</sup>, J. Gmür<sup>213</sup>, J. Mayer<sup>214</sup>.</p>", "<title>Funding</title>", "<p>This work was funded by European LeukemiaNet (ELN) Foundation.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par38\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Flow chart.</title><p><bold>a</bold> Patients with ACA, <bold>b</bold> patients with BC, and <bold>c</bold> transplanted patients with high-risk ACA. BC blast crisis, AP accelerated phase, SCT stem cell transplantation.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Impact of high- vs. low-risk ACA on survival.</title><p>The left-side panel shows the impact of +8 (<bold>a</bold>), +Ph (<bold>b</bold>), complex ACA (<bold>c</bold>), and of chromosome 3, 7, 17, 19, and 21 aberrations combined (<bold>d</bold>) on survival in patients with primary imatinib treatment after the emergence of ACA. Suvival after emergence of low-risk ACA in imatinib-treated patients serves as control. The right-side panel shows the impact of rare high-risk ACA of chromosomes 3, 7, 17, 19, and 21 on survival (<bold>e</bold>–<bold>i</bold>).</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Hazard to die with high-risk and low-risk ACA compared to no ACA dependent on blast counts.</title><p>Hazard ratios for mortality in imatinib-treated patients with high-risk and low-risk ACA were determined in six different (but overlapping) patient groups (blast increase to 1–30%) together with 95% confidence intervals <bold>a</bold> in peripheral blood and <bold>b</bold> in bone marrow. The size of the circle correlates with the sample size. Thirty-seven patients with high-risk ACA and four patients with low-risk ACA died. In 34 patients with high-risk ACA, causes of death were known. Thirty-two of these (94%) died of progression, including progression-related transplantation in 21 patients. Two patients died of CML-unrelated causes. Causes of death were unknown in three patients. With low-risk ACA, causes of death were CML related in three patients and unknown in one patient.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td>Patients (cytogenetically evaluable), <italic>n</italic></td><td>1536 (1510)</td></tr><tr><td>Gender (%), male</td><td>60.2</td></tr><tr><td>Age at diagnosis of CML (years), median (range)</td><td>53 (16–88)</td></tr><tr><td>Hb (g/dl), median (range)</td><td>12.3 (4.7–19.1)</td></tr><tr><td>Platelets (×10<sup>12</sup>/μl), median (range)</td><td>375 (34–3020)</td></tr><tr><td>Patients with palpable splenomegaly</td><td>55.7%</td></tr><tr><td>ELTS-score (% low/intermediate/high)</td><td>57/30/13</td></tr><tr><td>WBC count at diagnosis (×10<sup>9</sup>/l) (median, range) with differential</td><td>76 (2.6–630)</td></tr><tr><td> Blasts (%)</td><td>1 (0–30)</td></tr><tr><td> Promyelocytes (%)</td><td>2 (0–34)</td></tr><tr><td> Basophils (%)</td><td>3 (0–66)</td></tr><tr><td>Median observation time (years)</td><td>8.6</td></tr><tr><td>10-Year survival</td><td>83%</td></tr><tr><td>Patients with ACA, high risk at diagnosis (<italic>n</italic>)</td><td>25</td></tr><tr><td>Patients with ACA, high risk in the course of disease (<italic>n</italic>)</td><td>66</td></tr><tr><td>High-risk ACA total (<italic>n</italic>)</td><td>91 (6%)</td></tr><tr><td>Patients with other-/low-risk ACA at diagnosis (<italic>n</italic>)</td><td>19</td></tr><tr><td>Patients with other-/low-risk ACA in the course of disease (<italic>n</italic>)</td><td>13</td></tr><tr><td>Low-risk ACA, total (<italic>n</italic>)</td><td>32 (2.1%)</td></tr><tr><td>ACA total (<italic>n</italic>)</td><td>123 (8.1%)</td></tr><tr><td>Patients with anemia (Hb &lt; 10) at first appearance of ACA (%)</td><td>26.7<sup>a</sup></td></tr><tr><td>Patients with thrombocytopenia (platelets &lt; 75 × 10<sup>9</sup>/l) at first appearance of ACA (%)</td><td>17.8<sup>a</sup></td></tr><tr><td>Patients with neutropenia (neutrophils &lt; 1.5 × 10<sup>6</sup>/l) at first appearance of ACA (%)</td><td>15.5<sup>a</sup></td></tr><tr><td>Patients with palpable splenomegaly (defined as spleen in cm below costal margin &gt;0) at first appearance of ACA (%)</td><td>39.2<sup>a</sup></td></tr><tr><td>Basophils at the time of ACA (%) (median, range)</td><td>1 (0–15)<sup>a</sup></td></tr><tr><td>Age at diagnosis of ACA (years) (median, range)</td><td>52 (18–89)</td></tr><tr><td>Age at diagnosis of high-risk ACA (years) (median, range)</td><td>52 (23–89)</td></tr><tr><td>Median interval diagnosis—ACA (years) (median, range)</td><td>n.r. (0–11.1)<sup>b</sup></td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Frequency of ACA.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Karyotypes</th><th>Single ACA</th><th>In combination with other ACA</th><th>Total</th></tr></thead><tbody><tr><td>High-risk ACA (<italic>n</italic> = 91)<sup>a</sup></td><td/><td/><td/></tr><tr><td>+8</td><td>19</td><td>19</td><td>38</td></tr><tr><td> At diagnosis</td><td>6</td><td>10</td><td>16</td></tr><tr><td> In the course of disease</td><td>13</td><td>9</td><td>22</td></tr><tr><td>+Ph</td><td>18</td><td>17</td><td>35</td></tr><tr><td> At diagnosis</td><td>7</td><td>6</td><td>13</td></tr><tr><td> In the course of disease</td><td>11</td><td>11</td><td>22</td></tr><tr><td>+19</td><td>0</td><td>11</td><td>11</td></tr><tr><td> At diagnosis</td><td>0</td><td>4</td><td>4</td></tr><tr><td> In the course of disease</td><td>0</td><td>7</td><td>7</td></tr><tr><td>+17/i(17q)</td><td>3</td><td>5</td><td>8</td></tr><tr><td> At diagnosis</td><td>1</td><td>3</td><td>4</td></tr><tr><td> In the course of disease</td><td>2</td><td>2</td><td>4</td></tr><tr><td>3q26.2</td><td>10</td><td>2</td><td>12</td></tr><tr><td> At diagnosis</td><td>1</td><td>0</td><td>1</td></tr><tr><td> In the course of disease</td><td>9</td><td>2</td><td>9</td></tr><tr><td>−7/7q abnormalities</td><td>5</td><td>4</td><td>9</td></tr><tr><td> At diagnosis</td><td>1</td><td>0</td><td>1</td></tr><tr><td> In the course of disease</td><td>4</td><td>4</td><td>8</td></tr><tr><td>+21</td><td>2</td><td>3</td><td>5</td></tr><tr><td> At diagnosis</td><td>1</td><td>1</td><td>2</td></tr><tr><td> In the course of disease</td><td>1</td><td>2</td><td>3</td></tr><tr><td>11q23</td><td>1</td><td>0</td><td>1</td></tr><tr><td> At diagnosis</td><td>0</td><td>0</td><td>0</td></tr><tr><td> In the course of disease</td><td>1</td><td>0</td><td>1</td></tr><tr><td>Complex karyotypes</td><td/><td>25</td><td>25</td></tr><tr><td> At diagnosis</td><td/><td>11</td><td>11</td></tr><tr><td> In the course of disease</td><td/><td>14</td><td>14</td></tr><tr><td>Low-risk ACA</td><td>32</td><td>32</td><td/></tr><tr><td> At diagnosis</td><td>19</td><td>19</td><td/></tr><tr><td> In the course of disease</td><td>13</td><td>13</td><td/></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Hazard to die in IM-treated patients with high- and low-risk ACA dependent on blast increase to 1–30% (Cox model).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>HR</th><th>Lower 95% CI</th><th>Upper 95% CI</th><th><italic>P</italic> value</th><th><italic>n</italic></th></tr></thead><tbody><tr><td colspan=\"6\">Peripheral blood (PB)</td></tr><tr><td colspan=\"6\"> 1% blasts in PB</td></tr><tr><td>   Presence of high-risk ACAs</td><td>3.65</td><td>2.32</td><td>5.75</td><td>&lt;0.001</td><td>224</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.92</td><td>1.06</td><td>8.07</td><td>0.039</td><td/></tr><tr><td>  5% blasts in PB</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td/><td>1.11</td><td>2.86</td><td>0.016</td><td>117</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.77</td><td>0.68</td><td>4.66</td><td>0.244</td><td/></tr><tr><td>  10% blasts in PB</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>1.39</td><td>0.87</td><td>2.21</td><td>0.167</td><td>107</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.38</td><td>0.53</td><td>3.60</td><td>0.506</td><td/></tr><tr><td>  15% blasts in PB</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>1.37</td><td>0.86</td><td>2.19</td><td>0.189</td><td>104</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.32</td><td>0.51</td><td>3.42</td><td>0.568</td><td/></tr><tr><td>  20% blasts in PB</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>0.84</td><td>0.50</td><td>1.40</td><td>0.502</td><td>79</td></tr><tr><td>   Presence of low-risk ACAs</td><td>0.74</td><td>0.20</td><td>2.71</td><td>0.652</td><td/></tr><tr><td>  30% blasts in PB</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>0.83</td><td>0.50</td><td>1.39</td><td>0.479</td><td>78</td></tr><tr><td>   Presence of low-risk ACAs</td><td>0.76</td><td>0.20</td><td>2.89</td><td>0.689</td><td/></tr><tr><td colspan=\"6\">Bone marrow (BM)</td></tr><tr><td> 1% blasts in BM</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>6.12</td><td>4.08</td><td>9.17</td><td>&lt;0.001</td><td>1033</td></tr><tr><td>   Presence of low-risk ACAs</td><td>2.71</td><td>0.99</td><td>7.44</td><td>0.053</td><td/></tr><tr><td>  5% blasts in BM</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>5.46</td><td>3.58</td><td>8.33</td><td>&lt;0.001</td><td>588</td></tr><tr><td>   Presence of low-risk ACAs</td><td>3.21</td><td>1.16</td><td>8.85</td><td>0.024</td><td/></tr><tr><td>  10% blasts in BM</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>2.21</td><td>1.37</td><td>3.56</td><td>0.001</td><td>134</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.68</td><td>0.61</td><td>4.60</td><td>0.311</td><td/></tr><tr><td>  15% blasts in BM</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>1.77</td><td>1.11</td><td>2.83</td><td>0.017</td><td>115</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.66</td><td>0.63</td><td>4.37</td><td>0.309</td><td/></tr><tr><td>  20% blasts in BM</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>1.24</td><td>0.74</td><td>2.06</td><td>0.416</td><td>87</td></tr><tr><td>   Presence of low-risk ACAs</td><td>1.11</td><td>0.29</td><td>4.18</td><td>0.882</td><td/></tr><tr><td>  30% blasts in BM</td><td/><td/><td/><td/><td/></tr><tr><td>   Presence of high-risk ACAs</td><td>0.89</td><td>0.53</td><td>1.49</td><td>0.665</td><td>79</td></tr><tr><td>   Presence of low-risk ACAs</td><td>0.81</td><td>0.21</td><td>3.07</td><td>0.760</td><td/></tr></tbody></table></table-wrap>", "<table-wrap id=\"Taba\"><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>No.</th><th>Sex</th><th>Age<sup>a</sup></th><th>Interval<sup>b</sup></th><th>Karyotype</th></tr></thead><tbody><tr><td colspan=\"5\">High-risk ACA in Ph+ cells</td></tr><tr><td> 1</td><td>F</td><td>54</td><td>11</td><td>46,XX,inv(3)(q21q26),t(7;9;22)(q31;q34;q11) [##REF##25931274##20##]</td></tr><tr><td/><td/><td/><td>16</td><td>46,XX,inv(3)(q21q26),t(7;9;22)(q31;q34;q11) [##REF##26416462##25##]</td></tr><tr><td/><td/><td/><td>20</td><td>46,XX,inv(3)(q21q26),t(7;9;22)(q31;q34;q11) [##REF##26416462##25##]</td></tr><tr><td> 2</td><td>F</td><td>45</td><td>28</td><td>46,XX,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##25888368##19##]</td></tr><tr><td/><td/><td/><td>32</td><td>46,XX,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td>  3</td><td>F</td><td>61</td><td>14</td><td><p>46,XX,t(9;22)(q34;q11) [##REF##3855376##3##]/46,XX,der(7)del(7)(p11)del(7)(q11),t(9;22)(q34;q11) [##REF##1065618##2##]/</p><p>45,XX,der(3;9;17)der(7)del(7)(p11)del(7)(q11),+der(9)ins(9;17)(p11;??),t(9;22)(q34;q11),der(9;17)dic(9;17)</p><p>(q11;p11)del(9)(p13p24),der(14)t(1;14)(q21;q21),+17,der(22)t(9;22)(q34;q11) [##REF##1065618##2##]/45,X,r(X),der(3;9;17)</p><p>(3pter?....)der(7)del(7)(p11)del(7)(q11),+der(9)ins(9;17)(p11;??),t(9;22)(q34;q11),der(9;17)dic(9;17)(q11;p11)del(9)(p13p24),der(14)(q21;q21),+17,der(22)t(9;22)(q34;q11) [##REF##7723396##4##]</p></td></tr><tr><td> 4</td><td>M</td><td>42</td><td>0</td><td>46,XY,inv(3)(p13q25),t(9;22)(q34;q11) [##REF##27006386##17##]</td></tr><tr><td> 5</td><td>F</td><td>32</td><td>12</td><td>46,XX,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##16527898##15##]</td></tr><tr><td> 6</td><td>M</td><td>43</td><td>8</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##28804124##24##]</td></tr><tr><td> 7</td><td>F</td><td>37</td><td>6</td><td>46,XX,t(3;21)(q26;q22),t(9;22)(q34;q11) [##REF##16527898##15##]</td></tr><tr><td/><td/><td/><td>7</td><td>46,XX,t(3;21)(q26;q22),t(9;22)(q34;q11) [##REF##27006386##17##]</td></tr><tr><td/><td/><td/><td>8</td><td>46,XX,t(3;21)(q26;q22),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 8</td><td>M</td><td>37</td><td>11</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##22653972##9##]</td></tr><tr><td/><td/><td/><td>14</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td/><td/><td/><td>17</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>24</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td/><td/><td/><td>32</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>36</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>44</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td/><td/><td/><td>45</td><td>46,XY,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 9</td><td>M</td><td>60</td><td>6</td><td><p>48–49,XY,t(5;9)(q31;q22),−7,der(7)del(7)(p11)del(7)(q11),+8,</p><p>der(9)t(7;9)(q11;p23)t(9;22)(q34;q11),+19,der(22)t(9;22)(q34;q11),+der(22)t(9;22)[cp12]</p></td></tr><tr><td> 10</td><td>M</td><td>71</td><td>12</td><td><p>46,XY,der(5)t(5;9)(q11;?),−7,der(9)t(9;22)(q34;q11)t(9;9)(p13;?)t(7;9)(?;?),</p><p>der(9)del(9)(p22)t(9;7)(q?;?)t(5;7)(q?;?),der(22)t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##12651263##6##]</p></td></tr><tr><td> 11</td><td>M</td><td>45</td><td>13</td><td>46,XY,r(7)(p11q32)del(7)(q11q22),del(9)(p12p24),t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td> 12</td><td>M</td><td>36</td><td>25</td><td>43–44,XY,−8,t(9;22)(q34;q11),−11,−15,−17,−18,−19,−20,−22,+2–3mar[cp10]</td></tr><tr><td> 13</td><td>M</td><td>44</td><td>3</td><td><p>46,XY,t(9;22)(q34;q11) [##UREF##0##11##]/51,XY,+X,dup(6)(p22p25),+der(6)t(4;6)(q24;q16),t(9;22)(q34;q11),+14,+21,</p><p>+der(22)t(9;22)(q34;q11) [##REF##7723396##4##]/51,XY,+X,der(4)t(4;8)(p15;q22),dup(6)(p22p25),+der(6)t(4;6)(q24;q16),+14,+21,</p><p>+der(22)t(9;22)(q34;q11) [##REF##1065618##2##]</p></td></tr><tr><td> 14</td><td>F</td><td>68</td><td>10</td><td>46,XX,t(9;22)(q34;q11) [##REF##12651263##6##]/46,XX,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td/><td/><td/><td>13</td><td>46,XX,inv(3)(q21q26),t(9;22)(q34;q11) [##REF##1065618##2##]/45,XX,inv(3)(q21q26),−7,t(9;22)(q34;q11) [##REF##16463391##10##]</td></tr><tr><td> 15</td><td>M</td><td>69</td><td>9</td><td><p>46,XY,t(9;22)(q34;q11)/48,XY,+8,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##3855376##3##]/</p><p>50,XY+8,+8,t(9;22)(q34;q11),+19,+der(22)t(9;22)(q34;q11) [##REF##7723396##4##]</p></td></tr><tr><td/><td/><td/><td>19</td><td>45,XY,t(3;12)(q26;q24),−7,t(9;22)(q34;q11) [##UREF##0##11##]</td></tr><tr><td> 16</td><td>F</td><td>54</td><td>45</td><td>46,XX,t(3;21)(q26;q22),t(9;22)(q34;q11) [##REF##26416462##25##]</td></tr><tr><td/><td/><td/><td>48</td><td>46,XX,t(3;21)(q26;q22),t(9;22)(q34;q11) [##REF##26416462##25##]</td></tr><tr><td> 17</td><td>M</td><td>64</td><td>22</td><td><p>51,XY,+6,+8,+8,der(9)t(9;10)(q34;q22),der(10)t(10;15)(q22;q15),</p><p>+der(11)t(11;17)(p11.2;q11.2),der(15)t(15;22)(q15;q11.2),der(22)t(9;22)(q34;q11.2),+der(22)t(9;22)(q34;q11.2) [##REF##27006386##17##]</p></td></tr><tr><td> 18</td><td>M</td><td>24</td><td>10</td><td><p>45,X,−Y,t(9;22)(q34;q11) [##REF##369590##8##]/46,X,−Y,t(9;22)(q34;q11.2),+der(22)t(9;22)(q34;q11.2) [##REF##7723396##4##]/</p><p>47,X,−Y,−7,t(9 ;22)(q34;q11.2),+der(22)t(9;22)(q34;q11.2),+mar,+r [##REF##369590##8##]</p></td></tr><tr><td> 19</td><td>M</td><td>61</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##20592475##14##]/46,XY,der(9)t(9;22)(q34;q11),ider(22)(q10)t(9;22)(q34;q11) [##UREF##0##11##]</td></tr><tr><td> 20</td><td>M</td><td>51</td><td>44</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td> 21</td><td>M</td><td>61</td><td>46</td><td>46,XY,t(9;22)(q34;q11),+der(22)idic(9)(q34)t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11)x3,+mar[cp20]</td></tr><tr><td/><td/><td/><td>64</td><td>49,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11)x3 [##REF##3855376##3##]</td></tr><tr><td> 22</td><td>F</td><td>60</td><td>2</td><td>46,XX,der(7)t(7;22)(q36;q11)del(7)(q11q22),der(9)t(7;9)(q36;q34),der(22)t(9;22)(q34;q11) [##REF##29296906##21##]</td></tr><tr><td> 23</td><td>M</td><td>46</td><td>0</td><td>47,XY,der(9)t(9;22)(q34;q11),del(9)(q33q34),del(22)(q11q12),+der(22)t(9;22)(q34;q11) [##REF##26416462##25##]</td></tr><tr><td> 24</td><td>F</td><td>59</td><td>6</td><td>46,XX,der(9)t(9;22;15)(q34;q11;q26),der(15)t(9;22;15),+22,der(22)idic(22)(p11)t(9;22) [##REF##16527898##15##]</td></tr><tr><td> 25</td><td>M</td><td>34</td><td>3</td><td>48,XY,+X,der(5)t(1;5)(q21;q31),t(9;22;21)(q34;q11;q22),+der(22)t(9;22) [##REF##16527898##15##]</td></tr><tr><td> 26</td><td>M</td><td>40</td><td>41</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>76</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##8558198##5##]</td></tr><tr><td/><td/><td/><td>78</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##369590##8##]</td></tr><tr><td/><td/><td/><td>79</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##8558198##5##]</td></tr><tr><td> 27</td><td>M</td><td>36</td><td>72</td><td>46,XY,t(9;22)(q34;q11)/47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 28</td><td>M</td><td>54</td><td>9</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td> 29</td><td>M</td><td>44</td><td>29</td><td>46,XY,t(9;22)(q34;q11) [##REF##8558198##5##]/47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##UREF##0##11##]</td></tr><tr><td/><td/><td/><td>33</td><td>46,XY,t(9;22)(q34;q11) [##REF##3855376##3##]/47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##22653972##9##]</td></tr><tr><td> 30</td><td>M</td><td>32</td><td>18</td><td><p>47,XY,t(1;14)(p3?1;q?32),del(9)(p22p24),t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##22653972##9##]/</p><p>46,XY,del(9)(p22p24),t(9;22)(q34;q11),del(12)(p11.2p13) [##REF##12651263##6##]</p></td></tr><tr><td> 31</td><td>M</td><td>78</td><td>13</td><td>45,X,−Y,del(9)(q22),+der(22)t(9;22)(q34;q11)t(9;9)(q34;q22) [##REF##26464170##13##]</td></tr><tr><td> 32</td><td>M</td><td>38</td><td>27</td><td>47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##20592475##14##]</td></tr><tr><td> 33</td><td>M</td><td>23</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##26243778##18##]/47,idem,+der(22)t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 34</td><td>M</td><td>68</td><td>19</td><td>46,XX,t(9;22)(q34;q11) [##REF##8558198##5##]/48,XX,t(9;22)(q34;q11),+19,+der(22)t(9;22)(q34;q11) [##REF##369590##8##]</td></tr><tr><td> 35</td><td>F</td><td>69</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##369590##8##]/47,idem,+ider(22)(q10)t(9;22)(q34;q11) [##REF##12560227##7##]</td></tr><tr><td/><td/><td/><td>3</td><td>46,XX,t(9;22)(q34;q11) [##REF##12560227##7##]/47,idem,ider(22)(q10)t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 36</td><td>M</td><td>51</td><td>27</td><td>46,XY,t(9;22)(q34;q11) [##REF##20592475##14##]/48,idem,+19,+der(22)t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 37</td><td>F</td><td>60</td><td>35</td><td>45,XX,−7,t(9;22)(q34;q11) [##REF##12560227##7##]</td></tr><tr><td/><td/><td/><td>41</td><td>46,XX,r(7)(p13q11),t(9;22)(q34;q11) [##REF##22653972##9##]/45,XX,−7,t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>44</td><td>46,XX,r(7)(p13q11),t(9;22)(q34;q11) [##REF##12560227##7##]/45,XX,−7,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 38</td><td>M</td><td>39</td><td>12</td><td>46,XY,t(9;22)(q34;q11) [##REF##12560227##7##]/47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]/48,XY,+8,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td/><td/><td/><td>88</td><td>46,XY,t(9;22)(q34;q11) /47,XY,+8,t(9;22)(q34;q11) /48,XY,+8,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##REF##22039253##16##]</td></tr><tr><td> 39</td><td>F</td><td>58</td><td>7</td><td>55,XX,+X,+5,+6,+7,+8,t(9;22)(q34;q11),+14,+19,+22,+der(22)t(9;22)(q34;q11)[cp5]</td></tr><tr><td> 40</td><td>M</td><td>38</td><td>15</td><td>47,XY,t(9;22)(q34;q11),+8 [##REF##1065618##2##]</td></tr><tr><td> 41</td><td>M</td><td>39</td><td>0</td><td><p>47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]/47,XY,+8,t(9;22)(q34;q11),i(17)(q10) [##REF##1065618##2##]/48,XY,+8,t(9;22)(q34;q11),i(17)(q10),+22,</p><p>del(22)q11) [##REF##16463391##10##]</p></td></tr><tr><td> 42</td><td>M</td><td>64</td><td>10</td><td>46,XY,t(9;22)(q34;q11) [##REF##22039253##16##]/50,XY,+8,+12,+18,+21,t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 43</td><td>M</td><td>34</td><td>5</td><td>46,XY,t(9;22)(q34;q11) [##REF##26464170##13##]/47,XY,+8,t(9;22)(q34;q11) [##REF##8558198##5##]</td></tr><tr><td/><td/><td/><td>117</td><td>46,XY,t(9;22)(q34;q11) [##REF##22653972##9##]/47,XY,+8,t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td> 44</td><td>F</td><td>54</td><td>73</td><td>46,XX,t(9;22)(q34;q11) [##REF##1065618##2##]/47,XX,+8,t(9;22)(q34;q11) [##REF##19965667##12##]</td></tr><tr><td/><td/><td/><td>81</td><td>47,XX,+der(8),t(1;8)(q22;p22),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 45</td><td>F</td><td>42</td><td>51</td><td>46,XX,t(9;22)(q34;q11) [##REF##27006386##17##]/47,XX,+8,t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 46</td><td>M</td><td>72</td><td>0</td><td>47,XY,+8,t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td/><td/><td/><td>7</td><td>46,XY,t(9;22)(q34;q11) [##REF##12651263##6##]/47,XY,+8,t(9;22)(q34;q11) [##REF##26243778##18##]</td></tr><tr><td> 47</td><td>M</td><td>40</td><td>0</td><td>47,XY,+8,t(9;22)(q34;q11) [##REF##27006386##17##]/49,XY,+8,t(9;22)(q34;q11),+19,+20 [##REF##12560227##7##]</td></tr><tr><td> 48</td><td>M</td><td>25</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##29122769##22##]/47,XY,+8,t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 49</td><td>F</td><td>24</td><td>23</td><td>46,XX,t(9;22)(q34;q11) [##REF##7723396##4##]/47,XX,+8,t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>29</td><td>46,XX,t(9;22)(q34;q11) [##UREF##0##11##]/47,XX,+8,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>36</td><td>46,XX,t(9;22)(q34;q11) [##REF##3855376##3##]/47,XX,+8,t(9;22)(q34;q11) [##REF##26243778##18##]</td></tr><tr><td/><td/><td/><td>40</td><td>47,XX,+8,t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td/><td/><td/><td>44</td><td>47,XX,+8,t(9;22)(q34;q11) [##REF##8558198##5##]</td></tr><tr><td> 50</td><td>M</td><td>31</td><td>0</td><td><p>46,XY,t(9;22;10)(q34;q11;p15) [##REF##26464170##13##]/55,XY,+3,+8,t(9;22;10)(q34;q11;p15),+12,+13,+14,+18,+19,+21,+der(22)</p><p>t(9;22(q34;q11) [##REF##12560227##7##]</p></td></tr><tr><td> 51</td><td>M</td><td>28</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##16463391##10##]/47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34:q11) [##REF##16463391##10##]</td></tr><tr><td/><td/><td/><td>3</td><td>46,XY,t(9;22)(q34;q11) /47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34:q11) [##REF##25931274##20##]</td></tr><tr><td> 52</td><td>M</td><td>55</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##12560227##7##]/49,XY,+8,+10,t(9;22)(q34;q11),+der(22)t(9;22)(q34:q11) [##REF##26464170##13##]</td></tr><tr><td/><td/><td/><td>6</td><td>46,XY,t(9;22)(q34;q11) [##REF##3855376##3##]/49,XY,+8,+10,t(9;22)(q34;q11),+der(22)t(9;22)(q34:q11) [##REF##19965667##12##]</td></tr><tr><td> 53</td><td>F</td><td>68</td><td>0</td><td>48,XX,+8,t(9;18)(q34;q21),+der(22)ins(22;9)(q11;34q34) [##REF##8558198##5##]</td></tr><tr><td/><td/><td/><td>6</td><td>48,XX,+8,t(9;18)(q34;q21),+der(22)ins(22;9)(q11;34q34) [##REF##7723396##4##]</td></tr><tr><td> 54</td><td>M</td><td>38</td><td>0</td><td>47,XY,+8,t(9;22)(q34;q11) [##REF##3855376##3##]/48,XY,+8,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) [##UREF##0##11##]</td></tr><tr><td> 55</td><td>M</td><td>53</td><td>0</td><td>50,XY,+8,+8,i(17)(q10),+19,+der(22)t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td> 56</td><td>M</td><td>28</td><td>10</td><td>45,XY,t(8;21)(q22;q22),t(9;22)(q34;q11),−11/93,XXYY,+8,+8,t(8;21)(q22;q22),t(9;22)(q34;q11),−11 [##REF##369590##8##]</td></tr><tr><td> 57</td><td>M</td><td>70</td><td>19</td><td>46,XY,t(9;22)(q34;q11) [##REF##25888368##19##]/47,XY,+8,t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>64</td><td>46,XY,t(9;22)(q34;q11) [##REF##7723396##4##]/47,XY,+8,t(9;22)(q34;q11) [##REF##22039253##16##]</td></tr><tr><td> 58</td><td>M</td><td>55</td><td>0</td><td>46,XY,t(1;12)(p34;q24),t(1;9;22)(p36;q34;q11) [##REF##29296906##21##]/48,idem,+8,+9 [##REF##1065618##2##]</td></tr><tr><td> 59</td><td>M</td><td>46</td><td>52</td><td>46,XY,t(9;22)(q34;q11) [##REF##369590##8##]/47,XY,+8,t(9;22)(q34;q11) [##REF##12560227##7##]</td></tr><tr><td/><td/><td/><td>72</td><td>46,XY,t(9;22)(q34;q11) [##REF##25888368##19##]/47,XY,+8,t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>80</td><td>47,XY,+8,t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>86</td><td>46,XY,t(9;22)(q34;q11) [##REF##7723396##4##]/47,XY,+8,t(9;22)(q34;q11) [##REF##26464170##13##]</td></tr><tr><td> 60</td><td>M</td><td>64</td><td>42</td><td>46,XY,t(9;22)(q34;q11) [##REF##27006386##17##]/47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>53</td><td>46,XY,t(9;22)(q34;q11) [##REF##27006386##17##]/47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>57</td><td>46,XY,t(9;22)(q34;q11) [##REF##8558198##5##]/47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>65</td><td>46,XY,t(9;22)(q34;q11) [##REF##7723396##4##]/47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td/><td/><td/><td>69</td><td>47,XY,+8,t(9;22)(q34;q11) [##REF##1065618##2##]/46,XY,del(7)(q11;q22) [##REF##12651263##6##]</td></tr><tr><td> 61</td><td>M</td><td>57</td><td>0</td><td>48,XY,+8,t(9;22)(q34;q11),+19 [##REF##26416462##25##]</td></tr><tr><td> 62</td><td>M</td><td>50</td><td>76</td><td>46,XY,t(9;22)(q34;q11)/47,XY,+8,t(9;22)(q34;q11),idic(17)(p11) [##REF##28804124##24##]</td></tr><tr><td> 63</td><td>M</td><td>46</td><td>102</td><td>47,XY,+8,t(9;22)(q34;q11),i(17)(q10) [##REF##12651263##6##]</td></tr><tr><td> 64</td><td>M</td><td>41</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##16463391##10##]/44,XY,t(9;22)(q34;q11),−14,i(17)(q10),−18 [##REF##16527898##15##]</td></tr><tr><td> 65</td><td>F</td><td>62</td><td>10</td><td>46,XX,t(1;9;22)(p36;q34;q11) [##REF##369590##8##]/47,XX,t(1;9;22)(p36;q34;q11),+8 [##REF##1065618##2##]</td></tr><tr><td> 66</td><td>F</td><td>76</td><td>8</td><td>47,XX,+8,t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 67</td><td>M</td><td>44</td><td>20</td><td>46,XY,t(3;21)(q26;q22),t(9;22)(q34;q11) [##REF##22039253##16##]/46,XY [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>34</td><td>49,XY,t(3;21)(q26;q22),+8,t(9;22)(q34;q11),+12,+der(22)t(9;22)(q34;q11) [##REF##7723396##4##]/46,XY [##REF##25888368##19##]</td></tr><tr><td> 68</td><td>F</td><td>52</td><td>0</td><td>47,XX,+8,t(9;22)(q34;q11),i(17)(q10) [##REF##16463391##10##]</td></tr><tr><td/><td/><td/><td>3</td><td>47,XX,+8,t(9;22)(q34;q11),idic(17)(p12) [##REF##7723396##4##]/46,XX [##REF##29296906##21##]</td></tr><tr><td/><td/><td/><td>8</td><td>50,XX,+8,+8,+8,t(9;22)(q34;q11),idic(17)(p12),+19 [##REF##12560227##7##]/46,XX [##REF##25931274##20##]</td></tr><tr><td> 69</td><td>F</td><td>56</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##29296906##21##]/47,XX,+8,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 70</td><td>M</td><td>48</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##12651263##6##]/47,XY,+8,t(9;22)(q34;q11) [##REF##19965667##12##]</td></tr><tr><td> 71</td><td>M</td><td>51</td><td>0</td><td>46,XY,t(9;22)((q34;q11) [##REF##12560227##7##]/47,XY,+8,t(9;22)(q34;q11),i(17)(q10) [##REF##26243778##18##]</td></tr><tr><td> 72</td><td>F</td><td>75</td><td>24</td><td>47,XX,t(9;22)(q34;q11),+21 [##REF##7723396##4##]</td></tr><tr><td> 73</td><td>F</td><td>39</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##3855376##3##]/46,idem,t(20;21)(q10;q10) /47,idem,t(20;21)(q10;q10),+21 [##UREF##0##11##]</td></tr><tr><td> 74</td><td>M</td><td>42</td><td>19</td><td>47,XY,t(9;22)(q34;q11),+17 [##REF##25931274##20##]</td></tr><tr><td/><td/><td/><td>21</td><td>47,XY,t(9;22)(q34;q11),+17 [##REF##16463391##10##]</td></tr><tr><td> 75</td><td>F</td><td>62</td><td>11</td><td>46,XX,t(9;11)(p21–22;q23),t(9;22)(q34;q11) [##REF##26416462##25##]</td></tr><tr><td> 76</td><td>M</td><td>51</td><td>38</td><td>46,XY,t(9;22)(q34;q11),inv(16)(p13q22),+der(22)t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 77</td><td>F</td><td>59</td><td>32</td><td>48,XX,t(9;15;22) (q34;q26;q11),+8,+19 [##REF##3855376##3##]</td></tr><tr><td> 78</td><td>F</td><td>51</td><td>7</td><td>45,XX,der(7;9)(q10;q10)t(9;22)(q34;q11) [##REF##369590##8##]</td></tr><tr><td> 79</td><td>M</td><td>24</td><td>8</td><td><p>46,XY,der(9)t(9;22)(q34;q11),der(19)t(9;19)(q34;p13);der(22)t(9;22)(q34;q11)</p><p>t(9;19)(q34,p13) [##REF##1065618##2##]</p></td></tr><tr><td> 80</td><td>M</td><td>53</td><td>0</td><td><p>46,XY,der(9)t(9;22)(q34;q11),idicder(22)(q11)t(9;22)(q34;q11),</p><p>idicder(22)(q11)t(9;22)(q34;q11) [##REF##3855376##3##]</p></td></tr><tr><td> 81</td><td>M</td><td>55</td><td>0</td><td><p>46,XY,der(7)t(7;9)(q11.2;q34),der(9)t(9;22)(q34;q11.2),der(22)t(9;22)(q34;q11.2)</p><p>t(7;9)(q11.2q34) [##REF##26464170##13##]</p></td></tr><tr><td> 82</td><td>M</td><td>40</td><td>0</td><td>47,XY,+8,t(9;22)(q34;q11) [##REF##22039253##16##]</td></tr><tr><td> 83</td><td>F</td><td>68</td><td>13</td><td>46,XX,t(3;11)(q26;q23),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 84</td><td>F</td><td>54</td><td>45</td><td>46,XX,t(3;21)(q26;q22),t (9;22)(q34;q11) [##REF##26416462##25##]</td></tr><tr><td> 85</td><td/><td>42</td><td>40</td><td><p>47,XY,der(8)t(8;9)(p21;p13),+der(8)t(8;9)(p21;p13),del(9)(p13p24),</p><p>der(9)t(8;9)(p21;p11)t(9;10)(q34;q24),der(10)t(10;22)(q24;q11),</p><p>t(14;21)(q22;q21),der(22)t(9;22)(q34;q11) [##REF##16463391##10##]</p></td></tr><tr><td> 86</td><td>M</td><td>60</td><td>22</td><td>46,XY,inv(7)(p22q32),+8,t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 87</td><td>M</td><td>44</td><td>0</td><td><p>46,XY,der(9)t(9;10)(q22;q23)t(10;22)(q25;q11),der(10)t(9;10)(q22;q23)t(9;15)(q34;q21),der(15)t(10;15)(q26;q15)</p><p>t(10;22)(q25;q11),der(22)t(9;22)(q34;q11) [##REF##29296906##21##]</p></td></tr><tr><td/><td/><td/><td>4</td><td><p>46,XY,der(9)t(9;10)(q22;q23)t(10;22)(q25;q11),der(10)t(9;10)(q22;q23)t(9;15)(q34;q21),der(15)t(10;15)(q26;q15)</p><p>t(10;22)(q25;q11),der(22)t(9;22)(q34;q11) [##REF##369590##8##]</p></td></tr><tr><td/><td/><td/><td>7</td><td><p>46,XY,der(9)t(9;10)(q22;q23)t(10;22)(q25;q11),der(10)t(9;10)(q22;q23)t(9;15)(q34;q21),der(15)t(10;15)(q26;q15)</p><p>t(10;22)(q25;q11),der(22)t(9;22)(q34;q11) [##REF##3855376##3##]</p></td></tr><tr><td/><td/><td/><td>10</td><td><p>46,XY,der(9)t(9;10)(q22;q23)t(10;22)(q25;q11),der(10)t(9;10)(q22;q23)t(9;15)(q34;q21),der(15)t(10;15)(q26;q15)</p><p>t(10;22)(q25;q11),der(22)t(9;22)(q34;q11) [##REF##3855376##3##]</p></td></tr><tr><td> 88</td><td>F</td><td>38</td><td>0</td><td>46,XX,del(1)(q21),der(9)t(9;22)(q34;q11)t(1;22)(q44;q11),der(22)t(9;22)(q34;q11)t(1;9)(q21;q34) [##REF##25931274##20##]</td></tr><tr><td> 89</td><td>F</td><td>66</td><td>0</td><td>46,XX,del(1)(q32),der(9)t(1;9)(q32;q34)t(1;22)(q44;q11),der(22)t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 90</td><td>M</td><td>55</td><td>0</td><td>46,XY,t(7;11),del(7q),der(9)t(9;22)(q34;q11),der(11),del(16q),der(17),der(22)t(9;22)(q34;q11) [##REF##16463391##10##]</td></tr><tr><td> 91</td><td>F</td><td>47</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##1065618##2##]/46,XX,der(7;11)ins(7;11)(p14;p11q25)t(7;11)(p22;p11),t(9;22)(q34;q11) [##REF##26464170##13##]</td></tr><tr><td/><td/><td/><td>3</td><td><p>46,XX,t(9;22)(q34;q11) [##REF##8558198##5##]/45,XX,der(7;11)ins(7;11)(p14;p11q25)t(7;11)(p22;p11),t(9;22)(q34;q11) [##REF##20592475##14##]/</p><p>46,XX,t(9;22)(q34;q11),del(11)(p13) [##REF##1065618##2##]/46,XX,del(4)(q31),der(11)t(4;11)(q?;p13)t(11;13)(q21;q14),</p><p>der(12)t(11;12)(p13;q24),der(13)t(11;13)(q21;q14) [##REF##1065618##2##]</p></td></tr><tr><td/><td/><td/><td>6</td><td>46,XX,t(9;22)(q34;q11) [##REF##1065618##2##]/45,XX,der(7;11)ins(7;11)(p14;p11q25)t(7;11)(p22;p11),t(9;22)(q34;q11) [##REF##22653972##9##]</td></tr><tr><td colspan=\"5\">Low-risk ACA in Ph+ cells</td></tr><tr><td> 92</td><td>M</td><td>59</td><td>0</td><td>46,XY,t(9;22)(q34;q11),del(15)(q22),add(17)(p11) [##REF##369590##8##]</td></tr><tr><td/><td/><td/><td>4</td><td>46,XY,t(9;22)(q34;q11),del(15)(q22),add(17)(p11) [##REF##12560227##7##]</td></tr><tr><td/><td/><td/><td>7</td><td>46,XY,t(9;22)(q34;q11),del(15)(q22),add(17)(p11) [##REF##16463391##10##]</td></tr><tr><td/><td/><td/><td>10</td><td>46,XY,t(9;22)(q34;q11),del(15)(q22),add(17)(p11) [##REF##12560227##7##]</td></tr><tr><td> 93</td><td>M</td><td>48</td><td>0</td><td>46,XY,t(4;6)(q21;p23),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 94</td><td>M</td><td>36</td><td>12</td><td>46,XY,t(9;22)(q34;q11) [##REF##369590##8##]/46,XY,t(9;22)(q34;q11),i(9)(p10),der(17)t(9;17)(q11;p11) [##REF##19965667##12##]</td></tr><tr><td> 95</td><td>F</td><td>58</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##12651263##6##]/92,XXXX,t(9;22)(q34;q11)x2 [##REF##7723396##4##]</td></tr><tr><td> 96</td><td>M</td><td>44</td><td>0</td><td>46,XY,der(10),t(9;22)(q34;q11) [##REF##26416462##25##]</td></tr><tr><td> 97</td><td>M</td><td>46</td><td>0</td><td>46,XY,t(9;22)(q34;q11),t(14;17)(p11;q11) [##REF##25931274##20##]</td></tr><tr><td> 98</td><td>M</td><td>40</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##1065618##2##]/46,XY,del(5)(q11q14),t(9;22)(q34;q11) [##REF##25888368##19##]</td></tr><tr><td/><td/><td/><td>6</td><td>46,XY,del(5)(q11q14),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 99</td><td>F</td><td>51</td><td>7</td><td>46,XX,t(9;22)(q34;q11) [##REF##7723396##4##]/45,XX,der(7;9)(q10;q10)t(9;22)(q34;q11),der(22)t(9;22)(q34;q11) [##REF##369590##8##]</td></tr><tr><td> 100</td><td>M</td><td>27</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##25931274##20##]/45,XY,t(9;22)(q34;q11),−21 [##REF##7723396##4##]</td></tr><tr><td> 101</td><td>F</td><td>52</td><td>0</td><td>46,XX,t(2;16)(p2?3;p1?3),t(9;22)(q34;q11) [##REF##26385387##26##]</td></tr><tr><td> 102</td><td>F</td><td>61</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##29296906##21##]/46,XX,del(6)(q15q23),t(9;22)(q34;q11) [##REF##7723396##4##]</td></tr><tr><td> 103</td><td>F</td><td>68</td><td>0</td><td>46,XX,t(9;22)(q34;q11) /46,XX,del(5)(q13q22),t(9;22)(q34;q11) [##REF##28804124##24##]</td></tr><tr><td> 104</td><td>F</td><td>64</td><td>0</td><td>46,XX,t(5;8)(q14;q23),t(9;22)(q34;q11) [##REF##25888368##19##]</td></tr><tr><td> 105</td><td>M</td><td>37</td><td>0</td><td>46,XY,t(9;22)(q34;q11),t(15;20)(q13;p12) [##REF##25931274##20##]</td></tr><tr><td> 106</td><td>F</td><td>45</td><td>39</td><td>46,XX,t(9;22)(q34;q11.2) [##REF##22039253##16##]/46,XX,t(9;22)(q34;q11.2),add(20)(p11.2) [##REF##22653972##9##]</td></tr><tr><td> 107</td><td>M</td><td>19</td><td>0</td><td>46,XY,der(1)t(1;9)(q21;q34)t(9;22)(q34:q11),der(9)t(1;9)(q21;q34)t(9;22)(q34:q11),der(22)t(9;22)(q34;q11) [##REF##3855376##3##]</td></tr><tr><td> 108</td><td>F</td><td>65</td><td>86</td><td>46,XX,t(9;22)(q34;q11) [##REF##27006386##17##]/46,XX,del(X)(p?21),t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td> 109</td><td>M</td><td>64</td><td>10</td><td>46,XY,add(9)(q34),add(9)(q32–34),der(22)t(9;22)(q34;q11) [##REF##16463391##10##]</td></tr><tr><td/><td/><td/><td>14</td><td>46,XY,t(1;3)(p36;q2?6),add(9)(q34),add(9)(q32–34),der(22)t(9;22)(q34;q11) [##REF##16463391##10##]</td></tr><tr><td> 110</td><td>F</td><td>56</td><td>0</td><td>46,XX,t(1;21)(q21;q22),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td/><td/><td/><td>12</td><td>46,XX,t(1;21)(q21;q22),t(9;22)(q34;q11) [##REF##16527898##15##]</td></tr><tr><td/><td/><td/><td>24</td><td>46,XX,t(1;21)(q21;q22),t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 111</td><td>M</td><td>36</td><td>0</td><td>46,XY,t(9;22)(q34;q11),t(11;19)(q14.1;q13) [##REF##25931274##20##]</td></tr><tr><td> 112</td><td>M</td><td>62</td><td>0</td><td>46,XY,t(9;22)(q34;q11) [##REF##1065618##2##]/46,idem,add(8)(q24) [##REF##1065618##2##]/45,idem,der(18)t(10;18)(q11;p11) [##REF##16463391##10##]</td></tr><tr><td> 113</td><td>M</td><td>61</td><td>0</td><td>46,XY,t(1;9)(q24;q31),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 114</td><td>F</td><td>46</td><td>0</td><td>46,XX,der(2)t(2;4)(q37;q21)del(4)(q21),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 115</td><td>M</td><td>70</td><td>0</td><td>46,XY,der(9)t(9;22)(q34;q11),t(10;22)(q25;q13) [##REF##27006386##17##]</td></tr><tr><td/><td/><td/><td>4</td><td>46,XY,der(9)t(9;22)(q34;q11),t(10;22)(q25;q13) [##REF##3855376##3##]</td></tr><tr><td/><td/><td/><td>7</td><td>45,X,−Y [##REF##3855376##3##]/46,XY,der(9)t(9;22)(q34;q11),t(10;22)(q25;q13) [##REF##1065618##2##]</td></tr><tr><td> 116</td><td>F</td><td>69</td><td>94</td><td>46,XX,t(9;22)(q34;q11),?del(17)(p12) [##REF##7723396##4##]</td></tr><tr><td> 117</td><td>F</td><td>49</td><td>0</td><td>46,XX,del(3)(p11p2?1)or(p21),t(9;22)(q34;q11) [##REF##19965667##12##]/46,XX,del(3)(p11p2?1)or(p21),del(5)(q15q31),t(9;22)(q34;q11) /46,XX,t(9;22)(q34;q11) [##REF##1065618##2##]</td></tr><tr><td> 118</td><td>F</td><td>76</td><td>0</td><td>46,XX,t(9;22)(q34;q11) [##REF##12560227##7##]/46,XX,t(9;22)(q34;q11),der(19)t(19;?)(p13.3;?) [##REF##26464170##13##]</td></tr><tr><td/><td/><td/><td>4</td><td>46,XX,t(9;22)(q34;q11) [##REF##12560227##7##]/46,XX,t(9;22)(q34;q11),der(19)t(17;19)(q22;p13) [##REF##26464170##13##]</td></tr><tr><td> 119</td><td>M</td><td>40</td><td>3</td><td>46,XY,t(6;15),t(9;22)(q34;q11) [##REF##12651263##6##]</td></tr><tr><td> 120</td><td>M</td><td>66</td><td>11</td><td>46,XY,t(9;22)(q34;q11) [##REF##22653972##9##]/46,XY,der(6)t(6;17)(p21;q11),t(9;22)(q34;q11) [##UREF##0##11##]</td></tr><tr><td> 121</td><td>F</td><td>43</td><td>0</td><td>46,XX,t(7;7)(p22;q22),t(9;22;9)(q34;q11;p24) [##REF##22039253##16##]</td></tr><tr><td/><td/><td/><td>60</td><td>40–43,XX,t(7;7)(p22;q22),t(9;22;9)(q34;q11;p24),inc [cp3]</td></tr><tr><td> 122</td><td>M</td><td>24</td><td>6</td><td>46,XY,t(2;12)(q33;p13),t(9;22)(q34;q11) [##REF##25931274##20##]</td></tr><tr><td> 123</td><td>F</td><td>31</td><td>11</td><td>46,XX,t(9;22)(q34;q11) [##REF##26243778##18##]/ 46,XX,t(9;22)(q34;q11),ins(11;11)(p15;p11.2p13) [##REF##7723396##4##]</td></tr><tr><td/><td/><td/><td>25</td><td>46,XX,t(9;22)(q34;q11) [##REF##22653972##9##]/ 46,XX,t(9;22)(q34;q11),ins(11;11)(p15;p11.2p13) [##REF##8558198##5##]</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
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[ "<table-wrap-foot><p><sup>a</sup>Only values up to 4 weeks in advance or 1 week after the first appearance of ACAs were counted (<italic>n</italic> ≥ 74).</p><p><sup>b</sup><italic>Maximum number</italic> = emergence of last ACA, <italic>n.r.</italic> not reported.</p></table-wrap-foot>", "<table-wrap-foot><p><sup>a</sup>Multiple listings possible.</p></table-wrap-foot>", "<table-wrap-foot><p><sup>a</sup>Age at diagnosis (years).</p><p><sup>b</sup>Interval between diagnosis and emergence of ACA (months).</p></table-wrap-foot>", "<fn-group><fn><p>Members of the SAKK and the German CML Study Group are listed below Acknowledgements.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_826_Fig1_HTML\" id=\"d32e3047\"/>", "<graphic xlink:href=\"41375_2020_826_Fig2_HTML\" id=\"d32e3688\"/>", "<graphic xlink:href=\"41375_2020_826_Fig3_HTML\" id=\"d32e4169\"/>" ]
[]
[{"label": ["11."], "surname": ["Hehlmann", "Hochhaus", "Baccarani"], "given-names": ["R", "A", "M"], "article-title": ["Chronic myeloid leukemia"], "source": ["The Lancet"], "year": ["2007"], "volume": ["370"], "fpage": ["342"], "lpage": ["50"], "pub-id": ["10.1016/S0140-6736(07)61165-9"]}]
{ "acronym": [], "definition": [] }
36
CC BY
no
2024-01-13 23:35:07
Leukemia. 2020 May 7; 34(8):2074-2086
oa_package/34/cf/PMC7387244.tar.gz
PMC7387246
32433508
[ "<title>Introduction</title>", "<p id=\"Par2\">Copy Number Variation (CNV), i.e. gains or losses of entire chromosomes or of specific genomic regions, are hallmarks of cancer. Understanding the impact of CNVs in tumor development is challenging since they can sometimes alter the dosage of hundreds or even thousands of genes simultaneously, thereby modifying mRNA and protein abundance, ultimately impacting cellular fitness. In hematological cancer, numerous CNVs are found and they are markedly different to those found in solid tumors. Gain of chromosome 21 (+21) is one of the most frequent CNVs observed in hematological malignancies [##UREF##0##1##, ##REF##23124507##2##]. Using large cohorts, it has been shown that +21 is rarely seen in solid tumors and that trisomy 21, the most common type of +21, is found in nearly all subtypes of hematological malignancies, ranging from 2.2% in chronic lymphocytic leukemia (CLL) to nearly 15% in acute lymphoblastic leukemia (ALL) (Mitelman database, available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://mitelmandatabase.isb-cgc.org\">https://mitelmandatabase.isb-cgc.org</ext-link>). It is rarely observed as a sole cytogenetic abnormality and its prognostic value varies depending on the cohort analyzed and the type of hematological malignancy [##UREF##0##1##, ##REF##10572083##3##]. Children with acute megakaryoblastic leukemia (AMKL) and B-cell precursor ALL (B-ALL) harbor +21 most frequently, occurring in approximately one-third of cases [##REF##17971484##4##, ##REF##22122069##5##]. To date, reasons for such an association remain elusive, but strongly suggest that somatic +21 is clonally selected during leukemia development and that megakaryocytic and B-cell progenitor/precursors are profoundly susceptible to the increased dosage of chromosome 21 genes.</p>", "<p id=\"Par3\">Individuals with Down syndrome (DS) harbor constitutive trisomy 21 [##REF##13662687##6##] and are predisposed to childhood acute leukemia [##REF##27031084##7##]. Constitutive trisomy 21 is the most common cytogenetic abnormality seen at birth (1 in 700–1000 newborns), and predominantly results from nondisjunction of chromosome 21 during meiosis (95%) [##REF##12699967##8##–##REF##16192705##10##]. Although this mechanism is different to somatic gain of chromosome 21, studying the predisposing and leukemia promoting role of trisomy 21 in DS human specimens, cell lines and murine models has been instrumental in undertanding the role of +21 alone and in cooperation with other secondary genetic alterations. Evidence indicates that trisomy 21 (or gain of chromosome 21), regardless of whether constitutive or acquired, is a promoting event in hematological malignancies.</p>", "<p id=\"Par01\">This review will present a snapshot of our knowledge on DS-associated leukemia, integrating studies addressing clinical features, therapy and molecular mechanisms of leukemogenesis, and will consider them in parallel to other hematological malignancies harboring somatic +21 with the view to emphasize similarities and differences at the clinical and biological level.</p>" ]
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[ "<p id=\"Par1\">Structural and numerical alterations of chromosome 21 are extremely common in hematological malignancies. While the functional impact of chimeric transcripts from fused chromosome 21 genes such as TEL-AML1, AML1-ETO, or FUS-ERG have been extensively studied, the role of gain of chromosome 21 remains largely unknown. Gain of chromosome 21 is a frequently occurring aberration in several types of acute leukemia and can be found in up to 35% of cases. Children with Down syndrome (DS), who harbor constitutive trisomy 21, highlight the link between gain of chromosome 21 and leukemogenesis, with an increased risk of developing acute leukemia compared with other children. Clinical outcomes for DS-associated leukemia have improved over the years through the development of uniform treatment protocols facilitated by international cooperative groups. The genetic landscape has also recently been characterized, providing an insight into the molecular pathogenesis underlying DS-associated leukemia. These studies emphasize the key role of trisomy 21 in priming a developmental stage and cellular context susceptible to transformation, and have unveiled its cooperative function with additional genetic events that occur during leukemia progression. Here, using DS-leukemia as a paradigm, we aim to integrate our current understanding of the role of trisomy 21, of critical dosage-sensitive chromosome 21 genes, and of associated mechanisms underlying the development of hematological malignancies. This review will pave the way for future investigations on the broad impact of gain of chromosome 21 in hematological cancer, with a view to discovering new vulnerabilities and develop novel targeted therapies to improve long term outcomes for DS and non-DS patients.</p>", "<title>Subject terms</title>" ]
[ "<title>Clinical features, therapy, and outcome of DS-associated leukemia</title>", "<title>Clinical features</title>", "<p id=\"Par5\">Common clinical features seen in individuals with DS are intellectual disability, congenital heart defects, Alzheimer’s disease and immunodeficiency, among many others [##REF##12699967##8##]. Incidence and severity of these phenotypes can vary due to trisomy 21 itself (complete or segmental trisomy) and/or potential modifier genes that remain elusive to date. Individuals with DS have a unique pattern of malignancies compared with the general population, characterized by a decreased incidence of solid tumors in adults and predisposition to leukemia during childhood, predominantly myeloid leukemia (ML–DS, &gt;100-fold increased risk), which has a high prevalence of AMKL, and ALL (DS–ALL) [##REF##27031084##7##].</p>", "<p id=\"Par6\">Indicative of an intrinsic effect of trisomy 21 on hematopoiesis, almost all neonates with DS have quantitative and/or qualitative disorders of the myeloid compartment such as macrocytosis, dysplastic platelets, leukocytosis, and on average 4% blasts in the peripheral blood [##REF##24021668##11##]. Approximately 10% develop transient myeloproliferative disorder (TMD), which is classically defined by the presence of megakaryoblasts in the peripheral blood, liver and bone marrow [##REF##26835364##12##]. TMD spontaneously resolves within the first months of life, suggesting that mechanisms regulated by the fetal microenvironment maintain perturbed hematopoiesis. Up to 30% of children with DS who have classically defined TMD develop ML–DS before 5 years of age. This occurs through stepwise pathogenesis with an incremental acquisition of genetic alterations (including GATA1 mutations, see below). However, ~20% of neonates with DS have been identified as having ‘silent TMD,’ defined by a peripheral blast count of ≤10% and detection of a <italic>GATA1</italic> gene mutation by next generation sequencing [##REF##24021668##11##]. This discovery has implications for the population at risk of transforming to ML–DS. Children with ML–DS have remarkably good prognosis compared with non-DS children with acute myeloid leukemia (AML), with 5-years overall and event-free survival of 89–93% and 87–90% respectively [##REF##28389462##13##, ##REF##28400376##14##].</p>", "<p id=\"Par7\">Children with DS have a 27-fold increased risk of developing ALL [##REF##27031084##7##]. A recent study has emphasized that these are almost exclusively B-cell phenotype, with T-cell ALL identified in only 5 of 653 children with DS [##REF##24222333##15##]. In contrast to ML–DS, children with DS–ALL have an inferior outcome compared with non-DS children with ALL due to higher relapse rates, increased risk of infection, treatment-related mortality and induction failure [##REF##24222333##15##, ##REF##27161549##16##].</p>", "<title>Therapy and outcome</title>", "<p id=\"Par8\">Significant progress has been made in the treatment of children with DS and leukemia, with outcomes summarized in Table ##TAB##0##1##. Several studies have prospectively collected and published data on TMD in neonates with DS. These studies have been instrumental in identifying that the majority of patients undergo spontaneous remission, demonstrated a clear benefit for treating babies with high-risk features with low-dose cytarabine (which reduces TMD-related mortality but does not prevent progression to ML–DS) and indicated that persistence of minimal residual disease (MRD) can be used to predict risk for developing ML–DS [##UREF##1##17##–##REF##29959152##21##]. The next generation of trials should seek to uniformly define high-risk criteria for therapy, identify the optimal dose and schedule for cytarabine treatment and identify the molecular mechanisms that underpin progression to ML–DS. Given the rarity of the disease, consideration should be given to a unified international protocol, which will provide uniformity of outcomes and permit a greater number of questions to be answered.</p>", "<p id=\"Par9\">Prior to the 1980s, children with ML–DS were undertreated resulting in a high rate of treatment failure [##REF##19703908##22##]. ML–DS patients were subsequently registered on protocols used for non-DS AML. Increased survival was evident, with lower rates of induction failure and relapse, however treatment-related mortality was more frequent resulting in protocol adaptation to dose-reduce therapy or prolong the interval between chemotherapy courses [##REF##25231553##23##]. Over the last 20 years, children have been enrolled onto uniform ML–DS specific protocols (Table ##TAB##0##1##) [##REF##28389462##13##, ##REF##28400376##14##, ##REF##18048827##24##–##REF##26481183##27##]. These studies have been instrumental in highlighting the benefit for reduced-intensity ML–DS specific protocols, with outcomes for children with ML–DS significantly better than non-DS AML. Due to concerns regarding increased treatment-related toxicity in children with ML–DS [##REF##9193351##28##–##REF##26814618##30##], sequential protocols successfully reduced cumulative exposure to several agents including daunorubicin, etoposide, and intrathecal cytarabine without impacting on overall outcome [##REF##28389462##13##, ##REF##28400376##14##, ##REF##22392565##26##, ##REF##26481183##27##]. High-dose cytarabine was established as an important component of therapy, with early administration leading to improved outcomes [##REF##28389462##13##] and subsequent attempts to omit high-dose cytarabine in standard-risk patients resulting in significantly lower event-free survival [##UREF##2##31##]. Several studies have identified older age as an unfavorable independent prognostic feature [##REF##22392565##26##, ##REF##26481183##27##, ##REF##23935021##32##]. ML–DS diagnosed in children over 4 years of age has been shown to lack <italic>GATA1</italic> mutations and has a cytogenetic profile more akin to children with sporadic AML [##REF##18059480##33##]. Given that biologically the disease appears to represent sporadic AML occurring in children with DS rather than ML–DS, undertreatment on the less intensive ML–DS protocols may account for the adverse prognosis in this age group. As such, it has been suggested that children within this older age group may benefit from more intensive therapy corresponding to that given to children with sporadic AML [##REF##18059480##33##]. Monosomy 7 [##REF##18048827##24##], gain of chromosome 8 [##REF##28400376##14##], normal karyotype and high white blood cell count ≥20 × 10<sup>9</sup>/l at presentation [##REF##23935021##32##] have also been identified in individual reports as independent variables associated with an inferior outcome. Detection of MRD after induction therapy using both deep sequencing of GATA1 and flow cytometric methods has been identified as a significant prognostic factor for predicting relapse [##UREF##3##34##], with current trials implementing risk-adapted therapy according to MRD response (NCT02521493; jRCTs041190047). Future studies should prospectively establish whether additional clinical and biological features can be utilized in addition to MRD response assessment for risk-stratification, which may help further reduce therapy in low-risk patients without compromising outcome and enable treatment intensification for high-risk patients to prevent relapse.</p>", "<p id=\"Par10\">Children with DS–ALL are treated on standard ALL chemotherapeutic protocols [##REF##27285583##35##–##REF##31160295##38##]. Similar to ML–DS, sequential treatment protocols have identified that children with DS–ALL are more prone to treatment-related toxicity due to heightened sensitivity to chemotherapeutic agents, particularly methotrexate, and infectious complications, further contributing to their inferior outcome [##REF##27161549##16##]. Consequently, this has resulted in modification of treatment to reduce intensity and implementation of intensified supportive care measures for children with DS–ALL [##UREF##4##39##, ##REF##21933171##40##]. We are rapidly approaching a therapeutic plateau to which we can intensify conventional chemotherapeutic agents in order to balance the equilibrium between relapse and treatment-related toxicity, indicating the need to investigate novel agents in children with DS–ALL. Integration of the bispecific T-cell engager, blinatumomab, into the treatment backbone is being investigated in the current upfront COG (NCT03914625) and AIEOP-BFM (NCT03643276) studies and children with high-risk DS–ALL who are MRD positive at the end of consolidation are eligible for the single arm, phase 2 study of tisagenlecleucel (NCT03876769).</p>", "<p id=\"Par11\">Outcomes for relapsed/refractory leukemia in children with DS are extremely poor [##REF##28389462##13##, ##REF##23594030##41##, ##REF##22776818##42##]. Stem cell transplantation has been associated with high rates of relapse and treatment-related mortality in children with DS [##REF##8879614##43##–##REF##24391118##45##]. CAR T-cell therapy may provide an alternative option to stem cell transplantation in this setting. Children with relapsed/refractory DS–ALL have been included in studies using tisagenlecleucel, with preliminary results identifying comparable safety and efficacy to children without DS [##REF##28912293##46##]. Such findings highlight the importance of including children with DS in studies of new agents for the treatment of leukemia to identify innovative approaches to reduce rates of relapse and treat relapsed disease [##REF##30724029##47##]. Novel therapeutic strategies targeting the somatic events found in DS leukemia or the mechanisms altered by trisomy 21 as the initiating event in DS leukemogenesis should also be explored, which ultimately may be applicable to a broad spectrum of hematological malignancies that present with a similar genetic background.</p>", "<title>Genetic landscape of DS-associated leukemia</title>", "<p id=\"Par12\">Several studies have recently reported on the genetic landscape of DS-associated leukemia, providing new insights into leukemia development. Ongoing functional characterization of secondary alterations that have been identified may shed light on novel actionable targets that could be therapeutically exploited.</p>", "<title>The multi-step pathogenesis of ML–DS development</title>", "<p id=\"Par13\">Development of ML–DS, from predisposing trisomy 21 to pre-leukemia TMD to frank ML–DS, has been considered a model of sequential acquisition of secondary alterations for many years.</p>", "<p id=\"Par14\">Discovery of <italic>GATA1</italic> mutations in nearly all patients with TMD and ML–DS was the first major breakthrough in our understanding of ML–DS [##REF##12172547##48##–##REF##12747884##50##]. The transcription factor GATA1 is a master regulator of erythroid and megakaryocytic lineages. In TMD/ML–DS, <italic>GATA1</italic> mutations are almost exclusively localized in exon 2 (97% of cases) and are predominantly insertions, deletions or duplications [##REF##21715302##51##]. All <italic>GATA1</italic> mutations lead to the appearance of a premature stop codon. Consequently, a ‘short’ GATA1 isoform (GATA1s), truncated from its amino-terminal transactivation domain is expressed. Identical <italic>GATA1</italic> mutations are seen in TMD and at progression to ML–DS, regardless of whether they are present in the major clone [##REF##21715302##51##–##UREF##6##55##]. The majority of these clones have leukemia-initiating and self-renewal potential [##REF##20220775##56##, ##REF##23482930##57##]. To date, the type of <italic>GATA1</italic> mutation, the level of <italic>GATA1s</italic> expression, the TMD blast karyotype and the size of dominant GATA1-bearing clones have not been shown to predict progression from TMD to ML–DS [##REF##21715302##51##, ##REF##24056718##54##, ##UREF##6##55##]. However, features identified as being predictive of progression include the persistence of immunophenotypic TMD blasts (&gt;0.1%) or detection of <italic>GATA1</italic> mutations by quantitative PCR using patient-specific oligonucleotides at week 12 and the presence of pleural effusions at diagnosis of TMD [##REF##18182574##19##, ##REF##29959152##21##].</p>", "<p id=\"Par15\">Clonal evolution from TMD to ML–DS has been linked to acquisition of several secondary chromosomal and genetic alterations. There is a lower incidence of CNVs and chromosomal translocations in ML–DS compared with non-DS children with AML; the most common cytogenetic alterations being +8, dup(1q), and a fourth chromosome 21 [##REF##17971484##4##, ##REF##22122069##5##]. Next generation sequencing experiments have uncovered the mutational spectrum of TMD/ML–DS [##REF##24056718##54##, ##UREF##6##55##, ##REF##23733339##58##], revealing <italic>GATA1</italic> mutations as the only somatic mutations seen in TMD in most cases. During progression to ML–DS, two to five additional mutations are found. Among them, the most frequently altered genes encode signaling effectors (JAK1/2/3, MPL, RAS) in 56% of cases, members of the cohesin complex or associated components (STAG2, RAD21, SMC1A) in 48%, and epigenetic regulators (EZH2, SUZ12, and BCOR) in 38% (Fig. ##FIG##0##1a##). A novel clonal gain of function mutation affecting the <italic>CSF2RB</italic> gene, encoding the common beta chain of the IL3, IL5, and CSF cytokine receptors, was recently identified in almost 5% of patients with ML–DS and was mutually exclusive with <italic>JAK1-3</italic>, <italic>MPL</italic> or <italic>RAS</italic> mutations [##UREF##6##55##]. In ML–DS, somatic variants in signaling effectors and cohesin complex components are likely clonal and frequently co-occur, indicative of a potential cooperative effect in trisomic <italic>GATA1s</italic>-expressing hematopoietic progenitors [##REF##24056718##54##, ##UREF##6##55##]. To date, nearly 75–80% of patients with ML–DS have been shown to harbor secondary alterations, emphasizing the model of progression from the pre-leukemic TMD stage. The molecular bases of clonal evolution for the remainder remains obscure to date.</p>", "<p id=\"Par16\">Numerous in vitro and in vivo assays have reported on the role of somatic alterations found in TMD/ML–DS. <italic>GATA1s</italic> expression has been shown to promote megakaryoblastic progenitor expansion during fetal life but does not to lead to ML–DS [##REF##15895080##59##]. Most of the somatic alterations found in ML–DS are shared with non-DS leukemia, and have been shown to promote self-renewal, differentiation blockade, proliferation and survival of hematopoietic stem cells (HSC) and/or myeloid progenitor populations [##REF##26186939##60##, ##REF##31552185##61##]. To assess leukemia progression from <italic>GATA1s</italic>-expressing cells without trisomy 21, models of oncogenic cooperation have been developed (retroviral insertional mutagenesis, ectopic expression and ‘loss of function screening’ using a CRISPR/Cas9 strategy and transgenic models) [##UREF##6##55##, ##REF##20679399##62##, ##REF##22354171##63##]. On average, 2.7 alterations on top of <italic>Gata1s</italic> expression was sufficient for the development of an erythro-megakaryoblastic leukemia in vivo, resembling ML–DS but strongly biased toward the erythroid lineage (CD117+Ter119+ population) [##UREF##6##55##]. The most common alterations found in these recipients were variants found in signaling effectors (73.7%) and epigenetic regulators (79%). The loss of function mutations in genes encoding components of the cohesin complex were underrepresented (16% in murine recipients vs 48% in patients with ML–DS). Whether this is due to human versus murine differences, or indicative of the lack of trisomy 21 in this model remains unknown.</p>", "<p id=\"Par17\">In summary, observations in humans and mice have emphasized the essential role of trisomy 21, the fetal hematopoietic context of <italic>GATA1s</italic> expression and the requirement of additional genetic events predominantly affecting signaling effectors, epigenetic regulators and cohesin complex components to drive progression toward ML–DS.</p>", "<title>Somatic alterations found in DS–ALL</title>", "<p id=\"Par18\">DS–ALL is a heterogeneous subtype of B-ALL that has a distinct cytogenetic profile compared with other types of childhood B-ALL (non-DS–ALL). Approximately 40% of DS–ALL cases have a normal karyotype other than the constitutional trisomy 21, 8–10% express the ETV6-RUNX1 fusion transcript, while 9–11% have high hyperdiploidy (HeH) [##REF##17971484##4##, ##REF##24222333##15##]. The most frequent CNV in DS–ALL is gain of chromosome X (38% vs 21% in non-DS–ALL); a twofold increase of del(9p) is also found in DS–ALL [##REF##17971484##4##]. DS–ALL also has a higher proportion of rearrangements affecting the <italic>CRLF2</italic> locus (~50% in DS–ALL vs 4–5% in non-DS–ALL), with interstitial deletion of the pseudoautosomal region PAR1 (that fuses <italic>CRLF2</italic> to the first non-coding exon of <italic>P2RY8</italic>) or chromosomal translocation to the immunoglobulin heavy chain (IgH) locus [##REF##19641190##64##–##REF##19965641##66##]. These rearrangements lead to overexpression of the <italic>CRLF2</italic> gene, encoding a protein that heterodimerizes with IL7RA to form the thymic stromal lymphopoietin receptor. To date, there are no fusion proteins uniquely found in DS–ALL [##REF##28461505##67##, ##REF##31385395##68##].</p>", "<p id=\"Par19\">At the gene level, <italic>JAK2</italic> activating mutations are present in 20–40% of patients with DS–ALL and predominantly affect the arginine 683 residue, located in the pseudokinase domain of JAK2 [##REF##18805579##69##] (Fig. ##FIG##0##1b##). These <italic>JAK2</italic> mutations are virtually always found in <italic>CRLF2</italic>-overexpressing cases, indicative of a mechanism of oncogenic cooperation, as shown in experimental models [##REF##19838194##65##, ##REF##19965641##66##, ##REF##20018760##70##]. In non-<italic>JAK2</italic> mutated or non-<italic>CRLF2</italic>-overexpressing cases, gain of function mutations are found in <italic>CRLF2</italic> or <italic>IL7RA</italic> genes, strongly implicating cytokine signaling as a major transforming process in DS–ALL [##REF##19965641##66##, ##REF##20018760##70##, ##REF##21536738##71##]. Activating mutations affecting other signaling effectors, such as NRAS, KRAS, KIT, FLT3, and PTPN11, are also frequently found and are often mutually exclusive with <italic>JAK2</italic> mutations, highlighting the high incidence (&gt;60%) of constitutively active signaling pathways in DS–ALL [##REF##28461505##67##, ##REF##25105841##72##]. In line with these findings, a recent study revealed enrichment of a Philadelphia-like transcriptional signature in DS–ALL [##REF##31385395##68##]. Other common genetic abnormalities found in DS–ALL affect genes encoding cell cycle regulators (CDKN2A/B, RB1), transcription factors (PAX5, IKZF1, and ETV6) and epigenetic modifiers (EZH2, SETD2, and CREBBP) (Fig. ##FIG##0##1b##). Of interest, a recent genome-wide association meta-analysis explored inherited genetic susceptibility to ALL in children with DS, highlighting increased germline penetrance of the rs3731249 <italic>CDKN2A</italic> risk locus (9p21.3) [##REF##31350265##73##].</p>", "<p id=\"Par20\">To our knowledge, there is only one in vivo model of DS–ALL [##REF##24747640##74##] and several other models assessing the cooperation between secondary mutations commonly found in DS–ALL. One study hypothesized that loss of <italic>USP9X</italic> (seen in 4/17 <italic>CRLF2</italic>-rearranged cases), which encodes a deubiquitinase known to stabilize activated JAK2, may naturally buffer the toxic effect of JAK/STAT signaling hyperactivation in DS–ALL [##REF##28461505##67##]. In addition, activating mutations in signaling effectors have been shown to functionally cooperate with loss of <italic>Ink4a/Arf</italic> and <italic>Pax5</italic> alterations to drive B-cell leukemia development in vivo [##REF##29296886##75##, ##REF##30257940##76##].</p>", "<p id=\"Par21\">Together, these studies unravel the high genetic complexity observed in DS–ALL, raising critical questions regarding the role of somatic alterations in the context of trisomy 21 in leukemia development. New models of DS–ALL will be required in the future to better understand the impact of trisomy 21, the mechanisms of cooperation and develop novel targeted therapies to improve outcome.</p>", "<title>Constitutive trisomy 21 as a ‘priming’ and cooperating event</title>", "<p id=\"Par22\">Constitutive trisomy 21 (T21) results in altered hematopoiesis affecting many lineages, is developmental stage selective, but is not sufficient to lead to leukemia. Studying embryonic, fetal and adult hematopoiesis in individuals with DS and from genetically engineered models has unraveled the cellular and molecular bases of DS-leukemogenesis, and allowed identification of chromosome 21 dosage-sensitive genes.</p>", "<title>Insights from human DS fetal hematopoiesis</title>", "<p id=\"Par23\">Compared with gestation-matched controls, analyses of second trimester human DS fetal livers revealed that trisomy 21 disturbs fetal hematopoiesis [##REF##18812473##77##–##REF##23045701##79##]. There is an increased proportion of HSC and of megakaryocyte-erythroid progenitors (MEP) in T21 fetal livers, at the expense of granulocyte-monocyte progenitors (GMP) and committed B progenitors. This bias toward the erythro-megakaryocytic lineage has been confirmed by clonogenic assays, liquid cultures, transplantation assays in immunodeficient mice and transcriptional analyses. A recent study showed that engineered <italic>GATA1s</italic> expression in human fetal liver HSC cooperates with trisomy 21 to promote blast and megakaryocyte expansion in xenotransplantation models [##UREF##7##80##]. The marked impairment of B lymphopoiesis in T21 fetal livers is also associated with a reduced proportion of both preproB (CD34+CD19+CD10−) and proB (CD34+CD19+CD10+) cells, and a decreased expression of early lymphoid or key lymphoid specific genes (such as <italic>IKZF1</italic>, <italic>FLT3, PAX5</italic>, and <italic>IL7RA</italic>) in early lymphoid progenitors [##REF##23045701##79##]. Importantly, this reduced B-cell progenitor compartment is also seen in T21 fetal bone marrow, and is associated with impaired B-cell differentiation potential in vitro that may be linked to inflammatory signatures driven by the trisomic microenvironment [##UREF##8##81##].</p>", "<p id=\"Par24\">This perturbed fetal hematopoiesis may provide insight into the cellular context of susceptibility for transformation of the megakaryocytic and B-cell lineages. However, several questions remain regarding the dynamic changes of these phenotypes during gestation before and after the second trimester, if they are maintained postnatally and throughout the life of individuals with DS, and how they may be affected by their microenvironment (yolk-sac, fetal liver and fetal and adult bone marrow).</p>", "<title>Insights from human DS-induced pluripotent stem cells</title>", "<p id=\"Par25\">Through the establishment of DS-derived induced pluripotent stem cells (iPSCs), independent studies have shown that trisomy 21 alone significantly accelerates the early stages of hematopoiesis [##REF##23045682##82##–##REF##27134169##85##]. Regardless of whether primitive (yolk-sac type) or definitive (fetal liver type) hematopoiesis was induced, all groups reported an increase in the clonogenic potential of erythroid progenitors and enhanced erythroid differentiation. However, in contrast to primitive-like hematopoietic progenitors [##REF##23045704##83##], mimicking fetal hematopoiesis from trisomic iPSCs led to multi-lineage expansion compared with disomic cells, characterized by significant expansion of myeloid and megakaryocytic progenitors in colony-forming unit (CFU) assays [##REF##23045682##82##, ##REF##30518921##84##, ##REF##27134169##85##]. These observations were confirmed by silencing one chromosome 21 in DS-derived iPSCs using an inducible XIST strategy [##REF##30518921##84##]. Extensive analyses of trisomic/GATA1s iPSCs, established from primary patient TMD samples or engineered by genome editing, have shown that <italic>GATA1s</italic> expression correlates with defective embryonic erythropoiesis and confers a strong bias toward the myelo-megakaryocytic compartment, indicating that both events cooperate in the development of TMD during fetal life [##REF##27134169##85##, ##REF##25621499##86##].</p>", "<p id=\"Par26\">A recent study reported that trisomic CD34+ hematopoietic progenitors derived from embryoid bodies also have decreased ability to generate CD19+ B cells compared with isogenic controls [##REF##29789608##87##]. This phenotype has been linked to reduced expression of endothelin signaling, and provides potential explanation for the impaired B-cell differentiation in human trisomic fetal livers [##REF##23045701##79##], and for the decreased number of circulating B cells in individuals with DS [##REF##25472482##88##].</p>", "<p id=\"Par27\">Overall, these findings suggest that trisomy 21 perturbs early hematopoiesis to create permissive cellular contexts, for the development of both ML–DS and DS–ALL, through the acquisition of additional genetic alterations.</p>", "<title>Partially trisomic mice as models to study DS-leukemia</title>", "<p id=\"Par28\">In the mouse genome, syngeneic regions of human chromosome 21 (Hsa21) are located on the three murine chromosomes (Mmu) 16, 17, and 10. Several partially trisomic murine models, containing some or all these syngeneic regions, have been used to study the impact of trisomy 21 on hematopoiesis, alone or in cooperation with <italic>GATA1s</italic> expression. The phenotypes of these models have been extensively reviewed [##REF##26835364##12##, ##REF##19139078##89##]. Here, we will briefly describe the phenotypes of murine transgenic DS models that have been used to define a minimal trisomic region and identify Hsa21 dosage-sensitive genes.</p>", "<p id=\"Par29\">Ts65Dn is the most commonly used model to understand the phenotypes associated with DS [##REF##2147289##90##]. This strain contains 104 trisomic genes, all located on Mmu16. Ts65Dn mice display an increased number of HSC and GMP, as well as decreased MEP during adulthood [##REF##17901249##91##]. These mice develop progressive myeloproliferative disorder characterized by megakaryocytic hyperplasia, thrombocytosis, and myelofibrosis in the bone marrow and spleen. Ts65Dn mice also display a decreased proportion of common lymphoid progenitors associated with a lower level of IL7 receptor expression [##REF##21504363##92##].</p>", "<p id=\"Par30\">The Dp(16); Dp(17); Dp(10) strain is the only model trisomic for all syngeneic regions of human chromosome 21. These mice display macrocytic anemia and also develop myeloproliferative disorder; these phenotypes are conserved in the Dp(16) model alone [##REF##29435140##93##], strongly suggesting that the minimal region implicated in DS-myeloid disorders is contained within Mmu16. In contrast to the Ts65Dn strain, 15-month-old Dp(16); Dp(17); Dp(10) mice have an increased percentage of MEP and a decreased percentage of GMP in the bone marrow. The reason for these differences are not known to date but may result from differences in strain and/or modifier genes.</p>", "<p id=\"Par31\">The Cre/LoxP system has been used to develop trisomy of genes contained in the Down syndrome critical region (DSCR): creating the.... Ts1Rhr model [##REF##15499018##94##]. The DSCR was triplicated on Mmu16 from the CBR1 to FAM3B genes and contains 31 protein coding genes and 2 antisense RNAs [##REF##15499018##94##]. The Ts1Rhr model has been extensively used to assess the role of trisomy of the DSCR in leukemia predisposition and development. Triplication of these 33 regulatory elements alone has no major effect on fetal hematopoiesis, apart from a significant increase in phenotypic HSC. Adult Ts1Rhr mice develop a phenotype similar to the Ts65Dn strain, indicating that trisomy of the DSCR is the minimally required region associated with these myeloid phenotypes [##REF##22354171##63##]. Expression of <italic>Gata1s</italic> in Ts1Rhr led to increased size of CFU-megakaryocyte colonies and transient thrombocytosis mimicking features of TMD. Reproducing the multi-step pathogenesis seen in patient samples by adding a third event in the Ts1Rhr/<italic>Gata1s</italic> model has provided insight on the role of trisomy 21 in TMD/ML–DS development. First, endogenous expression of <italic>JAK3</italic> activating mutations enhance a TMD phenotype during fetal hematopoiesis alone, reinforcing the concept of developmental stage selectivity [##REF##29986854##95##]. Moreover, bone marrow transplantation assays revealed that MPL<sup>W515L</sup> overexpression functionally cooperates with Gata1s and Ts1Rhr to drive megakaryocytic hyperplasia presenting with phenotypic features of DS–AMKL [##REF##22354171##63##].</p>", "<p id=\"Par32\">The Ts1Rhr strain has also been used to assess the impact of trisomy 21 on B-cell lineage. Compared with wild-type littermates, trisomic mice display a decreased proportion of bone marrow B220+CD43+ early B-cell progenitors, especially Hardy’s fractions B and C (proB cells), and increased clonogenic potential of CFU-preB colonies [##REF##24747640##74##]. Similar to the megakaryocytic lineage, these mice will not spontaneously develop B-cell leukemia and four additional events found in DS–ALL samples (<italic>CRLF2</italic> overexpression, Jak2<sup>R683G</sup>, <italic>Pax5</italic> haploinsufficiency and expression of the dominant negative Ikaros isoform IK6) are required to drive a B-ALL phenotype, although not to full penetrance [##REF##24747640##74##]. As a surrogate, Ts1Rhr was shown to cooperate with p210 BCR-ABL overexpression to develop B-ALL in vivo with shorter latency and complete penetrance, thus demonstrating the impact of trisomy of the DSCR in B-cell leukemogenesis.</p>", "<p id=\"Par33\">In summary, the use of partially trisomic murine models clearly emphasize the role of trisomy 21 in leukemia predisposition and development. While these models can be limited by potential differences between species, they provide the relevant genetic background to study fetal, neonatal and adult hematopoiesis, to investigate the impact of the microenvironment on leukemia predisposition and progression, to identify specific chromosome 21 genes and assess their cooperation with secondary mutations found in human DS-leukemia samples.</p>", "<title>Dosage-sensitive Hsa21 genes and mechanisms altered by trisomy 21 in DS-leukemia</title>", "<p id=\"Par34\">The combination of genetically engineered iPSCs, partially trisomic animal models and characterization of cases with segmental trisomy 21 in patients with DS-leukemia, have been instrumental in identifying dosage-sensitive genes implicated in leukemia predisposition and development in children with DS (Fig. ##FIG##1##2## and Table ##TAB##1##2##) [##REF##19597142##96##, ##REF##26138905##97##].</p>", "<title>Transcription factors</title>", "<p id=\"Par35\">The chromosome 21 <italic>ERG</italic> oncogene, which encodes a transcription factor from the E-twenty-six (ETS) family, is overexpressed in DS and de novo AMKL [##REF##16140924##98##]. In mice, <italic>ERG</italic> overexpression promotes the expansion of fetal megakaryocytic progenitors, cooperates with <italic>Gata1s</italic> expression and leads to AMKL in vivo [##REF##19487285##99##–##REF##23719302##101##]. Moreover, the loss of one copy of <italic>Erg</italic> (ERG<sup>mld2</sup> mice) reverts the myeloproliferative phenotype seen in the Ts65Dn model [##REF##20007548##102##]. Possible mechanisms of cooperation between ERG and the secondary alterations found in DS-leukemia include increased chromatin accessibility for ERG (along with RUNX1; another transcription factor encoded by chromosome 21), through the alteration of cohesin complex components [##REF##26607380##103##], and molecular interplay between ERG and the RAS/MAPK pathway, in which ERG induces the transcriptional signature of RAS/MAPK activation and RAS/MAPK regulates ERG activity [##REF##23974202##104##, ##REF##27055868##105##]. Whether this feed-forward loop participates in leukemia development and maintenance in both ML–DS and DS–ALL remains to be investigated. A study using genetically engineered DS-iPSCs suggests that trisomy of <italic>ERG</italic>, together with trisomy of <italic>ETS2</italic> and <italic>RUNX1</italic>, enhances early hematopoiesis and cooperates with <italic>GATA1s</italic> expression [##REF##27134169##85##].</p>", "<title>Signaling effectors</title>", "<p id=\"Par36\">Trisomy of <italic>DYRK1A</italic> has been shown to promote TMD/DS–AMKL development in human and murine models [##REF##22354171##63##]. <italic>DYRK1A</italic> encodes the dual-specificity tyrosine phosphorylation regulated kinase 1A; a kinase that has multiple targets, thus regulating diverse functions in a cellular context-dependent manner [##REF##27567487##106##]. In murine cells, increased dosage of <italic>Dyrk1a</italic> cooperates with <italic>Gata1s</italic> expression to increase megakaryocytic expansion through inhibition of the calcineurin/NFAT pathway [##REF##22354171##63##]. Another chromosome 21 gene, <italic>RCAN1</italic> (also known as <italic>DSCR1</italic>), encodes a negative regulator of the NFAT pathway, that also contributes to megakaryopoiesis [##REF##23446734##107##]. Since increased dosage of both <italic>DYRK1A</italic> and <italic>RCAN1</italic> cooperate to inhibit neo-angiogenesis in solid tumor development through calcineurin/NFAT pathway inhibition [##REF##19458618##108##], it may be reasonable to assume that a similar additive effect contributes to ML–DS.</p>", "<p id=\"Par37\">Interestingly, <italic>Dyrk1a</italic> has also been shown to regulate B lymphopoiesis [##REF##26008897##109##]. Genetic disruption or pharmacological inhibition of <italic>Dyrk1a</italic> completely abolished CFU-preB colony formation. Dyrk1a also controls the transition between proliferative large preB to quiescent small preB, by triggering cyclin D3 degradation required to exit the cell cycle. Together, these phenotypes indicate that trisomy of <italic>DYRK1A</italic> may promote B-cell leukemia and warrants further investigation.</p>", "<p id=\"Par38\">Over-activation and hypersensitivity to interferon (IFN) signaling, resulting from increased expression of IFN-related genes located on Hsa21 (including <italic>IFNAR1</italic>, <italic>IFNAR2</italic>, <italic>IFNGR2</italic> and <italic>IL10RB</italic>) outside of the DSCR region, has been observed in multiple cell types in individuals with DS [##UREF##9##110##]. During adulthood, individuals with DS display a perturbed immune system, consistent with a state of chronic inflammation [##REF##29093484##111##, ##UREF##10##112##]. IFN and inflammatory response transcriptional signatures have been recently observed in murine and human trisomic hematopoietic progenitors, as well as in DS–ALL samples [##UREF##8##81##] (Laurent A. and Malinge S., unpublished observations), and may be partly mediated by the microenvironment. These observations emphasize the link between IFN signaling, inflammation and immune deficiency in DS. Whether this is implicated in DS–ALL development and reflects the higher rate of B-cell leukemia in children with DS by increasing the risk of infections, which has been suggested as a causal factor for childhood ALL [##REF##26408659##113##, ##REF##29784935##114##], is a promising area for investigation. Moreover, since interferon α signaling has an anti-proliferative effect on DS-associated myeloid disorders in adult bone marrow but not during fetal life [##UREF##11##115##], this potential interplay between IFN signaling and DS-associated leukemia may also be time and spatially dependent.</p>", "<title>Epigenetic regulators</title>", "<p id=\"Par39\"><italic>HMGN1</italic> encodes the high mobility group nucleosome-binding protein N1, which modulates accessibility of the histone H3 tail to other epigenetic regulators. Decreased expression or loss of one copy of <italic>Hmgn1</italic> reverts the CFU-preB colonies seen in the Ts1Rhr model, indicative of its key role in leukemia predisposition [##REF##24747640##74##, ##REF##30428356##116##]. Mechanistically, increased dosage of <italic>Hmgn1</italic> increases global H3K27ac and is associated with upregulation of B-cell specific transcriptional signatures [##REF##30428356##116##], which are conserved in DS–ALL and normally associated with a H3K27me3 repressive mark in non-trisomic samples. Another chromatin associated protein encoded by chromosome 21 is CHAF1B, a component of the CAF-1 complex known to drive the first step of nucleosome formation after replication. <italic>CHAF1B</italic> has been shown to be overexpressed in AML, where it maintains the leukemic cells in an undifferentiated state by interfering with the occupancy of the regulator of myeloid differentiation CEBPA [##REF##30423293##117##]. <italic>CHAF1B</italic> is also overexpressed in DS–AMKL [##REF##22354171##63##], but whether it has a similar extra-canonical function in DS-associated leukemia remains elusive to date.</p>", "<title>Other mechanisms</title>", "<p id=\"Par40\">Chromosome 21 encodes microRNA (miR) and alters several other mechanisms that may impact leukemia development in children with DS. miR-125b-2 is overexpressed in several leukemia subtypes including DS–AMKL and B-ALL, but not in DS–ALL [##REF##20194440##118##, ##REF##19890372##119##]. miR-125b-2 overexpression has been shown to enhance proliferation and self-renewal of megakaryocytic progenitors and synergize with <italic>Gata1s</italic> expression to enhance the DS–AMKL phenotype [##REF##20194440##118##].</p>", "<p id=\"Par41\">Trisomy 21 has also been associated with DNA hypomethylation in TMD/ML–DS, with downregulation of endothelin signaling and over-activity of insulin-like growth factor (IGF) signaling in DS-leukemia primary patient samples, iPSCs and murine models [##REF##20679399##62##, ##REF##30518921##84##, ##REF##29789608##87##, ##REF##23980066##120##], although the chromosome 21 genes and the underlying mechanisms associated with these molecular features remain largely unknown.</p>", "<title>Gain of chromosome 21 in non-DS leukemia</title>", "<p id=\"Par42\">As a somatic event, +21 is one of the most common alterations in hematological cancer (Table ##TAB##2##3##). Whether findings associated with +21 in DS-leukemia can be extrapolated to other subtypes of leukemia with +21 remains to be seen. Here, we present several subtypes of non-DS leukemia or blood cancer harboring +21, to consider whether studying leukemogenesis in children with DS can have broader application.</p>", "<title>Gain of chromosome 21 in pediatric non-DS AMKL</title>", "<p id=\"Par43\">Analyses of large cohorts have revealed that +21 is often seen in AMKL, occurring most frequently in children with AMKL [##REF##22122069##5##]. In de novo AMKL, trisomy 21 has been found in all cytogenetically defined subtypes: ETO2-GLIS2, OTT-MAL, NUP98-KDM5A, HOX rearranged and Other [##REF##28112737##121##]. Strikingly, 9.2% of childhood AMKL has a genetic background similar to DS–AMKL (DS–AMKL-<italic>like</italic>: i.e. alterations in genes encoding GATA1, cohesin complex components and signaling effectors together with acquired trisomy 21), with excellent outcome also seen for this subgroup of non-DS children [##REF##28112737##121##]. DS–AMKL-<italic>like</italic> development has been reported in a patient with Cornelia de Lange syndrome, characterized by a mutation in <italic>NIPBL</italic> (encoding a cohesin complex component), with acquired trisomy 21, a <italic>GATA1</italic> mutation, along with EZH2 and JAK/RAS alterations [##REF##29217785##122##]. Moreover, underlying the impact of +21 in TMD development, rare TMD-<italic>like</italic> cases (occurrence of TMD in children without DS) have been described [##REF##18989165##123##–##REF##25266042##125##]. These studies on TMD/DS–AMKL-<italic>like</italic> disorders not only emphasize the cooperative role of trisomy 21, but also raise the question regarding the order of acquisition of these somatic events. Whether trisomy 21 is a founder alteration that is required to ‘prime’ the cellular context susceptible for <italic>GATA1s</italic> expression as highlighted in DS–TMD, or whether +21 cooperates with other genetic alterations in non-DS leukemia, regardless of whether it is an early or late event, remains unknown.</p>", "<title>Gain of chromosome 21 in pediatric B-ALL</title>", "<p id=\"Par44\">B-ALL is the most common type of childhood cancer, and accounts for approximately two-thirds of all pediatric acute leukemia. Complete or partial gain of chromosome 21 is one of the most frequent chromosomal alterations in childhood B-ALL, found in nearly 30% of cases compared with ~11% in adults. It is predominantly seen in HeH, which comprises 25–30% of all pediatric B-ALL, where more than 90% of cases harbor between one and three additional copies of whole chromosome 21, with the majority being tetrasomic [##REF##21098271##126##]. Intra-chromosomal amplification of chromosome 21 (iAMP21) occurs in 2% of pediatric B-ALL. iAMP21 is characterized by a rearranged chromosome 21 patterned with amplified and deleted genomic regions [##REF##24166298##127##, ##REF##24670643##128##]. As in DS–ALL, there is a high incidence of genetic alterations affecting signaling effectors (NRAS, KRAS, FLT3, and SH2B3) in HeH and iAMP21 subtypes [##REF##25917266##129##–##UREF##12##131##]. However, the molecular bases of this possible oncogenic cooperation are currently unknown. The observation that a minimal region of amplification on chromosome 21, found in both HeH and iAMP21 and overlapping with the DSCR [##REF##21098271##126##, ##REF##24670643##128##, ##REF##21527530##132##] (Fig. ##FIG##1##2##), indicates that increased dosage of specific chromosome 21 genes located in this region, regardless of whether they are amplified by a constitutive or somatic alteration, may play a role in B-cell leukemogenesis.</p>", "<title>Gain of chromosome 21 in other hematological malignancies</title>", "<p id=\"Par45\">Trisomy 21 is found in 4–5% of AML overall (8.5% in children and 3.9% in adults) [##REF##20439644##133##, ##REF##20385793##134##] (Table ##TAB##2##3##). This incidence may be slightly underestimated since focal amplifications of specific regions of chromosome 21, such as band 21q22 that contains the <italic>ERG</italic> oncogene, are also found in AML [##REF##15007164##135##–##REF##27686867##137##]. In human and animal models, <italic>ERG</italic> overexpression leads to the development of lymphoid and myeloid leukemia, and promotes a stem cell and progenitor signature [##REF##22936051##138##, ##REF##25306899##139##]. This may provide a susceptible context for oncogenic cooperation with additional alterations that have been found in primary patient samples (cohesin complex components and RAS/MAPK signaling). Overexpression of the chromosome 21 gene <italic>CHAF1B</italic> has been shown to maintain a stem cell signature in murine AML models [##REF##30423293##117##]. Whether other genes located in the 21q22 region are involved in AML development or maintenance remains unknown.</p>", "<p id=\"Par46\">Complete or partial gain of chromosome 21 is also frequently seen in adult hematological cancers such as lymphoma of B or T-cell origin (Table ##TAB##2##3##). Trisomy 21 has been shown to be an independent risk factor in follicular lymphoma [##REF##30417943##140##]. Gain of chromosome 21 is seen in 8.5% of cutaneous T-cell lymphoma (CTCL) [##REF##26551667##141##, ##REF##26192916##142##], and its role in CTCL development has been assessed in vivo in cooperation with <italic>JAK3</italic> activating mutations, through establishment of a trisomic murine model of CTCL [##REF##29986854##95##]. However, the chromosome 21 genes involved in these disorders and the associated molecular mechanisms require further investigation.</p>", "<title>Perspective</title>", "<p id=\"Par47\">A significant number of studies have been reported over the last decade. From the clinical aspect, we are now reaching a chemotherapeutic plateau and new therapies are required to further improve outcome for DS–ALL and relapsed ML–DS. Studying the molecular basis of leukemia predisposition and development in children with DS has been instrumental in dissecting the role of trisomy 21 on hematopoiesis, both alone and in cooperation with other genetic events, and led to the identification of several key dosage-sensitive chromosome 21 genes. Integration of fundamental research will provide new actionable targets to improve the outcome for children with DS. However, several important questions remain: Are the same chromosome 21 genes/mechanisms implicated in leukemia predisposition, development and maintenance? Does +21 have a role in response to treatment? Could we therapeutically target chromosome 21 proteins or the mechanisms of cooperation between +21 and somatic alterations? As +21 is frequently seen in non-DS-associated blood cancer, pursuing study of +21 in DS-leukemia may have high translational potential to ultimately provide clinical benefit for many patients with hematological malignancies.</p>" ]
[ "<title>Acknowledgements</title>", "<p>APL was supported by Cancéropôle Ile-de-France and Fondation pour la Recherche Médicale. RSK is supported by a Fellowship from the National Health and Medical Research Council of Australia (NHMRC APP1142627) and the Children’s Leukaemia and Cancer Research Foundation (CLCRF, Australia). SM is supported by the Children’s Leukaemia and Cancer Research Foundation (CLCRF, Australia), Fondation Jérôme Lejeune (#1806, France), and by a Fellowship from Cancer Council Western Australia (CCWA).</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par48\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Somatic alterations found in DS-associated leukemia.</title><p><bold>a</bold> Four of the most common types of alteration found in ML–DS, in addition to constitutive trisomy 21. <bold>b</bold> Four of the most common types of alteration found in DS–ALL, in addition to constitutive trisomy 21.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Trisomy 21 and chromosome 21 genes in DS-leukemia.</title><p><bold>a</bold> Minimal region of amplification of chromosome 21 in TMD/ML–DS, HeH, iAMP21 and their overlap with the DSCR. <bold>b</bold> Known cellular functions altered by increased dosage of chromosome 21 genes associated with DS-leukemogenesis.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of clinical trials for children with Down syndrome and leukemia.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"11\"><bold>Transient myeloproliferative disorder (TMD)</bold></th></tr><tr><th>Group</th><th>Study</th><th>Year</th><th colspan=\"2\">Evaluable patients</th><th>SR (%)</th><th>TMD-related death (%)</th><th>Developed ML–DS (%)</th><th>EFS (%)</th><th>OS (%)</th><th>Reference</th></tr></thead><tbody><tr><td>POG</td><td>9481</td><td>1996–1999</td><td colspan=\"2\">47</td><td>89.4</td><td>–</td><td>17.0</td><td>–</td><td>–</td><td>[##REF##16469874##18##]</td></tr><tr><td>COG</td><td>2971</td><td>1999–2004</td><td colspan=\"2\">135</td><td>78.5</td><td>10.4</td><td>15.6</td><td>57 (3-year)</td><td>77 (3-year)</td><td>[##REF##21849481##20##]</td></tr><tr><td>BFM</td><td>AML–BFM studies</td><td>1993–2006</td><td colspan=\"2\">146</td><td>66.4</td><td>8.9</td><td>19.9</td><td>63 (5-year)</td><td>85 (5-year)</td><td>[##REF##18182574##19##]</td></tr><tr><td>BFM/DCOG</td><td>TMD07</td><td>2007–2015</td><td colspan=\"2\">102</td><td>–</td><td>4.9</td><td>16.7</td><td>72 (5-year)</td><td>91 (5-year)</td><td>[##REF##29959152##21##]</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"11\"><bold>M</bold><bold>yeloid leukemia (ML–DS)</bold></th></tr><tr><th>Group</th><th>Study</th><th>Year</th><th>Evaluable patients</th><th>Prior TMD</th><th>CR</th><th>TRM (%)</th><th>Relapses (%)</th><th>EFS (%)</th><th>OS (%)</th><th>Reference</th></tr></thead><tbody><tr><td>COG</td><td>2971</td><td>1999–2003</td><td>132</td><td>57</td><td>91/108</td><td>2.3</td><td>–</td><td>79 (5-year)</td><td>84 (5-year)</td><td>[##REF##22392565##26##]</td></tr><tr><td><p>Japanese Childhood AML</p><p>Cooperative Study Group</p></td><td>AML99</td><td>2000–2004</td><td>72</td><td>9</td><td>70/72</td><td>1.4</td><td>12.5</td><td>83.3 (4-year)</td><td>83.7 (4-year)</td><td>[##REF##18048827##24##]</td></tr><tr><td>JCCLSG</td><td>AML 9805</td><td>1998–2006</td><td>24</td><td>7</td><td>21/24</td><td>12.5</td><td>4.2</td><td>82.6 (5-year)</td><td>87.5 (5-year)</td><td>[##REF##21557456##25##]</td></tr><tr><td>JPLSG</td><td>AML-D05</td><td>2008–2010</td><td>72</td><td>35</td><td>69/72</td><td>1.4</td><td>13.9</td><td>83.3 (3-year)</td><td>87.5 (3-year)</td><td>[##REF##26481183##27##]</td></tr><tr><td>COG</td><td>AAML0431</td><td>2007–2011</td><td>204</td><td>63</td><td>177/202</td><td>1.0</td><td>6.9</td><td>89.9 (5-year)</td><td>93.0 (5-year)</td><td>[##REF##28389462##13##]</td></tr><tr><td>BFM/DCOG/NOPHO</td><td>ML–DS 2006</td><td>2006–2015</td><td>170</td><td>43</td><td>–</td><td>2.9</td><td>5.3</td><td>87 (5-year)</td><td>89 (5-year)</td><td>[##REF##28400376##14##]</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"11\"><bold>A</bold><bold>cute lymphoblastic leukemia (DS–ALL</bold>)<sup>a</sup></th></tr><tr><th>Group</th><th>Study</th><th>Year</th><th colspan=\"2\">Evaluable patients</th><th>CR</th><th>TRM (%)</th><th>Relapses (%)</th><th>EFS (%)</th><th>OS (%)</th><th>Reference</th></tr></thead><tbody><tr><td>French Leukaemia Registry</td><td>Registry</td><td>1990–2008</td><td colspan=\"2\">92</td><td>88/92</td><td>9.8</td><td>26.1</td><td>64.1 (5-year)</td><td>73.6 (5-year)</td><td>[##REF##26457881##143##]</td></tr><tr><td>PPLLSG</td><td>ALL IC-BFM 2002</td><td>2003–2010</td><td colspan=\"2\">41</td><td>–</td><td>9.8</td><td>19.5</td><td>–</td><td>86 (5-year)</td><td>[##REF##29040012##36##]</td></tr><tr><td>DFCI</td><td>DFCI 00-001 and DFCI 05-001</td><td>2000–2011</td><td colspan=\"2\">38</td><td>38/38</td><td>0.0</td><td>10.5</td><td>91 (5-year)</td><td>97 (5-year)</td><td>[##REF##29878490##37##]</td></tr><tr><td>CCG</td><td>CCG 1991</td><td>2000–2005</td><td colspan=\"2\">75<sup>b</sup></td><td>–</td><td>1.3</td><td>9.3</td><td>86.9 (10-year)</td><td>91.1 (10-year)</td><td>[##REF##31160295##38##]</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>List of Hsa21 genes that have a potential role in DS-leukemia.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Hsa21 genes</th><th>Known function in hematopoiesis/leukemogenesis</th><th>Reference</th></tr></thead><tbody><tr><td><italic>ERG</italic></td><td>Promotes megakaryoblastic expansion and cooperates with GATA1s in AMKL</td><td>[##REF##27134169##85##, ##REF##16140924##98##–##REF##23719302##101##]</td></tr><tr><td><italic>ETS2</italic></td><td>Cooperates with GATA1s to enhance early hematopoiesis and expansion of fetal megakaryocytic progenitors</td><td>[##REF##27134169##85##, ##REF##19168790##100##]</td></tr><tr><td><italic>RUNX1</italic></td><td>Cooperates with ERG, ETS2 and GATA1s to enhance early hematopoiesis</td><td>[##REF##27134169##85##]</td></tr><tr><td><italic>DYRK1A</italic></td><td>Promotes TMD/DS–AMKL development in human and murine models; Cooperates with GATA1s to increase megakaryocytic expansion; Controls CFU-preB colony formation and B-cell differentiation</td><td>[##REF##22354171##63##, ##REF##26008897##109##]</td></tr><tr><td><italic>RCAN1</italic></td><td>Promotes megakaryopoiesis, Inhibits NFAT pathway</td><td>[##REF##23446734##107##]</td></tr><tr><td><italic>HMGN1</italic></td><td>Increases H3K27ac, associated with upregulation of B-cell specific transcriptional signatures</td><td>[##REF##24747640##74##, ##REF##30428356##116##]</td></tr><tr><td><italic>CHAF1B</italic></td><td>Interferes with myeloid transcription factor CEBPA and maintains undifferentiated state of leukemic cells</td><td>[##REF##30423293##117##]</td></tr><tr><td>miR-125b-2</td><td>Enhances proliferation and self-renewal of megakaryocytic progenitors, Cooperates with GATA1s</td><td>[##REF##20194440##118##]</td></tr><tr><td>IFN-genes: <italic>IFNAR1, IFNAR2, IFNGR2</italic> and <italic>IL10RB</italic></td><td>Over-activated in DS blood cells and in fetal hematopoietic progenitors</td><td>[##UREF##8##81##, ##UREF##9##110##–##UREF##10##112##]</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Incidence of gain of chromosome 21 in hematological cancer.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Subtype</th><th>Pediatric/Adult</th><th>Total cases (<italic>n</italic>)</th><th>Total +21 (<italic>n</italic>)</th><th>% of +21</th><th>Reference</th></tr></thead><tbody><tr><td colspan=\"6\"><bold>Myeloid disorders</bold></td></tr><tr><td>MPN</td><td>Adult</td><td>938</td><td>36</td><td>3.8</td><td>[##REF##21531982##144##] and MDB</td></tr><tr><td>MDS</td><td>Adult</td><td>3577</td><td>151</td><td>4.2</td><td>[##REF##17954704##145##] and MDB</td></tr><tr><td>AML</td><td>Pediatric</td><td>3758</td><td>319</td><td>8.5</td><td>[##REF##20439644##133##] and MDB</td></tr><tr><td/><td>Adult</td><td>17769</td><td>695</td><td>3.9</td><td>[##REF##20385793##134##] and MDB</td></tr><tr><td>AMKL</td><td>Pediatric</td><td>372</td><td>126</td><td>33.9</td><td>[##REF##28112737##121##] and MDB</td></tr><tr><td/><td>Adult</td><td>203</td><td>15</td><td>7.4</td><td>[##REF##28112737##121##] and MDB</td></tr><tr><td colspan=\"6\"><bold>L</bold><bold>ymphoid disorders</bold></td></tr><tr><td>T-ALL</td><td>Pediatric</td><td>1431</td><td>43</td><td>3.0</td><td>[##REF##11001909##146##] and MDB</td></tr><tr><td/><td>Adult</td><td>495</td><td>30</td><td>6.1</td><td>MDB</td></tr><tr><td>B-ALL</td><td>Pediatric</td><td>3973</td><td>1086</td><td>27.3</td><td>MDB</td></tr><tr><td/><td>Adult</td><td>1290</td><td>141</td><td>10.9</td><td>MDB</td></tr><tr><td>CLL</td><td>Adult</td><td>1432</td><td>32</td><td>2.2</td><td>MDB</td></tr><tr><td>FL</td><td>Adult</td><td>906</td><td>87</td><td>9.6</td><td>[##REF##30417943##140##] and MDB</td></tr><tr><td>CTCL</td><td>Adult</td><td>246</td><td>21</td><td>8.5</td><td>[##REF##25306899##139##, ##REF##30417943##140##] and MDB</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p><italic>TMD</italic> transient myeloproliferative disorder, <italic>ML–DS</italic> myeloid leukemia associated with Down syndrome, <italic>DS-ALL</italic> acute lymphoblastic leukemia associated with Down syndrome, <italic>SR</italic> spontaneous remission, <italic>EFS</italic> event-free survival, <italic>OS</italic> overall survival, <italic>CR</italic> complete remission, <italic>TRM</italic> treatment-related mortality, <italic>POG</italic> Pediatric Oncology Group, <italic>COG</italic> Children’s Oncology Group, <italic>BFM</italic> Berlin–Frankfurt–Münster study group, <italic>DCOG</italic> Dutch Childhood Oncology Group, <italic>JCCLSG</italic> Japanese Children’s Cancer and Leukemia Study Group, <italic>JPLSG</italic> Japanese Pediatric Leukemia/Lymphoma Study Group, <italic>NOPHO</italic> Nordic Society of Pediatric Hematology and Oncology, <italic>PPLLSG</italic> Polish Pediatric Leukemia and Lymphoma Study Group, <italic>DFCI</italic> Dana-Farber Cancer Institute, <italic>CCG</italic> Children’s Cancer Group.</p><p><sup>a</sup>Trials published subsequent to Lee et al. [##REF##27285583##35##].</p><p><sup>b</sup>Randomized patients.</p></table-wrap-foot>", "<table-wrap-foot><p><italic>MDB</italic> Mitelman database (updated on 15/10/2019, available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://mitelmandatabase.isb-cgc.org\">https://mitelmandatabase.isb-cgc.org</ext-link>), <italic>MPN</italic> myeloproliferative neoplasms, <italic>MDS</italic> myelodysplastic syndrome, <italic>AML</italic> acute myeloid leukemia, <italic>AMKL</italic> acute megakaryoblastic leukemia, <italic>T-ALL</italic> T-cell acute lymphoblastic leukemia, <italic>B-ALL</italic> B-cell acute lymphoblastic leukemia, <italic>CLL</italic> chronic lymphocytic leukemia, <italic>FL</italic> follicular lymphoma, <italic>CTCL</italic> cutaneous T-cell lymphoma.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_854_Fig1_HTML\" id=\"d32e1139\"/>", "<graphic xlink:href=\"41375_2020_854_Fig2_HTML\" id=\"d32e1507\"/>" ]
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[{"label": ["1."], "surname": ["Mitelman", "Heim", "Mandahl"], "given-names": ["F", "S", "N"], "article-title": ["Trisomy 21 in neoplastic cells"], "source": ["Am J Med Genet."], "year": ["1990"], "volume": ["7"], "issue": ["(Suppl.)"], "fpage": ["262"], "lpage": ["6"]}, {"label": ["17."], "surname": ["Yamato", "Muramatsu", "Watanabe", "Deguchi", "Iwamoto", "Hasegawa"], "given-names": ["G", "H", "T", "T", "S", "D"], "article-title": ["Predictive factors of the development of leukemia in patients with transient abnormal myelopoiesis and Down syndrome: The Jccg Study JPLSG TAM-10"], "source": ["Blood."], "year": ["2019"], "volume": ["134"], "issue": ["Supplement_1"], "fpage": ["3833"]}, {"label": ["31."], "surname": ["Hitzler", "Berman", "Gerbing", "Beckman", "Hirsch", "Raimondi"], "given-names": ["JK", "J", "RB", "A", "BA", "SC"], "article-title": ["High-dose cytarabine is indispensable for the survival of children with myeloid leukemia in Down syndrome Despite negative minimal residual disease Post-induction."], "source": ["Blood."], "year": ["2019"], "volume": ["134"], "issue": ["Supplement_1"], "fpage": ["118"]}, {"label": ["34."], "surname": ["Taga", "Tanaka", "Terui", "Iwamoto", "Hiramatsu", "Miyamura"], "given-names": ["T", "S", "K", "S", "H", "T"], "article-title": ["Post-induction minimal residual disease measured by flow cytometry and deep sequencing of mutant GATA1 are both significant prognostic factors for children with myeloid leukemia and Down syndrome: a nationwide prospective study of the Japanese Pediatric Leukemia/Lymphoma Study Group."], "source": ["Blood."], "year": ["2019"], "volume": ["134"], "issue": ["Supplement_1"], "fpage": ["3848"]}, {"label": ["39."], "mixed-citation": ["Kroll M, Kaupat-Bleckmann K, Moricke A, Alten J, Schewe DM, Stanulla M, et al. Methotrexate-associated toxicity in children with Down syndrome and acute lymphoblastic leukemia during consolidation therapy with high dose methotrexate according to ALL-BFM treatment regimen. Haematologica. 2020;105:1013\u201320."]}, {"label": ["44."], "surname": ["Hitzler", "He", "Doyle", "Cairo", "Camitta", "Chan"], "given-names": ["JK", "W", "J", "M", "BM", "KW"], "article-title": ["Outcome of transplantation for acute myelogenous leukemia in children with Down syndrome"], "source": ["Biol Blood Marrow Transpl"], "year": ["2013"], "volume": ["19"], "fpage": ["893"], "lpage": ["7"]}, {"label": ["55."], "mixed-citation": ["Labuhn M, Perkins K, Matzk S, Varghese L, Garnett C, Papaemmanuil E, et al. Mechanisms of progression of myeloid preleukemia to transformed myeloid leukemia in children with Down syndrome. Cancer Cell. 2019;36:123\u201338e10."]}, {"label": ["80."], "surname": ["Wagenblast", "Gan", "Azkanaz", "Smith", "Ara\u00fajo", "Shakib"], "given-names": ["E", "OI", "M", "SA", "J", "L"], "article-title": ["Understanding pre-leukemia in trisomy 21 human HSC and modeling progression towards Down syndrome associated leukemia Using CRISPR/Cas9 at single cell resolution"], "source": ["Blood."], "year": ["2019"], "volume": ["134"], "issue": ["Supplement_1"], "fpage": ["2531"]}, {"label": ["81."], "surname": ["O\u2019Byrne", "Elliott", "Buck", "Rice", "O\u2019Connor", "Oswald"], "given-names": ["SI", "N", "G", "S", "D", "J"], "article-title": ["Trisomy 21 driven pro-inflammatory signalling in fetal bone marrow may play a role in perturbed B-Lymphopoiesis and acute lymphoblastic leukemia of Down syndrome"], "source": ["Blood."], "year": ["2019"], "volume": ["134"], "issue": ["Supplement_1"], "fpage": ["1206"]}, {"label": ["110."], "mixed-citation": ["Sullivan KD, Lewis HC, Hill AA, Pandey A, Jackson LP, Cabral JM, et al. Trisomy 21 consistently activates the interferon response. eLife. 2016;5:e16220."]}, {"label": ["112."], "mixed-citation": ["Waugh KA, Araya P, Pandey A, Jordan KR, Smith KP, Granrath RE, et al. Mass cytometry reveals global immune remodeling with multi-lineage hypersensitivity to Type I interferon in Down Syndrome. Cell Rep. 2019;29:1893\u2013908 e4."]}, {"label": ["115."], "mixed-citation": ["Woo AJ, Wieland K, Huang H, Akie TE, Piers T, Kim J, et al. Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation. J Clin Invest. 2013;123:3292\u2013304."]}, {"label": ["131."], "mixed-citation": ["Sinclair PB, Ryan S, Bashton M, Hollern S, Hanna R, Case M, et al. SH2B3 inactivation through CN-LOH 12q is uniquely associated with B-cell precursor ALL with iAMP21 or other chromosome 21 gain. Leukemia. 2019;33:1881\u201394."]}]
{ "acronym": [], "definition": [] }
146
CC BY
no
2024-01-13 23:35:09
Leukemia. 2020 May 20; 34(8):1984-1999
oa_package/11/7c/PMC7387246.tar.gz
PMC7387292
32042082
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[ "<title>Subject terms</title>" ]
[ "<p id=\"Par1\">The authors would like to thank the GFCH for their commentary on the recommendations for cytogenomic testing [##UREF##0##1##]. We are pleased to note that they are in agreement for the majority of aspects of our recommendations and appreciate where the GFCH have expanded or provided clarification to our statements.</p>", "<p id=\"Par2\">In particular we were pleased that the GFCH concur with the authors of the importance and value of chromosome-banding analysis in the diagnostic pathway of haematological neoplasms as well as the complementary nature of different testing strategies. We note that the GFCH has a few concerns and these are addressed individually below.</p>", "<p id=\"Par3\">In replying to their comments, it is important to restate that the recommendation document is a consensus document of working practices for cytogenomic testing in a number of different European countries and describes the minimum testing required, and that national policies should be taken into consideration [##REF##30696948##2##]. We acknowledge that more extensive testing is undertaken in some countries particularly relating to the number of metaphases analysed and the pathological entities tested by chromosome-banding analysis. The aim of the recommendations was to provide the practical advice to help laboratories prioritise and rationalise cytogenomic testing where health care resources are restricted and where the extent of testing is limited by testing reimbursement.</p>", "<p id=\"Par4\">Choice of sample: We agree with this recommendation and thank them for the extra clarification provided to our statement regarding the use of peripheral blood samples for chromosome-banding analysis in this section.</p>", "<p id=\"Par5\">For cell culture of AML the authors agree that these rearrangements can be detected in 24 h cultures. The authors state that a 48 h culture should be considered but this was not intended to be a requirement. This observation referred mainly to historic data of t(15;17) cases when FISH or RT-PCR were not widely available.</p>", "<p id=\"Par6\">Choromosome banding analysis: We agree that the full ISCN 2016 definition of a clone includes the example cited but this is also covered by the statement provided in our paper.</p>", "<p id=\"Par7\">We agree that ideally 20 metaphases would be analysed regardless of the result, and indeed are aware that many laboratories do systematically analyse this number. However, a minimum of ten metaphases are considered acceptable when an abnormal clone is detected, unless there is suspicion of clonal evolution (e.g., one metaphase with additional abnormality) where a more extensive analysis should be performed.</p>", "<p id=\"Par8\">Recommended testing: We state that chromosome-banding analysis is mandatory for ALL in Table 2 [##REF##30696948##2##]. However, for information, we refer to the publication of the International Berlin–Frankfurt–Münster study group on recommendations for the detection of prognostically relevant genetic abnormalities in childhood B-cell precursor acute lymphoblastic leukaemia [##REF##20701601##3##]. The vast majority of laboratories undertake chromosome analysis of childhood B-cell ALL but our experience through EQA has also shown that some laboratories are only following this strategy when cases require risk stratification. Of note, for the new childhood ALLTogether trial karyotyping is not mandatory in all cases provided sufficient extensive first line testing using FISH, PCR, and SNP-array is performed.</p>", "<p id=\"Par9\">FISH in AML: Regarding <italic>NUP98</italic> in AML, the authors are in agreement with this approach. We recognise that the text should have stated 17 months-18 years not 5 years.</p>", "<p id=\"Par10\">Waldenström macrogobulinemia (WM): The authors consider that cytogenomic testing, including chromosome analysis, for recurrent abnormalities should be undertaken as the detection of disease-specific recurrent abnormalities can be beneficial in a differential diagnosis case. Many laboratories would not undertake routine chromosome analysis for all WM cases although we recognise that country-specific guidelines may recommend this.</p>", "<p id=\"Par11\">High grade B-cell lymphomas: We are in concordance with the overall choice of probes to be used for this disease entity and agree that as <italic>MYC</italic> breakpoints are located across a large genomic region the choice of adequate FISH probe(s) is paramount. It is therefore important that laboratories understand the limitations of FISH probes when they select which probes to use.</p>", "<p id=\"Par12\">Concerning chromosome analysis, many laboratories do not undertake routine chromosome analysis of high grade B-cell lymphoma where FISH is the priority testing for these cases. We agree that karyotyping is useful, however, this is not always possible. In addition, when karyotyping is performed, this is also often supplemented by FISH analysis. Many laboratories no longer undertake chromosome analysis but instead perform FISH on FFPE sections to detect the most significant abnormalities.</p>", "<p id=\"Par13\">Reporting time: We are aware that a large number of examinations are requested, and that laboratories can find it difficult to adhere to recommended turnaround times but it is important that laboratories organise the work flow so that reporting times can be met. Concerning prioritisation of testing, we recommend that this is assigned according to clinical need as reporting results in follow-up samples can also be urgent since targeted therapy is frequently used in a relapse or emerging relapse setting.</p>", "<p id=\"Par14\">In summary, the authors are grateful for the response by the GFCH to our recommendations as this has enabled us to clarify certain points and address a few omissions. In addition, both the recommendations and the GFCH underline the importance of cytogenetics and a combined approach to testing. Finally, the authors recognise that different countries will have different approaches to ensure that full testing is addressed and adoption of these recommendations, together with this clarification, will assist in further harmonisation of genetic testing.</p>" ]
[ "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par15\">The authors declare that they have no conflict of interest.</p>" ]
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[ "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[]
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[{"label": ["1."], "mixed-citation": ["Nguyen-Khac F, Bidet B, Veronese L, Daudignon A, Penther D, Troadec M-B, et al. Recommendations for cytogenomic analysis of hematologic malignancies: comments from the Francophone Group of Hematological Cytogenetics (GFCH). Leukemia. 2020, in press."]}]
{ "acronym": [], "definition": [] }
3
CC BY
no
2024-01-13 23:35:06
Leukemia. 2020 Feb 7; 34(8):2262-2264
oa_package/7a/be/PMC7387292.tar.gz
PMC7387295
32071430
[]
[]
[]
[]
[]
[ "<title>Subject terms</title>" ]
[ "<p id=\"Par1\">Pediatric patients with relapsed/refractory (R/R) mature B-cell non-Hodgkin lymphoma (B-NHL) have a 2-year overall survival rate with chemoimmunotherapy (CIT) of 15–33% [##REF##18816698##1##, ##REF##29984828##2##]. Rituximab plus ifosfamide, carboplatin, and etoposide (RICE) is widely used in R/R children with NHL [##REF##30613948##3##] and rituximab plus vincristine, ifosfamide, carboplatin, idarubicin, and dexamethasone (RVICI) has been used in Europe [##UREF##0##4##, ##UREF##1##5##].</p>", "<p id=\"Par3\">In preclinical studies, ibrutinib, a Bruton’s tyrosine kinase inhibitor approved to treat adults with various B-cell malignancies in the United States and the European Union, among other countries [##REF##26348626##6##], inhibited Burkitt lymphoma (BL; the predominant pediatric mature B-NHL) and diffuse large B-cell lymphoma (DLBCL) tumor cell growth, and prolonged survival in BL xenografted mice [##REF##30546948##7##–##REF##23045577##10##].</p>", "<p id=\"Par4\">We report safety, pharmacokinetics, and preliminary efficacy findings from the run-in stage (part 1; December 2016–December 2018) of an ongoing phase 3 trial (SPARKLE).</p>", "<p id=\"Par5\">Of 21 patients with mature B-NHL (median age, 8 years [range, 3–17]; Table ##TAB##0##1##), 11 received ibrutinib plus modified RICE (RICE modified with dexamethasone) and 10 received ibrutinib plus RVICI (Supplementary Fig. ##SUPPL##1##1##). Detailed methods are presented in the ##SUPPL##0##Supplementary Appendix##, and Supplementary Tables ##SUPPL##0##1## and ##SUPPL##0##2## show the dosing and administration schedule for these treatment regimens. In ibrutinib plus modified RICE and ibrutinib plus RVICI groups, respectively, zero and four (40.0%) patients had central nervous system (CNS) disease, three (27.3%) and four (40.0%) had bone marrow involvement, and one (9.1%) and six (60.0%) received &gt;1 prior line of therapy (&gt;1 relapse).</p>", "<p id=\"Par6\">In ibrutinib plus modified RICE and ibrutinib plus RVICI groups, respectively, median number of treatment cycles was 3.0 (range, 1–4) and 2.0 (range, 1–4), and eight and three patients completed ≥3 cycles (Supplementary Fig. ##SUPPL##1##1##).</p>", "<p id=\"Par7\">Despite limitations of cross-trial comparisons, including small patient numbers and trial design differences, safety of combined therapies was consistent with known ibrutinib, RVICI, or RICE safety profiles and experience with ibrutinib plus RICE or rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone in adults with DLBCL [##UREF##2##11##]. All patients had 1 or more grade ≥3 treatment-emergent adverse events (TEAEs), with &gt;50% of patients in either group having hematologic, gastrointestinal, infectious, and metabolism- or nutrition-associated events (Supplementary Table ##SUPPL##0##3##). Ten (90.9%) patients in ibrutinib plus modified RICE and nine (90.0%) in ibrutinib plus RVICI groups had serious TEAEs (all grade ≥3). Most frequent grade ≥3 ibrutinib-related TEAEs (i.e., those considered related by the investigator) were thrombocytopenia (42.9%), neutropenia (38.1%), anemia (33.3%), and febrile neutropenia (19.0%; Supplementary Table ##SUPPL##0##4##).</p>", "<p id=\"Par8\">Two patients in ibrutinib plus modified RICE and three in ibrutinib plus RVICI groups had major hemorrhage events (grade 3/4; in the setting of thrombocytopenia; Supplementary Table ##SUPPL##0##5##). Two major hemorrhage events (intestinal and intracranial) in the ibrutinib plus RVICI arm were considered ibrutinib related. Major hemorrhage has occurred in 4% of 2838 ibrutinib-exposed patients in 27 previous clinical trials [##UREF##3##12##].</p>", "<p id=\"Par9\">Hematologic TEAEs were expected, particularly with background CIT. In a study of 20 children with R/R B-NHL receiving RICE alone, rituximab-related AEs, infections, and hematologic toxicities (e.g., neutrophil- and platelet-related events) occurred frequently, and one child discontinued because of prolonged myelosuppression [##REF##18816698##1##].</p>", "<p id=\"Par10\">As expected with the CIT regimen [##REF##18816698##1##], bone marrow suppression, reported as laboratory abnormality of low hemoglobin, platelets, or neutrophils, was common (##SUPPL##0##Supplementary Appendix##). Hematologic events were generally manageable, most recovering before the next cycle without delaying treatment.</p>", "<p id=\"Par11\">Pharmacokinetic data were assessed for &lt;12 years and ≥12 years. For patients aged 12–17 years (<italic>n</italic> = 6), area under plasma concentration–time curves (AUCs) were within target range (250–1500 ng × h/mL), supporting the 329 mg/m<sup>2</sup>/day dose (not exceeding 560 mg daily). However, in younger patients (<italic>n</italic> = 14), AUCs at 329 mg/m<sup>2</sup>/day were lower than expected, and 440 mg/m<sup>2</sup>/day was required to obtain exposures mostly within target range (Fig. ##FIG##0##1##). No AUC values were &gt;1500 ng × h/mL regardless of age or dose. AUC value on cycle 1 day 7 was notably lower than day 1.</p>", "<p id=\"Par12\">Although treatment was scheduled to avoid overlapping ibrutinib and mesna dosing, this was not always possible given prolonged mesna infusion during days 3–9 with RVICI. Mass spectrometric and radiometric analysis of 14C-labeled ibrutinib and mesna, or ifosfamide with metabolizing enzymes, did not show ibrutinib adduct formation or decreased ibrutinib concentrations. Inter- and intraindividual AUC value variability occurred and exceeded variability in adults. Inter-sample discrepancies were potentially introduced by cytochrome P450 subtype 3A inhibitors/inducers (one patient used CYP3A inhibitor, one used CYP3A inducer) before/during ibrutinib treatment (considered a study protocol deviation), ibrutinib administration via nasogastric tube in some patients, or time and content of food intake differences pre- and post-ibrutinib administration.</p>", "<p id=\"Par13\">Physiology-based pharmacokinetic simulations using an adult ibrutinib model [##UREF##4##13##] suggested body surface area–based dosing would result in adult range exposures (Loeckie de Zwart, May 28, 2015, unpublished data). Although the youngest patient was almost 4 years old, a predicted increase in patients aged &lt;6 years was uncorroborated. Exposures were still at the low end of the target range, but above observed minimum exposure in adults at 560 mg/day (shown as efficacious in adult B-cell malignancies).</p>", "<p id=\"Par14\">In the ibrutinib plus modified RICE group, there were three progressive disease (PD)-related deaths (one of which was due to multiple organ failure within 30 days of the last dose) and one AE (septic shock)-related death within 30 days of the last dose occurred. In the ibrutinib plus RVICI group, three TEAE-related deaths (unrelated to ibrutinib; within 30 days of last dose; two due to sepsis and one because of neutropenic sepsis) and six PD-related deaths occurred. Most of these patients had multiply relapsed disease and were heavily pretreated pre-enrollment; therefore, they had poor hematologic reserve and were highly susceptible to complications due to therapy.</p>", "<p id=\"Par15\">Preliminary efficacy findings were promising. As prognosis for this pediatric patient population is poor, achieving a complete response (CR) to proceed to high-dose consolidative therapy is important [##REF##18816698##1##]. Of 21 patients who received ≥1 dose of ibrutinib, 12 (57.1%) experienced a response. In the ibrutinib plus modified RICE group, eight (72.7%) were responders, among whom three (27.3%) had a CR, including one unconfirmed CR (one DLBCL, one BL, one Burkitt leukemia [B-AL]), and five (45.5%) had a partial response (PR; one BL, two DLBCL, two Burkitt-like lymphoma). In the ibrutinib plus RVICI group, four (40.0%) were responders, among whom two (20%) had a CR (both B-AL) and two had a PR (one B-AL, one high-grade B-cell lymphoma). Across both groups, all responders received treatment at first relapse, except one with high-grade B-cell lymphoma in the ibrutinib plus RVICI arm who achieved PR in second relapse.</p>", "<p id=\"Par16\">Among 14 patients treated after first relapse, 11 (78.6%) were responders (five CR, six PR), and one of seven (14.3%) treated after second relapse was a responder (one PR). Supplementary Table ##SUPPL##0##6## presents response by histology. Although this part of the study was not designed to compare efficacy of either regimen, overall response and CR rates were higher with ibrutinib plus RICE than ibrutinib plus RVICI. At an 18-month median follow-up, median investigator-assessed event-free survival (EFS) was unreached in the ibrutinib plus modified RICE group and 2.4 months in the ibrutinib plus RVICI group (Supplementary Fig. ##SUPPL##2##2##), potentially because more patients treated with the latter had received &gt;1 prior line of therapy. Treatment at first relapse (versus second relapse) was associated with a higher response rate and longer EFS possibly because patients tolerated therapy better and did not discontinue prematurely due to AEs. In addition, unlike those in the ibrutinib plus RICE group, some patients in the ibrutinib plus RVICI arm had CNS involvement, and therefore a poorer overall prognosis. Given the exploratory nature of the EFS analysis and small sample size, it is difficult to ascertain whether the addition of ibrutinib to CIT is beneficial and results should be interpreted with caution.</p>", "<p id=\"Par17\">Seven patients subsequently received hematopoietic stem cell transplantation (HSCT; four in the ibrutinib plus modified RICE group and two in the ibrutinib plus RVICI arm after completing ≥3 treatment cycles, and one after completing two cycles of ibrutinib plus RVICI and one cycle of ibrutinib monotherapy; Supplementary Fig. ##SUPPL##1##1##).</p>", "<p id=\"Par18\">Results of part 1 of the trial support the continued assessment of ibrutinib with modified RICE/RVICI in this patient population. Part 2 is assessing the efficacy of ibrutinib (329 mg/m<sup>2</sup>/day in patients aged ≥12 years and 440 mg/m<sup>2</sup>/day in patients aged &lt;12 years) plus modified RICE/RVICI versus modified RICE/RVICI CIT alone in a randomized fashion. Pharmacokinetic sampling in part 2 is on day 15 instead of day 7 of cycle 1 to avoid potential interactions. Because preliminary efficacy findings were better in patients treated upon first relapse, the protocol was amended to only include patients in first relapse. Due to the very poor prognosis for pediatric patients with R/R mature B-NHL, trials such as this, while challenging to conduct, are critically important.</p>", "<title>Supplementary information</title>", "<p>\n\n\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0749-5) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>Sponsored by Janssen Research &amp; Development, LLC. Support for pharmacokinetic and pharmacodynamic assessments was provided by Martine Neyens and Sriram Balasubramanian, respectively, Janssen Research &amp; Development, LLC. Additional data quality control and manuscript review were provided by Madeliene Curtis, Janssen Research &amp; Development, LLC. Writing assistance was provided by Liqing Xiao and Sally Hassan of Parexel and funded by Janssen Global Services, LLC. The authors would also like to thank the patients who participated in this trial, and their families, as well as the investigators, study coordinators, study teams, and nurses.</p>", "<title>Author contributions</title>", "<p>GAAB, AB, BB, MC, VM-C, AH, JdJ, KN, and MS designed the study. EK, MC, BB, KN, and MS collected and assembled data. VM-C, EK, FGP, and NT provided study material or patients. GAAB, AB, BB, MC, DB, REN, AH, JdJ, GL, XWdT, KN, and MS analyzed and interpreted data. All authors wrote and/or critically revised the manuscript for important intellectual content, provided final approval of the manuscript, and are accountable for all aspects of the work.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par19\">GAAB has received consultancy fees from Janssen, Merck, Takeda, and Roche. BB did not receive personal fees, but her institution received consultancy fees from Janssen, Roche, ADC Therapeutics, Novartis, and Celgene. AH, JdJ, KN, and MS hold Johnson &amp; Johnson stock and are employed by Janssen R&amp;D. GL and XWdT are also employees of Janssen. MC has received research funding from Janssen. AB, DB, VM-C, REN, EK, FGP, and NT have no conflicts of interest to disclose.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Ibrutinib exposures with 240–440 mg/m<sup>2</sup>/day doses.</title><p>Box/whisker plots of estimated AUC on pharmacokinetic occasions by cycle and day for each dose for (<bold>a</bold>) AUC<sub>τ</sub> (estimated AUC of 24-h dosing interval) and (<bold>b</bold>) AUC<sub>∞</sub> (dose*F/CL; predicted AUC at steady state). Solid line represents median, box represents 25/75%, and whiskers represent 10/90% confidence interval. Individual symbols represent outliers. (<bold>c</bold>) AUC<sub>τ</sub> versus age and (<bold>d</bold>) versus body surface area. Vertical lines represent individual patients, color represents dose, and symbol represents pharmacokinetic occasion. Target AUC range based on adult exposures was 250–1500 ng × h/mL. <italic>AUC</italic> area under the plasma concentration–time curve.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient demographics and baseline characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Ibrutinib plus modified RICE (<italic>n</italic> = 11)</th><th>Ibrutinib plus RVICI (<italic>n</italic> = 10)</th></tr></thead><tbody><tr><td>Median age (range), years</td><td>11.0 (3–17)</td><td>8.0 (4–15)</td></tr><tr><td colspan=\"3\">Age group, years, <italic>n</italic> (%)</td></tr><tr><td>  1–5</td><td>2 (18.2)</td><td>2 (20.0)</td></tr><tr><td>  6–11</td><td>4 (36.4)</td><td>6 (60.0)</td></tr><tr><td>  12–17</td><td>5 (45.5)</td><td>2 (20.0)</td></tr><tr><td colspan=\"3\">Sex, <italic>n</italic> (%)</td></tr><tr><td>  Female</td><td>3 (27.3)</td><td>1 (10.0)</td></tr><tr><td>  Male</td><td>8 (72.7)</td><td>9 (90.0)</td></tr><tr><td colspan=\"3\"> Ethnicity, <italic>n</italic> (%)</td></tr><tr><td>  Hispanic or Latino</td><td>2 (18.2)</td><td>0</td></tr><tr><td>  Not Hispanic or Latino</td><td>6 (54.5)</td><td>9 (90.0)</td></tr><tr><td>  Unknown</td><td>1 (9.1)</td><td>0</td></tr><tr><td>  Not reported</td><td>2 (18.2)</td><td>1 (10.0)</td></tr><tr><td colspan=\"3\">Race, <italic>n</italic> (%)</td></tr><tr><td>  White</td><td>9 (81.8)</td><td>10 (100.0)</td></tr><tr><td>  Asian</td><td>1 (9.1)</td><td>0</td></tr><tr><td>  Not reported</td><td>1 (9.1)</td><td>0</td></tr><tr><td colspan=\"3\">Time from initial diagnosis to first dose of study drug (months)</td></tr><tr><td>  Mean (SD)</td><td>11.7 (10.94)</td><td>10.5 (6.22)</td></tr><tr><td>  Median (range)</td><td>7.0 (4–37)</td><td>8.2 (5–25)</td></tr><tr><td colspan=\"3\">Type of mature B-cell NHL at initial diagnosis, <italic>n</italic> (%)</td></tr><tr><td>  Burkitt-like lymphoma</td><td>2 (18.2)</td><td>1 (10.0)</td></tr><tr><td>  Burkitt lymphoma</td><td>4 (36.4)</td><td>3 (30.0)</td></tr><tr><td>  Burkitt leukemia</td><td>2 (18.2)</td><td>4 (40.0)</td></tr><tr><td>  Diffuse large B-cell lymphoma</td><td>3 (27.3)</td><td>0</td></tr><tr><td>  Primary mediastinal B-cell lymphoma</td><td>0</td><td>1 (10.0)</td></tr><tr><td>  High-grade B-cell lymphoma</td><td>0</td><td>1 (10.0)</td></tr><tr><td>  Other</td><td>0</td><td>0</td></tr><tr><td colspan=\"3\">Extra nodal sites, <italic>n</italic> (%)</td></tr><tr><td>  Central nervous system</td><td>0</td><td>4 (40.0)</td></tr><tr><td>  Bone marrow</td><td>3 (27.3)</td><td>4 (40.0)</td></tr><tr><td>  Other</td><td>9 (81.8)</td><td>9 (90.0)</td></tr><tr><td colspan=\"3\">Prior lines of therapy, <italic>n</italic> (%)</td></tr><tr><td>  1</td><td>10 (90.9)</td><td>4 (40.0)</td></tr><tr><td>  &gt;1</td><td>1 (9.1)</td><td>6 (60.0)</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>" ]
[ "<table-wrap-foot><p><italic>NHL</italic> non-Hodgkin lymphoma, <italic>SD</italic> standard deviation, <italic>modified RICE</italic> rituximab, ifosfamide, carboplatin, and etoposide, with the addition of dexamethasone, <italic>RVICI</italic> vincristine, ifosfamide, carboplatin, idarubicin, that includes dexamethasone.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_749_Fig1_HTML\" id=\"d32e784\"/>" ]
[ "<media xlink:href=\"41375_2020_749_MOESM1_ESM.docx\"><caption><p>Supplementary Information</p></caption></media>", "<media xlink:href=\"41375_2020_749_MOESM2_ESM.jpg\"><caption><p>Supplementary Figure 1</p></caption></media>", "<media xlink:href=\"41375_2020_749_MOESM3_ESM.eps\"><caption><p>Supplementary Figure 2</p></caption></media>" ]
[{"label": ["4."], "surname": ["Woessmann", "Zimmermann", "Burkhardt", "Meinhardt", "Rosenbusch", "Vaillant"], "given-names": ["W", "M", "B", "A", "C", "V"], "article-title": ["Relapsed or refractory Burkitt lymphoma in children and adolescents after BFM-type first-line therapy-a BFM group report"], "source": ["Blood"], "year": ["2014"], "volume": ["124"], "fpage": ["1738"], "pub-id": ["10.1182/blood.V124.21.1738.1738"]}, {"label": ["5."], "mixed-citation": ["Woessmann W, Zimmermann M, Meinhardt A, M\u00fcller S, Hauch H, Kn\u00f6rr F, et al. Progressive or relapsed Burkitt lymphoma or leukemia in children and adolescents after BFM-type first-line therapy. Blood. 2020. 10.1182/blood.2019003591 [Epub ahead of print]."]}, {"label": ["11."], "surname": ["Sauter", "Verwys", "McCall", "Miller", "Courtien", "Schoder"], "given-names": ["CS", "SL", "SJ", "ST", "AI", "H"], "article-title": ["Phase I study combining ibrutinib with rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) in patients with relapsed or primary refractory diffuse large B-cell lymphoma (DLBCL): NCI-Cancer Therapeutics Evaluation Program (CTEP) #9588"], "source": ["Blood"], "year": ["2016"], "volume": ["128"], "issue": ["Suppl 1"], "fpage": ["4198"], "pub-id": ["10.1182/blood.V128.22.4198.4198"]}, {"label": ["12."], "mixed-citation": ["IMBRUVICA (ibrutinib). [Prescribing information]. Horsham, PA: Pharmacyclics LLC; Sunnyvale, CA: Janssen Biotech, Inc; 2018."]}, {"label": ["13."], "surname": ["de Zwart", "Snoeys", "De Jong", "Sukbuntherng", "Mannaert", "Monshouwer"], "given-names": ["L", "J", "J", "J", "E", "M"], "article-title": ["Ibrutinib dosing strategies based on interaction potential of CYP3A4 perpetrators using physiologically based pharmacokinetic modeling"], "source": ["Clin Pharm Ther"], "year": ["2016"], "volume": ["100"], "fpage": ["548"], "lpage": ["57"], "pub-id": ["10.1002/cpt.419"]}]
{ "acronym": [], "definition": [] }
13
CC BY
no
2024-01-13 23:35:07
Leukemia. 2020 Feb 18; 34(8):2271-2275
oa_package/83/57/PMC7387295.tar.gz
PMC7387297
32265500
[ "<title>Introduction</title>", "<p id=\"Par2\">Achieving early molecular response (EMR), defined as a reduction in <italic>BCR-ABL1</italic> transcripts to ≤10% (International Scale [IS]) at 3 or 6 months after initiating tyrosine kinase inhibitor (TKI) treatment, has been shown to improve the probability of achieving a subsequent deep molecular response (DMR; typically MR<sup>4.5</sup> or <italic>BCR-ABL1</italic> ≤ 0.0032% [IS]) and to be associated with superior progression-free and overall survival (OS) in chronic myeloid leukemia in chronic phase (CML-CP) [##UREF##0##1##, ##REF##27217448##2##]. The prognostic significance of EMR has been established for both imatinib and second-generation TKIs in the first-line setting. Patients treated with dasatinib or imatinib with EMR at 3 months in DASISION (the Dasatinib vs. Imatinib Study in Treatment-Naïve Chronic Myeloid Leukemia Patients Trial) had an increased likelihood of achieving complete cytogenetic response (CCyR), major molecular response (MMR), increased progression-free survival (PFS), and decreased likelihood of progression to CML in accelerated phase or blast crisis (CML-AP/BC) [##REF##27217448##2##]. Similar improvement in long-term outcomes has also been reported with other second-generation TKIs [##REF##24335106##3##, ##UREF##1##4##]. However, nearly one-third of patients with CML-CP treated with first-line imatinib fail to achieve EMR [##REF##27217448##2##, ##REF##24335106##3##] and, compared with imatinib, second-generation TKIs have been shown to be associated with a 96% reduction in the risk of a poor cytogenetic response at the 3-month timepoint [##REF##23620574##5##].</p>", "<p id=\"Par3\">In light of this growing body of evidence, the 2013 European LeukemiaNet (ELN) recommendations and the National Comprehensive Cancer Network Clinical Practice Guidelines (NCCN Guidelines®) now include EMR as a treatment milestone for patients with newly diagnosed CML-CP, and consider <italic>BCR-ABL1</italic> ≤ 10% (IS) at 3 months as the optimal molecular response [##UREF##0##1##, ##REF##23803709##6##]. Although the ELN recommendations consider <italic>BCR-ABL1</italic> &gt; 10% at 3 months a warning, a change in therapy is not conclusively recommended, reflecting the current lack of data from prospective clinical trials describing how an early change in therapy at this time may translate into a clinical benefit.</p>", "<p id=\"Par4\">DASCERN (Study of Dasatinib vs. Imatinib in Patients With Chronic Myeloid Leukemia Who Did Not Have Favorable Response to Imatinib; NCT01593254) is the first and only prospective randomized trial to explore the potential benefit of an early switch to dasatinib in patients with lack of EMR to first-line imatinib. Here we present the first results of this study, including response rates and 2-year survival outcomes.</p>" ]
[ "<title>Materials and methods</title>", "<title>Study design and eligibility</title>", "<p id=\"Par5\">DASCERN is an open-label, randomized, international, multicenter phase 2b trial of dasatinib vs. imatinib in patients with Philadelphia chromosome-positive (Ph+) CML-CP who had achieved complete hematologic response (CHR), but had <italic>BCR-ABL1</italic> &gt; 10% (IS) 3 months after starting first-line treatment with imatinib 400 mg once daily (QD) (Fig. ##FIG##0##1##). Molecular response assessments prior to enrollment were performed at a central laboratory, and patients with <italic>BCR-ABL1</italic> ≤ 10% (IS) were ineligible. Patients with <italic>BCR-ABL1</italic> &gt; 10% (IS) at 3 months were considered eligible and randomized 2:1 to receive dasatinib 100 mg QD (early switch) or continue on imatinib (at any dose selected by the enrolling investigator). Randomization occurred up to 8 weeks after the 3-month molecular assessment. Patients were randomized by means of an interactive voice response system, with randomization performed using permuted blocks within each stratum and stratified by Sokal score (high, intermediate, low, or unknown) and time between the 3-month molecular assessment and randomization (≤4 weeks vs. &gt;4 weeks). Patients randomized to imatinib who subsequently met ELN 2013 criteria for treatment failure [##REF##18716134##7##] were crossed over to dasatinib unless they had documented dasatinib-resistant <italic>BCR-ABL</italic> mutations (e.g., T315I/A, F317L, V299L) as assessed in a central laboratory. Assessment of prior mutations could be performed at local laboratories, but was not mandatory. Mutational analysis was performed following a suboptimal response, treatment failure, or progression, and at the end of treatment or prior to any change in therapy.</p>", "<p id=\"Par6\">Sample size for randomized patients was computed based on the following assumptions: 2:1 randomization ratio, two-sided superiority test with <italic>α</italic> = 0.05 and 90% power, and MMR at 12 months of 10% for imatinib and 25% for dasatinib.</p>", "<p id=\"Par7\">Eligible patients were aged ≥18 years and had started imatinib monotherapy within 6 months of the initial CML-CP diagnosis (PH+ or <italic>BCR-ABL1</italic> detection). Patients were required to be tolerating imatinib 400 QD (maximum of cumulative 2 weeks’ interruption within the prior 3 months permitted), and to have an Eastern Cooperative Oncology Group (ECOG) performance status 0–2, with adequate renal/hepatic function.</p>", "<p id=\"Par8\">Patients could receive dasatinib or imatinib for up to 60 months after randomization of the last patient, or until disease progression, treatment failure, unacceptable toxicity, withdrawal of consent, or discontinuation of the study. All patients provided written informed consent in accordance with the Declaration of Helsinki and institutional guidelines before study entry. The study protocol was approved by institutional review boards (and/or ethics committee) of each participating center, as well as the competent national authority.</p>", "<title>Study endpoints</title>", "<p id=\"Par9\">The primary endpoint in DASCERN was defined as the proportion of patients who achieved an MMR at 12 months after day 1 of first-line imatinib treatment in patients randomized at 3 months to dasatinib or imatinib (up to 9 months after randomization). Key secondary endpoints include time to MMR, time to MR<sup>4.5</sup>, PFS, and OS. Tertiary endpoints include development of <italic>BCR-ABL1</italic> mutations, safety and tolerability, molecular and cytogenetic response over time, and benefit of early switch to dasatinib (at 3 months) over a later switch/crossover at the time of imatinib failure (based on ELN 2013 failure criteria).</p>", "<title>Evaluations and study definitions</title>", "<p id=\"Par10\">Data from all evaluations, except for the primary endpoint, were from a 24-month data cut. For all evaluations, all patients were followed every 3 months for the first 24 months, then every 6 months until month 60; patients were then followed annually. Safety assessments and molecular analyses were conducted at month 4 or 5, and 6, and then every 3 months for up to 24 months. For patients continuing their assigned treatment beyond 24 months, safety assessments, as well as hematology and molecular analysis/quantitative polymerase chain reaction (qPCR) were conducted every 6 months, and cytogenetic assessments (conventional or fluorescence in situ hybridization [FISH; peripheral blood]) were conducted every year. A cytogenetic response was based on the prevalence of Ph+ cells in metaphase in bone marrow according to standard criteria (CCyR = 0% Ph+ cells) [##REF##18716134##7##]. MR<sup>4.5</sup> was considered ≤0.0032% <italic>BCR-ABL1</italic> (IS). Time to MMR or MR<sup>4.5</sup> was defined as the time from randomization until first PCR showing MMR or MR<sup>4.5</sup>. PFS was defined as time from randomization to transformation to CML-AP/BC or death from any cause during treatment. OS was defined as the time between the randomization date and death date. Adverse events (AEs) and serious AEs were assessed according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events version 4.0 [##UREF##2##8##].</p>", "<title>Statistical analysis</title>", "<p id=\"Par11\">The primary endpoint analysis was performed using the Cochran–Mantel–Haenszel (CMH) test [##UREF##3##9##], stratified by Sokal score and time from molecular analysis to randomization. The intent-to-treat (ITT) population included all patients initially randomized to each arm, irrespective of crossover. An exact 95% confidence interval (CI) for the difference in MMR rate at 12 months was computed. Time-to-event endpoints were estimated using the Kaplan–Meier, Brookmeyer–Crowley, and Fine and Gray methods (calculation of competing risk), and compared between treatment groups using a two-sided stratified log-rank test [##UREF##4##10##–##UREF##8##14##]. Competing risks for cumulative incidence of MMR were death or bone marrow transplantation. Patients who did not achieve MMR or MR<sup>4.5</sup> were censored at their last molecular assessment date. Differences in response rates were assessed using the CMH test. For primary endpoint analyses, any patient with treatment failure after randomization who discontinued from the study (any arm, for any reason) or crossed over to dasatinib from the imatinib arm was considered a nonresponder. A sensitivity analysis of PFS and OS was performed on randomized patients where patients who crossed over to dasatinib after failure on imatinib were censored at the date of crossover.</p>" ]
[ "<title>Results</title>", "<title>Baseline patient characteristics and patient disposition</title>", "<p id=\"Par12\">A total of 1126 patients were enrolled in the study from September 12, 2012, to November 8, 2016, of whom 260 patients with <italic>BCR-ABL1</italic> &gt; 10% were randomized (dasatinib, <italic>n</italic> = 174; imatinib, <italic>n</italic> = 86). Baseline patient characteristics for all randomized patients are shown in Table ##TAB##0##1##. Median age was 37 years (range 18–82) and 248 (95%) patients were &lt;65 years old. Sokal scores were evenly distributed (low, 28%; intermediate, 30%; high, 24%; unknown, 18%). Patients were predominantly male (78%) and Asian (73%), and most (84%) had an ECOG performance status of zero. All patients had b2a2 (e13a2) or b3a2 (e14a2) transcripts.</p>", "<p id=\"Par13\">After a minimum of 24 months’ follow-up, 135 (79%) patients randomized to dasatinib and 68 (79%) patients randomized to imatinib (three patients randomized to dasatinib were not treated) were continuing on treatment (Table ##TAB##1##2## and Supplementary Fig. ##SUPPL##0##S1##). Of the 45 patients randomized to imatinib who crossed over to dasatinib, 32 (71%) patients continued to receive dasatinib therapy at 24 months. The median daily dose was 100 mg (range 26–136) for dasatinib and 400 mg (range 129–801) for imatinib. Additional treatment exposure information can be found in Supplementary Table ##SUPPL##0##S1##.</p>", "<p id=\"Par14\">Of the 86 patients randomized to imatinib, 45 (52%) subsequently crossed over to dasatinib, with an overall median time to dasatinib crossover of 9 months (95% CI 6–12) after randomization (Supplementary Fig. ##SUPPL##0##S1##). Overall, 44 of 86 patients randomized to imatinib (51%) experienced treatment failure, and one patient (1%) had suboptimal response to imatinib. Patient characteristics were consistent between patients who crossed over to dasatinib and those who remained on imatinib after randomization.</p>", "<p id=\"Par15\">The median treatment duration was longer for patients randomized to dasatinib (33 months, range &lt;1–63) vs. imatinib (20 months, range 1–57). For patients receiving dasatinib after crossing over from imatinib (<italic>n</italic> = 45), the median treatment duration on dasatinib was 23 months (range &lt;1–48). For patients receiving imatinib who did not crossover to dasatinib (<italic>n</italic> = 41), the median treatment duration was 33 months (range 1–57). Dose interruptions, escalations, and reductions were experienced by 75 (44%), 23 (13%), and 10 (6%) patients randomized to dasatinib and 37 (43%), 10 (12%), and 7 (8%) patients randomized to imatinib, respectively. To date, 54 (21%) patients discontinued treatment; 12 (7%) and 4 (5%) patients randomized to dasatinib and imatinib, respectively, discontinued due to toxicity. The most common reasons for discontinuing dasatinib due to toxicity were hematological toxicity in six patients (grade 3–4) and pleural effusion in five patients (grade 2–3). Among patients randomized to imatinib, the most common reason for discontinuation due to toxicity was non-hematological toxicity in three patients (all grade 2).</p>", "<title>Efficacy</title>", "<p id=\"Par16\">The primary endpoint of MMR rate after 12 months was met, with a significantly higher MMR rate in the dasatinib vs. imatinib arm (29% vs. 13%, <italic>P</italic> = 0.005, Fig. ##FIG##1##2##). Further efficacy analyses described below were performed at a 2-year data cut. Overall, per ITT, 66% of patients randomized to dasatinib and 67% of patients randomized to imatinib achieved MMR (Fig. ##FIG##2##3a##). When accounting for competing risk, the cumulative incidence of MMR was higher in patients in the dasatinib arm than the imatinib arm (Fig. ##FIG##2##3b##); however, this finding was not statistically significant as these results were influenced by 30% of patients from the imatinib arm achieving MMR after crossing over to dasatinib. After taking this treatment crossover into account, 141 of 219 patients (64%) on dasatinib, including 115 (66%) initially randomized patients and 26 (58%) patients who crossed over from imatinib to dasatinib, achieved MMR by 24 months. When patients with treatment failure were censored at crossover, MMR was achieved in 35 of 86 patients (41%) on imatinib. Median time to MMR was 14 months (95% CI 12–18) in patients randomized to dasatinib vs. 20 months (95% CI 14–26) in patients who remained on imatinib (<italic>P</italic> = 0.130). In patients initially randomized to imatinib who crossed over to dasatinib, the median time to MMR was 19 months (95% CI 8–38). Cumulatively, 36 patients (21%) on dasatinib and 18 patients (21%) on imatinib, regardless of crossover status, achieved MR<sup>4.5</sup> by month 24. Patients randomized to dasatinib or imatinib who did not experience treatment failure had similar declines in <italic>BCR-ABL1</italic> transcript levels over time (Supplementary Table ##SUPPL##0##S2##). The decline in <italic>BCR-ABL1</italic> transcript levels was delayed in patients with suboptimal response to imatinib until crossover to dasatinib. Cumulatively, 147 (85%) patients randomized to dasatinib and 71 (83%) patients randomized to imatinib achieved CCyR. Notably, 29 of the 45 (64%) patients randomized to imatinib who experienced treatment failure achieved CCyR after crossover to dasatinib.</p>", "<p id=\"Par17\">In the ITT population, PFS at 24 months was 96% (95% CI 92–98) for patients randomized to dasatinib and 95% (95% CI 88–98) for patients randomized to imatinib (Fig. ##FIG##3##4a##). According to switch status, PFS at 24 months was 96% (95% CI 92–98) in patients initially randomized to dasatinib (early switch), 93% (95% CI 80–98) in patients initially randomized to imatinib who subsequently crossed over to dasatinib, and 98% (95% CI 84–100) in patients randomized to imatinib and without subsequent crossover (Fig. ##FIG##3##4b##). In the ITT population, OS at 24 months was 98% (95% CI 94–99) in patients randomized to dasatinib and 97% (95% CI 90–99) in patients randomized to imatinib (Fig. ##FIG##4##5a##). By switch status, 24-month OS was 98% (95% CI 94–99) in patients randomized to dasatinib (early switch), 96% (95% CI 83–99) in patients initially randomized to imatinib who later crossed over to dasatinib, and 98% (95% CI 84–100) in patients receiving imatinib without crossover (Fig. ##FIG##4##5b##). Six patients in the dasatinib arm progressed to CML-AP/BC, two of whom progressed after discontinuing dasatinib—one discontinued due to hematologic toxicity (progressed 38 days after last dose) and one discontinued due to treatment failure (progressed 11 months after last dose). One patient in the imatinib arm progressed at month 4, and two patients who crossed over to dasatinib progressed—one crossed over at month 12 (progressed at month 15) and one crossed over at month 24 (progressed at month 59).</p>", "<title>Safety</title>", "<p id=\"Par18\">Treatment-related AEs of any grade occurred in 141 (82%) patients randomized to dasatinib and 67 (78%) randomized to imatinib (Table ##TAB##2##3##). Grade 3/4 treatment-related AEs occurred in 60 (35%) and 36 (42%) patients in the dasatinib and imatinib arms, respectively. Any grade treatment-related AEs were reported in 38 of the 45 patients (84%) who crossed over to dasatinib from imatinib and in 29 of the 41 patients (71%) who remained on imatinib. Serious AEs (of any grade and cause) occurred in 33 (19%) patients on dasatinib and 16 (19%) patients on imatinib, including in eight (18%) patients who crossed over to dasatinib and eight (20%) patients on imatinib without crossover. Pleural effusion occurred in 15 (9%) patients on dasatinib (grade 3/4 severity in three patients), including in five patients (all grade 2–3) who discontinued due to study drug toxicity. In addition, five (11%) patients initially randomized to imatinib who crossed over to dasatinib developed pleural effusion (two cases were grade 3/4). The most common treatment-related non-hematologic AE was headache in patients randomized to dasatinib (15%) and hypophosphatemia in patients randomized to imatinib (13%, Table ##TAB##2##3##). Grade 3/4 headaches occurred in three (2%) patients in the dasatinib arm, and grade 3/4 hypophosphatemia was observed in five (6%) patients in the imatinib arm. In patients receiving imatinib who crossed over to dasatinib, headache was the most frequently observed treatment-related AE (13%). Treatment-related hematologic toxicity was comparable between both treatment arms, with grade 3/4 neutropenia occurring in 21 (12%) patients on dasatinib, 14 (16%) patients on imatinib, and in 13 (29%) patients on dasatinib after crossover (Table ##TAB##2##3##). The occurrence of anemia, thrombocytopenia, and leukopenia was also similar across treatment arms. Arterial occlusive events occurred in two patients treated with dasatinib (cardiac angina and cerebral ischemia) and in one patient treated with imatinib who crossed over to dasatinib (ischemic stroke). In total, nine (4%) patients died: five (3%) were randomized to dasatinib and four (5%) to imatinib (three of whom subsequently crossed over to dasatinib). Of the nine deaths, three were due to disease progression (one patient randomized to dasatinib, two patients randomized to imatinib, both of whom crossed over to dasatinib) and one due to study drug toxicity (imatinib with crossover to dasatinib).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par19\">Achievement of EMR may increase the likelihood of attaining a subsequent DMR and having favorable long-term outcomes, but it is not known whether patients without an EMR at 3 months will benefit from an early switch to a potent second-generation TKI. DASCERN is the first prospective trial to demonstrate the potential benefit of early switching to dasatinib in patients without EMR after 3 months of imatinib treatment. In this study, patients who switched to dasatinib at 3 months had a significantly higher MMR rate at 12 months than patients who remained on imatinib (29% vs. 13%, <italic>P</italic> = 0.005), and cumulatively, by month 24 more patients on dasatinib had achieved MMR (64% vs. 41%) once treatment crossover was accounted for. Cumulative incidence of CCyR was similar in the dasatinib and imatinib arms; however, 29 (64%) patients initially randomized to the imatinib arm achieved a CCyR after having a suboptimal response and crossing over to dasatinib. Overall, these findings support the need for early monitoring and intervention for newly diagnosed patients with CML-CP not receiving a second-generation TKI as first-line therapy, and indicate that patients who fail to achieve EMR with first-line imatinib benefit from switching to dasatinib at 3 months.</p>", "<p id=\"Par20\">Previous studies have shown that early intervention may be considered when patients have suboptimal cytogenetic responses on first-line imatinib [##REF##19402171##15##–##REF##29556695##20##]. Quintas-Cardama et al. retrospectively demonstrated that response rates and survival were most favorable when dasatinib was administered early after imatinib failure, with 72% of patients who received dasatinib after loss of major cytogenetic response (MCyR) to imatinib achieving CCyR, compared with 42% of patients who were treated after loss of both MCyR and CHR [##REF##19402171##15##]. In the same study, event-free survival (EFS) was higher after earlier vs. later dasatinib intervention; the EFS with early intervention was in line with previous reports for second-line imatinib after interferon failure [##REF##19402171##15##, ##REF##11870241##16##]. In the TIDEL (Therapeutic Intensification in De Novo Leukaemia)-II study, patients who started on imatinib and switched to nilotinib due to intolerance, treatment failure, or loss of response, achieved improved survival outcomes (including OS and transformation-free survival), although only a small number of patients (<italic>n</italic> = 54) switched to nilotinib, and the study included an assessment of imatinib plasma trough levels, which are not routinely assessed in clinical practice [##REF##25519749##17##]. In the LASOR (Imatinib Dose Optimization vs. Nilotinib in CML Patients With Suboptimal Response to Imatinib) trial, patients with suboptimal cytogenetic responses to imatinib were more likely to achieve CCyR and MMR after switching to nilotinib, although the difference was not statistically significant [##REF##27890073##18##]. It has also been shown that imatinib dose escalation fails to “rescue” those patients with suboptimal responses [##REF##18768781##19##]. Despite these observations, there has been a general lack of larger, prospective studies exploring the significance of early intervention for patients with suboptimal molecular responses to first-line imatinib.</p>", "<p id=\"Par21\">Attainment of improved and rapid molecular responses with TKI therapy could help decrease the probability of transformation and improve long-term outcomes. It has been demonstrated that patients treated with first-line imatinib who did not achieve an EMR had significantly lower 8-year probabilities of OS (57% vs. 93%), PFS, and complete molecular response than patients who achieved EMR at 3 months [##REF##22067393##21##]. Consequently, achieving EMR at the 3-month molecular milestone is considered an optimal response [##UREF##0##1##]. More patients treated with second-generation TKIs achieve these treatment goals compared with those treated with imatinib [##REF##27217448##2##, ##REF##24335106##3##]. In the ENESTnd study (Study of Imatinib vs. Nilotinib in Adult Patients With Newly Diagnosed Philadelphia Chromosome Positive Chronic Myelogenous Leukemia in Chronic Phase), 89–91% of patients who received nilotinib achieved EMR at 3 months, compared with 67% of patients who received imatinib [##REF##24335106##3##]. Similarly, in the DASISION study, 84% of patients achieved EMR at 3 months with dasatinib vs. 64% with imatinib [##REF##27217448##2##]. Furthermore, patients who achieved EMR with dasatinib had improved 5-year OS and PFS rates, as well as reduced rates of transformation [##REF##27217448##2##].</p>", "<p id=\"Par22\">In DASCERN, no differences in OS and PFS outcomes were observed between the treatment arms—a finding that was likely influenced by the short follow-up period of this study. However, an extended follow-up will be of interest as the crossover from the imatinib arm is expected to have an impact on the differences in long-term outcomes. Interestingly, PFS in DASCERN appears higher (96% [95% CI 92–98]) than has been previously reported for dasatinib [##REF##27217448##2##]. The seemingly favorable PFS may be related to the study definition of progression (transformation to CML-AP/BC or death from any cause since randomization), which differs from the definition of progression historically used in clinical trials with dasatinib. For example, progression was defined in DASISION as loss of CHR, MCyR, transformation to CML-AP/BC, death, or increasing white blood cell counts [##REF##27217448##2##, ##REF##20525995##22##]. In addition, patients who had progressed before 3 months were not eligible for this trial. Excluding these patients (although few) from the PFS calculation may also influence the overall PFS rate. Furthermore, ~50% of patients who were randomized to remain on imatinib experienced treatment failure and required crossover to dasatinib at a later time. In this subgroup of patients (later switch/crossover), a trend toward worse PFS was observed compared with those who were randomized to dasatinib at study entry (i.e., 3 months after start of imatinib; early switch), suggesting that a delayed treatment switch may have increased the risk of transformation or death in this patient population. Notably, a previous long-term follow-up study has shown that most progression events occur within the first 3 years of imatinib treatment [##REF##19282833##23##]. These observations highlight the need for early monitoring and intervention in patients with suboptimal responses to imatinib.</p>", "<p id=\"Par23\">The early switch to dasatinib in DASCERN did not increase the incidence of treatment-related events. In addition, the rates of treatment-related hematologic AEs in those who switched to dasatinib and in those who remained on imatinib were similar. Interestingly, the incidence of pleural effusion in this study (9%) was lower than that seen in other dasatinib studies [##REF##22160483##24##, ##REF##19924787##25##]. Indeed, in a 2-year follow-up of the DASISION study, pleural effusion was observed in 14.3% of patients, with a discontinuation rate of 1.9% [##REF##22160483##24##]. A similar rate (14%) was observed in the phase 3 dasatinib dose optimization study (CA180–034), in which most cases were managed with temporary dose interruption or reduction; only three (1.4%) patients required dasatinib discontinuation due to pleural effusion [##REF##19924787##25##]. The lower incidence of pleural effusion in DASCERN may be due to the relatively young age of the patients, as younger patients have been reported to be at a reduced risk of developing pleural effusion after initiating dasatinib therapy [##REF##30093398##26##]. However, a longer follow-up is required as pleural effusions may occur later in the course of therapy with dasatinib.</p>", "<p id=\"Par24\">In this first prospective randomized study to explore the benefit of early switching to dasatinib, the greater response rates with dasatinib and the observation that approximately half of patients who did not achieve EMR with imatinib subsequently met treatment failure criteria and crossed over to the dasatinib arm, provide further support for using strategies that increase the probability of achieving optimal responses early on in the treatment paradigm. As MMR rates are known to improve with longer treatment duration [##REF##30120281##27##], additional follow-up will help to determine if the rates continue to favor the use of dasatinib and whether this early benefit translates into a greater probability of achieving DMR and improved PFS and OS. In summary, initial findings from DASCERN provide new insight into the potential benefit of switching to dasatinib in patients failing to achieve important treatment milestones with first-line imatinib. Furthemore, these data support the importance of early monitoring for patients who do not receive a second-generation TKI as their first-line treatment, and suggest that pre-emptive switching to dasatinib in such instances may provide clinical benefit.</p>" ]
[]
[ "<p id=\"Par1\">Early molecular response is associated with improved probability of deep molecular response and superior survival in patients with CML-CP. However, ~1 in 3 patients on first-line imatinib do not achieve this threshold. The phase 2b DASCERN trial (NCT01593254) assessed the outcome of early switch to dasatinib in patients with suboptimal response to first-line imatinib. Adult patients with CML-CP were randomized (2:1) to receive 100 mg dasatinib (<italic>n</italic> = 174) or continue imatinib at ≥400 mg (<italic>n</italic> = 86). The primary endpoint was the rate of major molecular response (MMR) at 12 months, which was 29% (dasatinib) and 13% (imatinib; <italic>P</italic> = 0.005). After ≥2 years of follow-up, 45 patients (52%) randomized to continue imatinib had crossed over to dasatinib. Considering treatment crossover, the 2-year cumulative MMR rate was 64% with dasatinib and 41% with imatinib (66% and 67%, respectively by intent-to-treat). Adverse events were consistent with the established safety profiles of both drugs. The results of this first prospective study support early monitoring of patients treated with first-line imatinib, and suggest that switching to dasatinib in cases of suboptimal response may offer clinical benefit. Further follow-up is needed to assess the long-term clinical benefit of early switching.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0805-1) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>This study was sponsored and funded by Bristol Myers Squibb. We thank the patients who participated in this study and the clinical study teams. Bristol Myers Squibb policy on data sharing may be found at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bms.com/researchers-and-partners/independent-research/data-sharing-request-process.html\">https://www.bms.com/researchers-and-partners/independent-research/data-sharing-request-process.html</ext-link>. Medical writing and editorial support were provided by Jane Cheung, PhD, of Caudex, funded by Bristol Myers Squibb.</p>", "<title>Author contributions</title>", "<p>JEC, AH, JR, MS, and GS were part of the Steering Committee. QJ, JxW, JyW, HZ, and XL enrolled patients. DWK was part of the Steering Committee and enrolled patients. PMR and OS collected and interpreted data. RG proposed and reviewed the amended protocol. All authors provided directions and guidance to paper writing, and reviewed, edited, and approved the final paper.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par25\">JEC has served as a consultant to and received research funding from Bristol Myers Squibb, Novartis, Pfizer, and Takeda. QJ, JyW, HZ, and XL declare no conflicts to disclose. AH has received research funding from Bristol Myers Squibb, Incyte, Novartis, Pfizer, and Takeda. DWK has received research funding from Bristol Myers Squibb, IIyang, Novartis, and Pfizer. JR has served as a consultant to Bristol Myers Squibb, Novartis, Pfizer, and Takeda, and has received research funding from Novartis. JxW has received research funding from Celgene. MS has served as a consultant to, or served on the board of directors/advisors of Abbvie, Celgene, Incyte, Karyopharm, and Takeda, and received funding from Takeda and holds equity ownership in Karyopharm. PMR is an employee and holds equity ownership with Bristol Myers Squibb. OS and RG are employees of Bristol Myers Squibb. GS has served as a consultant to Ariad, Bristol Myers Squibb, Incyte, Novartis, and Pfizer.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Study design.</title><p>*Patients initially randomized to imatinib, meeting ELN 2013 failure criteria, and without dasatinib-resistant mutations, were crossed over to the dasatinib arm. BID twice daily, CHR complete hematologic response, ELN European LeukemiaNet, IS International Scale, LPFV last patient first visit, QD once daily.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>MMR at 12 months in the ITT population (primary endpoint).</title><p>Error bars represent 95% CI. CI confidence interval, ITT intent-to-treat, MMR major molecular response.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>MMR after a minimum follow-up of 24 months.</title><p><bold>a</bold> MMR according to study population and crossover. <bold>b</bold> Cumulative incidence of MMR accounting for competing risk. CI confidence interval, ITT intent-to-treat, MMR major molecular response.</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>PFS.</title><p>Kaplan–Meier estimate of PFS in ITT population (<bold>a</bold>) and by switch status (<bold>b</bold>). PFS was defined as the time from randomization to transformation to CML-AP/BC or death, whichever occurred first. All patients who discontinued study treatment were followed for progression and survival unless consent was withdrawn. CI confidence interval, ITT intent-to-treat, NE not evaluable, PFS progression-free survival.</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>OS.</title><p>Kaplan–Meier estimate of OS in ITT population (<bold>a</bold>) and by switch status (<bold>b</bold>). CI confidence interval, ITT intent-to-treat, NE not evaluable, OS overall survival.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Baseline patient characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Dasatinib<break/> (<italic>n</italic> = 174)</th><th>Imatinib<break/> (<italic>n</italic> = 86)</th><th>Total<break/> (<italic>N</italic> = 260)</th></tr></thead><tbody><tr><td>Age, median (range), years</td><td>35 (18–82)</td><td>40 (18–73)</td><td>37 (18–82)</td></tr><tr><td colspan=\"4\">Age categorization</td></tr><tr><td>  &lt;65 years</td><td>166 (95)</td><td>82 (95)</td><td>248 (95)</td></tr><tr><td>  ≥65 years</td><td>8 (5)</td><td>4 (5)</td><td>12 (5)</td></tr><tr><td>Male</td><td>133 (76)</td><td>70 (81)</td><td>203 (78)</td></tr><tr><td colspan=\"4\">Race</td></tr><tr><td>  White</td><td>36 (21)</td><td>15 (17)</td><td>51 (20)</td></tr><tr><td>  Black or African American</td><td>4 (2)</td><td>3 (4)</td><td>7 (3)</td></tr><tr><td>  Asian</td><td>127 (73)</td><td>63 (73)</td><td>190 (73)</td></tr><tr><td>  Other</td><td>7 (4)</td><td>5 (6)</td><td>12 (5)</td></tr><tr><td colspan=\"4\">Sokal score</td></tr><tr><td>  Low</td><td>47 (27)</td><td>26 (30)</td><td>73 (28)</td></tr><tr><td>  Intermediate</td><td>51 (29)</td><td>26 (30)</td><td>77 (30)</td></tr><tr><td>  High</td><td>44 (25)</td><td>19 (22)</td><td>63 (24)</td></tr><tr><td>  Unknown</td><td>32 (18)</td><td>15 (17)</td><td>47 (18)</td></tr><tr><td colspan=\"4\">ECOG performance status</td></tr><tr><td>  0</td><td>142 (82)</td><td>75 (87)</td><td>217 (84)</td></tr><tr><td>  1</td><td>27 (16)</td><td>10 (12)</td><td>37 (14)</td></tr><tr><td>  2</td><td>0</td><td>1 (1)</td><td>1 (&lt;1)</td></tr><tr><td>  Not reported</td><td>5 (3)</td><td>0</td><td>5 (2)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Patient disposition of treated patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Dasatinib<break/> (<italic>n</italic> = 171)</th><th>Imatinib<break/> (<italic>n</italic> = 86)</th><th>Total<break/> (<italic>N</italic> = 257)</th></tr></thead><tbody><tr><td>Continuing on treatment</td><td>135 (79)</td><td>68 (79)</td><td>203 (79)</td></tr><tr><td>Crossed over to dasatinib</td><td>–</td><td>45 (52)</td><td>45 (18)</td></tr><tr><td>  Imatinib failure</td><td>–</td><td>44 (51)</td><td>44 (17)</td></tr><tr><td>  Suboptimal response</td><td>–</td><td>1 (1)</td><td>1 (&lt;1)</td></tr><tr><td>Not continuing on treatment</td><td>36 (21)</td><td>18 (21)</td><td>54 (21)</td></tr><tr><td>  Disease progression</td><td>5 (3)</td><td>1 (1)</td><td>6 (2)</td></tr><tr><td>  Study drug toxicity</td><td>12 (7)</td><td>4 (5)</td><td>16 (6)</td></tr><tr><td>  Death</td><td>1 (1)</td><td>2 (2)</td><td>3 (1)</td></tr><tr><td>  Other</td><td>18 (11)</td><td>11 (13)</td><td>29 (11)</td></tr><tr><td>Continuing in the study</td><td>23 (13)</td><td>13 (15)</td><td>36 (14)</td></tr><tr><td>Not continuing in the study</td><td>13 (8)</td><td>5 (6)</td><td>18 (7)</td></tr><tr><td>  Withdrew consent</td><td>2 (1)</td><td>0</td><td>2 (1)</td></tr><tr><td>  Death</td><td>3 (2)</td><td>4 (5)</td><td>7 (3)</td></tr><tr><td>  Lost to follow-up</td><td>1 (1)</td><td>0</td><td>1 (&lt;1)</td></tr><tr><td>  Other</td><td>7 (4)</td><td>1 (1)</td><td>8 (3)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Any grade and grade 3/4 treatment-related AEs reported in ≥5% of all randomized patients in either arm.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Patients with AEs</th><th colspan=\"2\">Patients randomized to dasatinib<break/> (<italic>n</italic> = 171)</th><th colspan=\"2\">Patients randomized to imatinib<break/> (<italic>n</italic> = 86)</th><th colspan=\"2\">Patients on dasatinib after crossing over from imatinib<break/> (<italic>n</italic> = 45)</th><th colspan=\"2\">Patients on imatinib with no crossover to dasatinib<break/> (<italic>n</italic> = 41)</th></tr><tr><th/><th>Any grade</th><th>Grade 3–4</th><th>Any grade</th><th>Grade 3–4</th><th>Any grade</th><th>Grade 3–4</th><th>Any grade</th><th>Grade 3–4</th></tr></thead><tbody><tr><td>Total patients with an AE</td><td>141 (82)</td><td>60 (35)</td><td>67 (78)</td><td>36 (42)</td><td>38 (84)</td><td>25 (56)</td><td>29 (71)</td><td>11 (27)</td></tr><tr><td colspan=\"9\">Non-hematologic AEs</td></tr><tr><td>  Headache</td><td>26 (15)</td><td>3 (2)</td><td>9 (10)</td><td>0</td><td>6 (13)</td><td>0</td><td>3 (7)</td><td>0</td></tr><tr><td>  Diarrhea</td><td>16 (9)</td><td>0</td><td>8 (9)</td><td>0</td><td>4 (9)</td><td>0</td><td>4 (10)</td><td>0</td></tr><tr><td>  Hypophosphatemia</td><td>16 (9)</td><td>2 (1)</td><td>11 (13)</td><td>5 (6)</td><td>5 (11)</td><td>1 (2)</td><td>6 (15)</td><td>4 (10)</td></tr><tr><td>  Pleural effusion</td><td>15 (9)</td><td>3 (2)</td><td>5 (6)</td><td>2 (2)</td><td>5 (11)</td><td>2 (4)</td><td>0</td><td>0</td></tr><tr><td>  Rash</td><td>14 (8)</td><td>0</td><td>8 (9)</td><td>1 (1)</td><td>2 (4)</td><td>0</td><td>6 (15)</td><td>1 (2)</td></tr><tr><td>  Nausea</td><td>13 (8)</td><td>0</td><td>8 (9)</td><td>0</td><td>2 (4)</td><td>0</td><td>6 (15)</td><td>0</td></tr><tr><td>  URT infections</td><td>10 (6)</td><td>0</td><td>3 (3)</td><td>0</td><td>3 (7)</td><td>0</td><td>0</td><td>0</td></tr><tr><td>  Asthenia</td><td>8 (5)</td><td>0</td><td>3 (3)</td><td>0</td><td>2 (4)</td><td>0</td><td>1 (2)</td><td>0</td></tr><tr><td>  Dizziness</td><td>8 (5)</td><td>0</td><td>2 (2)</td><td>0</td><td>2 (4)</td><td>0</td><td>0</td><td>0</td></tr><tr><td>  Pain in extremity</td><td>6 (4)</td><td>0</td><td>6 (7)</td><td>0</td><td>2 (4)</td><td>0</td><td>4 (10)</td><td>0</td></tr><tr><td>  Upper abdominal pain</td><td>5 (3)</td><td>0</td><td>4 (5)</td><td>0</td><td>3 (7)</td><td>0</td><td>1 (2)</td><td>0</td></tr><tr><td>  Vomiting</td><td>4 (2)</td><td>0</td><td>4 (5)</td><td>0</td><td>2 (4)</td><td>0</td><td>2 (5)</td><td>0</td></tr><tr><td>  Fatigue</td><td>4 (2)</td><td>0</td><td>6 (7)</td><td>0</td><td>3 (7)</td><td>0</td><td>3 (7)</td><td>0</td></tr><tr><td>  Eyelid edema</td><td>2 (1)</td><td>0</td><td>8 (9)</td><td>0</td><td>4 (9)</td><td>0</td><td>4 (10)</td><td>0</td></tr><tr><td>  Hypocalcemia</td><td>2 (1)</td><td>0</td><td>6 (7)</td><td>0</td><td>4 (9)</td><td>0</td><td>2 (5)</td><td>0</td></tr><tr><td>  Muscle spasms</td><td>2 (1)</td><td>0</td><td>8 (9)</td><td>0</td><td>1 (2)</td><td>0</td><td>7 (17)</td><td>0</td></tr><tr><td colspan=\"9\">Hematologic AEs</td></tr><tr><td>  Neutropenia</td><td>37 (22)</td><td>21 (12)</td><td>25 (29)</td><td>14 (16)</td><td>19 (42)</td><td>13 (29)</td><td>6 (15)</td><td>1 (2)</td></tr><tr><td>  Anemia</td><td>39 (23)</td><td>11 (6)</td><td>21 (24)</td><td>3 (3)</td><td>13 (29)</td><td>3 (7)</td><td>8 (20)</td><td>0</td></tr><tr><td>  Thrombocytopenia</td><td>39 (23)</td><td>18 (11)</td><td>15 (17)</td><td>9 (10)</td><td>13 (29)</td><td>7 (16)</td><td>2 (5)</td><td>2 (5)</td></tr><tr><td>  Leukopenia</td><td>14 (8)</td><td>2 (1)</td><td>11 (13)</td><td>2 (2)</td><td>7 (16)</td><td>1 (2)</td><td>4 (10)</td><td>1 (2)</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>Values are <italic>n</italic> (%) unless otherwise noted.</p><p><italic>ECOG</italic> Eastern Cooperative Oncology Group.</p></table-wrap-foot>", "<table-wrap-foot><p>Values are <italic>n</italic> (%).</p></table-wrap-foot>", "<table-wrap-foot><p>Values are <italic>n</italic> (%).</p><p><italic>AE</italic> adverse event, <italic>URT</italic> upper respiratory tract.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"41375_2020_805_MOESM1_ESM.docx\"><caption><p>Supplementary information</p></caption></media>" ]
[{"label": ["1."], "mixed-citation": ["National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: chronic myeloid leukemia. 2019. "], "ext-link": ["https://www.nccn.org/professionals/physician_gls/PDF/cml.pdf"]}, {"label": ["4."], "surname": ["Brummendorf", "Kantarjian", "Gambacorti-Passerini", "Guilhot", "Akard", "Doshi"], "given-names": ["TH", "H", "C", "F", "L", "V"], "article-title": ["Assessment of early molecular response as a predictor of long-term clinical outcomes in the phase 3 BELA study"], "source": ["Blood."], "year": ["2012"], "volume": ["120"], "fpage": ["69"], "pub-id": ["10.1182/blood.V120.21.69.69"]}, {"label": ["8."], "mixed-citation": ["Health NIo. Common terminology criteria for adverse events (CTCAE) version 5.0. 2017. "], "ext-link": ["https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick_Reference_8.5x11.pdf"]}, {"label": ["9."], "surname": ["Cochran"], "given-names": ["WG"], "article-title": ["Some methods for strengthening the common X2 tests"], "source": ["Biometrics."], "year": ["1954"], "volume": ["10"], "fpage": ["417"], "lpage": ["51"], "pub-id": ["10.2307/3001616"]}, {"label": ["10."], "surname": ["Kaplan", "Meier"], "given-names": ["EK", "P"], "article-title": ["Nonparametric estimation from incomplete observations"], "source": ["J Am Stat Assoc"], "year": ["1958"], "volume": ["53"], "fpage": ["457"], "lpage": ["81."], "pub-id": ["10.1080/01621459.1958.10501452"]}, {"label": ["11."], "collab": ["Cox DR."], "article-title": ["Regression models and life tables"], "source": ["J R Stat Soc Ser B"], "year": ["1972"], "volume": ["34"], "fpage": ["187"], "lpage": ["220"]}, {"label": ["12."], "surname": ["Harrington", "Fleming"], "given-names": ["DP", "TR"], "article-title": ["A class of rank test procedures for censored survival data"], "source": ["Biometrika."], "year": ["1982"], "volume": ["69"], "fpage": ["133"], "lpage": ["43."], "pub-id": ["10.1093/biomet/69.3.553"]}, {"label": ["13."], "surname": ["Brookmeyer", "Crowley"], "given-names": ["R", "J"], "article-title": ["A confidence interval for the median survival time"], "source": ["Biometrics."], "year": ["1982"], "volume": ["38"], "fpage": ["29"], "lpage": ["41"], "pub-id": ["10.2307/2530286"]}, {"label": ["14."], "surname": ["Fine", "Gray"], "given-names": ["JP", "RJ"], "article-title": ["A proportional hazards model for the subdistribution of a competing risk"], "source": ["J Am Stat Assoc"], "year": ["1999"], "volume": ["94"], "fpage": ["496"], "lpage": ["509"], "pub-id": ["10.1080/01621459.1999.10474144"]}]
{ "acronym": [], "definition": [] }
27
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2024-01-13 23:35:06
Leukemia. 2020 Apr 7; 34(8):2064-2073
oa_package/a9/fc/PMC7387297.tar.gz
PMC7387299
32601376
[ "<title>Introduction</title>", "<p id=\"Par2\">For patients with Philadelphia chromosome-positive (Ph+) chronic-phase chronic myeloid leukemia (CML), four baseline prognostic scores were addressed by the most recent European LeukemiaNet (ELN) recommendations [##REF##32127639##1##]. First, in 1984, the Sokal score was developed to allocate chemotherapy-treated patients into three risk groups of approximately equal size predicting significantly different overall survival (OS) probabilities [##REF##25814087##2##, ##REF##6584184##3##]. In 1998, the Euro score was proposed to discriminate OS between three risk groups of patients treated with interferon alpha [##REF##25814087##2##, ##REF##9625174##4##]. Using data on patients who were treated with imatinib, in 2011 the European Treatment and Outcome Study for CML (EUTOS) score identified two risk groups with significantly different probabilities of complete cytogenetic response after 18 months of therapy [##REF##25814087##2##, ##REF##21536864##5##], and in 2016, the EUTOS Long-Term Survival (ELTS) score was introduced in order to distinguish three risk groups with significantly different probabilities of dying of CML [##REF##26416462##6##].</p>", "<p id=\"Par3\">Regarding its primary endpoint, the ELTS score was successfully validated in an independent patient sample and showed a superior risk group discrimination compared with the Sokal score [##REF##26416462##6##]. The Sokal score identified 41% of patients as low-risk and 23% as high-risk. The ELTS score, however, identified an absolute proportion of 20% more low-risk patients and 11% fewer high-risk patients [##REF##26416462##6##]. Ten years after the start of first-line imatinib treatment, probabilities of dying of CML were 6 and 8% according to Hehlmann et al. [##REF##28804124##7##] and Molica et al. [##REF##31079264##8##], respectively. These results are rather in line with 12% high-risk patients as suggested by the ELTS score than with 23% high-risk patients as defined by the Sokal score.</p>", "<p id=\"Par4\">The Sokal score has been particularly popular [##REF##32127639##1##]. This may have been due to the preference for risk groups of more equal size, but a more likely reason was lack of acceptance of newer scores. Accordingly, analyses established in major randomized trials continued to be risk stratified by the Sokal score [##REF##28273028##9##–##REF##29091516##13##]. Here, some association between Sokal risk group and clinical outcome was identified [##REF##28273028##9##–##REF##25196702##11##, ##REF##29091516##13##]. While the most recent ELN recommendations advise risk assessment with the ELTS score [##REF##32127639##1##], it is hence still essential to provide convincing data-based evidence when arguing for its preference over others.</p>", "<p id=\"Par5\">The aim of this work was to compare the prognostic discrimination between the Sokal score [##REF##6584184##3##] and the ELTS score [##REF##26416462##6##] and to provide an evidence-based recommendation of which score to apply. Although the focus was on the comparison between the enduringly popular Sokal score and the relatively new ELTS score, results for the Euro and the EUTOS score are also provided.</p>" ]
[ "<title>Patients and methods</title>", "<title>Patients</title>", "<p id=\"Par6\">In 2007, a registry of CML patients was established by the ELN and maintained within the EUTOS framework [##REF##21536864##5##]. This registry contains individual data on adult patients who were prospectively enrolled between 2002 and 2006, either within or outwith a clinical trial (in-study and out-study sections, respectively) [##REF##21536864##5##, ##REF##23752173##14##]. Further patient eligibility criteria for both registry sections were diagnosis of Ph+ and/or BCR-ABL1-positive CML in chronic phase, no transcript type other than b2a2 and/or b3a2, and any form of imatinib-based treatment within 6 months from diagnosis [##REF##21536864##5##, ##REF##23752173##14##]. In accordance with these criteria, 2205 patients with data on all variables of each score were retrieved from the in-study section [##REF##26416462##6##]. While data on the in-study patients remained unchanged for the present report, follow-up was updated in 2016 for most patients in the out-study section. Two of the 1120 cases reported earlier [##REF##26416462##6##] were identified as double data entries and were left out from further analyses. A third population-based component of the registry accumulated data on adult patients newly diagnosed between 2008 and 2013 [##REF##27568522##15##]. Apart from adulthood, Ph+ and/or BCR-ABL1-positive CML was the only inclusion criterion [##REF##27568522##15##]. For the population-based section, the same inclusion criteria were chosen as for the two other sections, except that the restriction on patients with first-line imatinib treatment within 6 months from diagnosis was relaxed. Of the 1831 patients finally included, 68 had received first-line dasatinib (4%) and 247 (14%) first-line nilotinib treatment; similarly for 78 patients (4%), treatment start was later than 6 months after diagnosis. Relaxation of the two criteria was based on the observation that both had no association with survival probabilities in the population-based section.</p>", "<p id=\"Par7\">At first, the score comparisons were based on the 2949 patients with data entirely independent of any score development. In a second step, data of the in-study sample used for the development of the ELTS score were added. Only after addition of these patients was the number of events sufficient in order to assess the adequacy of low- or high-risk categorization between the different scores.</p>", "<title>Definitions and endpoints</title>", "<p id=\"Par8\">OS time was calculated from the start date of tyrosine kinase inhibitor (TKI) treatment to death or to the latest follow-up date. Progression-free survival time was calculated like survival time but ended with the observation of progression. Progression was defined by the observation of accelerated phase or blast crisis, with both phases determined according to the ELN criteria [##REF##23803709##16##]. Chronic phase was defined by the absence of progression [##REF##23803709##16##]. Only death after recorded disease progression was regarded as “death due to CML”. Death without prior disease progression was rated as “death unrelated to CML”. For details regarding the calculation of the Sokal [##REF##6584184##3##], the ELTS [##REF##26416462##6##], the Euro [##REF##9625174##4##], and the EUTOS score [##REF##21536864##5##], see Supplementary Table ##SUPPL##0##1##.</p>", "<title>Statistical analysis</title>", "<p id=\"Par9\">OS probabilities were calculated by the Kaplan–Meier method, and the hazards ratios (HRs) for dying from any cause were calculated by the Cox regression model [##UREF##0##17##]. When differentiating competing causes of death, cumulative incidence probabilities of dying of CML were obtained using the Aalen–Johansen estimator [##UREF##1##18##, ##REF##11933038##19##] and the subdistribution hazards ratios (SHRs) for dying of CML were obtained using the Fine–Gray model [##UREF##2##20##]. Like the Aalen–Johansen estimator, the Fine–Gray model and its SHRs consider death unrelated to CML as the competing event to death due to CML, the event of interest. Both the hazards from the Cox model as well as the SHRs were compared by the Wald test. To assess discrimination of prognostic models, concordance probabilities were estimated using the truncated concordance index suggested by Wolbers et al. [##REF##24493091##21##]. For the description of discrimination ability over time, the truncation times 1, 5, and 10 years were considered. A higher concordance index hints at a better discrimination of the survival outcome. With indices greater than 50, a prognostic model provides clinically useful information different from chance; the closer to 100, the more supportive the model is.</p>", "<p id=\"Par10\">Lauseker and Zu Eulenburg elucidated that the use of the competing risk model leads to biased cumulative incidence probability estimates when the censoring mechanism differs between status, e.g., between patients in chronic- or progressive- phase [##REF##30680772##22##]. In the case of a status-dependent censoring mechanism, they showed that the progressive illness-death model should be preferred over the competing risk model (see Supplementary Fig. ##SUPPL##0##1## for a comparison of the models). Accordingly, in the presence of status-dependent censoring, the ability to discriminate probabilities of dying of CML was additionally investigated with the progressive illness-death model. For this, the associations between risk group and transition probabilities were considered [##UREF##3##23##].</p>", "<p id=\"Par11\">For the two-sided <italic>P</italic> values, the unadjusted significance level of 0.05 was applied for all statistical tests. Estimates were presented with 95% confidence interval (95% CI). More on statistical methods is given in the Supplementary appendix.</p>" ]
[ "<title>Results</title>", "<title>Prognostic discrimination in 2949 patients from the combined out-study and population-based sections</title>", "<p id=\"Par12\">For the out-study data, origin and details as well as the validation of a significant discrimination of the probabilities of dying of CML by the ELTS score have been previously described [##REF##26416462##6##, ##REF##23752173##14##].</p>", "<p id=\"Par13\">Adding the population-based section to the 1118 out-study patients, a validation sample of 2949 patients independent of any score development was achieved. The combined sample consisted of 52% males. Median age of the 2949 patients was 52 years (range: 18–91 years) and median follow-up was 3.3 years (range: 0.01–12.6 years). Altogether, 236 patients died, of whom 89 (38%) died of CML. Six-year OS probability in the 2949 patients was 88% (95% CI: 86–89%), and 6-year probability of death due to CML was 5% (95% CI: 4–6%).</p>", "<title>Prognostic discrimination of cumulative incidence probabilities of dying of CML</title>", "<p id=\"Par14\">The high-risk group of the Sokal score (<italic>n</italic> = 698, 24%), though not the intermediate-risk group (<italic>n</italic> = 1177, 40%), had significantly higher probabilities of dying because of CML than the low-risk group (<italic>n</italic> = 1074, 36%), <italic>P</italic> &lt; 0.0001 and <italic>P</italic> = 0.0835, respectively (Fig. ##FIG##0##1a##). The corresponding SHRs were 3.559 (95% CI: 2.030–6.240) and 1.668 (95% CI: 0.934–2.978). The concordance indices at 1, 5, and 10 years were 59.7, 62.4, and 63.3, respectively.</p>", "<p id=\"Par15\">With the ELTS score, both the intermediate- (<italic>n</italic> = 853, 29%; <italic>P</italic> = 0.0031) and the high-risk group (<italic>n</italic> = 408, 14%; <italic>P</italic> &lt; 0.0001) had significantly higher probabilities of dying because of CML than the low-risk group (<italic>n</italic> = 1688, 57%, Fig. ##FIG##0##1b##). The corresponding SHRs were 2.203 (95% CI: 1.306–3.718) and 5.646 (95% CI: 3.397–9.387). The concordance indices at 1, 5, and 10 years were 68.0, 66.0, and 68.1. Discrimination abilities were worse with the Euro and the EUTOS score (Supplementary Fig. ##SUPPL##0##2a–b##). The Euro score was not able to find a significant discrimination between the intermediate- and the low-risk group, and the EUTOS score was not able to find a significant discrimination between the low- and the high-risk group.</p>", "<title>State-dependent censoring: application of the progressive illness-death model</title>", "<p id=\"Par16\">In the combined out-study/population-based sample, 153 patients (5%) experienced progression. The cumulative hazard of censoring was significantly higher for patients in progressive phase (<italic>P</italic> &lt; 0.0001). Differences in the state occupation probabilities for death after progression were observed (Supplementary Fig. ##SUPPL##0##3##). After 8 years, the probability of death after progression was 7.3% with the progressive illness-death model and 5.7% with the competing risk model. In contrast, for death without progression probability differences were small (10.5 and 10.6%).</p>", "<p id=\"Par17\">The estimated associations between risk group and transition probabilities in the progressive illness-death model are shown in Supplementary Table ##SUPPL##0##2##. Compared with the ELTS score, none of the three other prognostic models displayed a better discrimination of transition probabilities (Supplementary Table ##SUPPL##0##2##).</p>", "<title>Prognostic discrimination of OS probabilities</title>", "<p id=\"Par18\">The intermediate- (HR: 2.256 [95% CI: 1.590–3.201] and high-risk groups (HR: 3.384 [95% CI: 2.359–4.852] of the Sokal score had significantly lower survival probabilities than the low-risk group with both <italic>P</italic> &lt; 0.0001 (Fig. ##FIG##1##2a##). The concordance indices at 1, 5, and 10 years were 62.9, 62.0, and 61.3, respectively.</p>", "<p id=\"Par19\">With slightly higher hazard ratios and concordance indices of 65.6, 64.0, and 64.0 at 1, 5, and 10 years, the same was observed for the intermediate- (HR: 2.479 [95% CI: 1.836–3.345] and high-risk groups (HR: 4.012 [95% CI: 2.884–5.582] of the ELTS score (Fig. ##FIG##1##2b##).</p>", "<p id=\"Par20\">While the HRs and the concordances indices of the Euro score were slightly less favorable than the ELTS score, the EUTOS score failed to discriminate risk groups (Supplementary Fig. ##SUPPL##0##4a–b##).</p>", "<title>Prognostic discrimination in 5154 patients from all three combined registry sections</title>", "<p id=\"Par21\">The sample of all three combined registry sections consisted of 5154 patients with 52% males and a median age of 52 years (range: 18–91 years). With a median follow-up of 5.3 years (range: 0.01–12.6 years), 429 deaths were recorded, 175 (41%) of which were due to CML. Six-year survival probability of all patients was 90% (95% CI: 89–81%) and 6-year probability of death due to CML was 4% (95% CI: 4–5%).</p>", "<title>Prognostic discrimination of cumulative incidence probabilities of dying of CML</title>", "<p id=\"Par22\">The intermediate- (<italic>n</italic> = 1975, 38%) and the high-risk groups of the Sokal score (<italic>n</italic> = 1197, 23%) had significantly higher cumulative incidence probabilities of dying due to CML than the low-risk group (<italic>n</italic> = 1982, 38%), <italic>P</italic> = 0.0088 and <italic>P</italic> &lt; 0.0001, respectively (Fig. ##FIG##2##3a##). The corresponding SHRs were 1.695 (95% CI: 1.142–2.515) and 3.161 (95% CI: 2.146–4.655). The concordance indices at 1, 5, and 10 years were 58.8, 62.1, and 62.2.</p>", "<p id=\"Par23\">Of the 1197 patients allocated to high-risk by the Sokal score, 671 (56%) were classified as non-high-risk by the ELTS score. Compared with the 526 patients identified as high-risk by both scores, the cumulative incidence probabilities of dying because of CML were significantly lower for the 671 ELTS non-high-risk patients (SHR: 0.415 [95% CI: 0.256–0.671], <italic>P</italic> = 0.0003, Fig. ##FIG##2##3b##). The concordance indices at 1, 5, and 10 years were 63.3, 60.8, and 59.9, respectively.</p>", "<p id=\"Par24\">Compared with the low-risk group (<italic>n</italic> = 3037, 59%), the cumulative incidence probabilities of dying because of CML were significantly higher in the intermediate- (<italic>n</italic> = 1449, 28%, SHR: 2.584 [95% CI: 1.795–3.721]) and the high-risk groups (<italic>n</italic> = 668, 13% SHR: 5.667 [95% CI: 3.912–8.209]) of the ELTS score, with both <italic>P</italic> &lt; 0.0001 (Fig. ##FIG##3##4a##). The concordance indices at 1, 5, and 10 years were 69.6, 66.8, and 67.3, respectively.</p>", "<p id=\"Par25\">Of the 3037 patients identified as low-risk by the ELTS score, the Sokal score allocated 1200 (40%) to non-low-risk. In relation to the low-risk patients, the cumulative incidence probabilities of dying of CML of the 1200 Sokal non-low-risk patients were hardly different (SHR: 1.129 [95% CI: 0.653–1.951], <italic>P</italic> = 0.6635, Fig. ##FIG##3##4b##).</p>", "<p id=\"Par26\">With reference to its low-risk group, the Euro score identified significantly higher cumulative incidence probabilities of dying because of CML in high-risk patients (<italic>P</italic> &lt; 0.0001) but failed to do so in patients with intermediate risk (<italic>P</italic> = 0.3768, Supplementary Fig. ##SUPPL##0##5a##). The EUTOS score found significantly higher cumulative incidence probabilities of dying in high-risk patients (<italic>P</italic> = 0.0002, Supplementary Fig. ##SUPPL##0##5b##).</p>", "<title>No state-dependent censoring in the 5154 patients from all three combined registry sections</title>", "<p id=\"Par27\">In the patient sample made up of data from all three registry sections, 275 patients had disease progression (5%). The cumulative hazard of censoring was not significantly different between the phases (<italic>P</italic> = 0.2868) and differences in the state occupation probabilities between the statistical models were not of any relevance (Supplementary Fig. ##SUPPL##0##6##).</p>", "<title>Prognostic discrimination of OS probabilities</title>", "<p id=\"Par28\">The intermediate- (HR: 2.049 [95% CI: 1.607–2.611]) and high-risk groups (HR: 2.596 [95% CI: 2.009–3.355]) of the Sokal score had significantly lower survival probabilities than the low-risk group, with both <italic>P</italic> &lt; 0.0001 (Fig. ##FIG##4##5a##). The concordance indices at 1, 5, and 10 years were 61.2, 60.6, and 59.7.</p>", "<p id=\"Par29\">The 526 high-risk patients according to both scores had significantly lower survival probabilities than the 671 non-high-risk patients identified by the ELTS score (<italic>P</italic> = 0.0041, Fig. ##FIG##4##5b##). The HR for non-high- to high-risk patients was 0.615 (95% CI: 0.442–0.857); concordance indices at 1, 5, and 10 years were 56.5, 55.6, and 55.4</p>", "<p id=\"Par30\">With reference to the low-risk group of the ELTS score, the HRs of the intermediate- and high-risk groups were 2.631 (95% CI: 2.116–3.273) and 3.675 (95% CI: 2.861–4.720), respectively (both <italic>P</italic> &lt; 0.0001, Fig. ##FIG##5##6a##) and the concordance indices at 1, 5, and 10 years were 66.6, 63.8, and 63.7.</p>", "<p id=\"Par31\">The 1200 non-low-risk patients identified by the Sokal score had significantly lower survival probabilities than the 1837 low-risk patients according to both scores (<italic>P</italic> = 0.0147, Fig. ##FIG##5##6b##). The corresponding hazard ratio was 1.490 (95% CI: 1.082–2.053). The concordance indices at 1, 5, and 10 years were 49.8, 54.4, and 54.4, respectively.</p>", "<p id=\"Par32\">Like the Sokal and ELTS scores, the Euro score suggested an intermediate- and a high-risk group with significantly lower OS probabilities compared with low-risk patients (both <italic>P</italic> &lt; 0.0001, Supplementary Fig. ##SUPPL##0##7a##) while the EUTOS score failed to discriminate significantly different OS probabilities (<italic>P</italic> = 0.0739, Supplementary Fig. ##SUPPL##0##7b##).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par33\">Although first described over 30 years ago, the Sokal score remains popular for risk group discrimination, despite suggesting that, at diagnosis, more than 20% of chronic-phase patients are at high-risk with respect to OS—even in the presence of TKIs—and despite the availability of the ELTS score developed in imatinib-treated patients [##REF##26416462##6##]. The main objective of this work is to provide evidence-based information on what score should be preferred, comparing prognostic discrimination performance between the Sokal and the ELTS score.</p>", "<p id=\"Par34\">To pay tribute to the improved survival evoked by TKI therapy, when developing the ELTS score, the focus was the probabilities of dying of CML (i.e., after progression) rather than dying of any cause. In 2949 patients independent of any score development, unlike the ELTS score, the Sokal score failed to recognize significantly different cumulative incidence probabilities of dying of CML between intermediate- and low-risk patients. Secondly, in relation to the low-risk group, the SHRs as well as the concordance indices were always higher with the ELTS score indicating a better discrimination than with the Sokal score. This result was also observed in the combined sample of 5154 patients from all three registries.</p>", "<p id=\"Par35\">A limitation of the prognostic discrimination comparisons in the combined out-study/population-based sample of 2949 patients was the probable state-dependent censoring. This led to slightly biased cumulative incidence probabilities for death after progression when compared with the gold standard of the progressive illness-death model. Applying the illness-death model, the significantly different hazards for the transitions into progression and into death in chronic phase confirmed a satisfactory discrimination between the risk groups of the ELTS score (Supplementary Table ##SUPPL##0##2##). No other score provided a better discrimination of risk groups.</p>", "<p id=\"Par36\">In the samples of 2949 and 5154 patients, for both the Sokal and the ELTS score, all pairwise risk group comparisons led to significant differences in OS probabilities. Again, in any case, the HRs as well as the concordance indices were always higher with the ELTS score than with the Sokal score.</p>", "<p id=\"Par37\">While the sample of 2949 independent patients guaranteed an unbiased comparison between the Sokal and the ELTS score, the sample of 5154 patients was needed for provision of event numbers high enough to investigate risk group classification differences between the scores. Of 1197 patients allocated to high-risk by the Sokal score, the ELTS score classified 56% as non-high-risk. Compared with the 526 high-risk patients according to both scores, the cumulative incidences of dying of CML were significantly lower and OS probabilities were significantly higher for the 671 ELTS non-high-risk patients. For 56% of 1197 patients the allocation of high-risk by the Sokal score was inappropriate. Of 3037 patients identified as low-risk by the ELTS score, the Sokal score classified 1200 (40%) as non-low-risk. The cumulative incidences of dying of CML were only slightly different from those of the remaining 1837 low-risk patients, pointing to another inappropriate classification by the Sokal score. However, in relation to the 1837 patients assessed as low-risk by both scores, OS was significantly lower. Nevertheless, at 92% after 6 years (95% CI: 90–94%) and 88% after 9 years (95% CI: 84–92%), OS probabilities were still high for the 1200 Sokal non-low-risk patients.</p>", "<p id=\"Par38\">HRs and concordance indices showed the best prognostic discrimination in the unbiased comparisons in the 2949 independent patients. Since the ELTS score was developed in the 2205 in-study patients, their inclusion in the total sample of 5154 patients meant some advantage for the ELTS score compared with the other scores. The extent of this limitation cannot be quantified, but in consideration of the very distinctive results, it is still fair to conclude that the risk of inappropriate classification is decidedly higher with the Sokal score.</p>", "<p id=\"Par39\">Successful validation of the ELTS score and superiority in comparison with other scores were also reported by Geelen et al [##REF##29743721##24##]. In 709 patients with first-line imatinib treatment, only the ELTS score was able to identify three pairwise significantly different risk groups with respect to OS and to achievement of a first major molecular response. With only 23 deaths after progression, the ELTS score also provided satisfactory differences in cumulative incidences of death due to CML, but numbers were too low to allow a reliable assessment of prognostic performance. Molica et al. compared the four prognostic systems in 459 individuals treated with imatinib as first-line TKI [##REF##31079264##8##]. Of 51 deaths, only ten were assessed as CML related. The authors judged that the ELTS score predicted probabilities of death better than the other scores. While Yang et al. observed the most distinct risk group discrimination of OS probabilities with the ELTS score in 462 imatinib-treated Chinese patients with a median follow-up of 69 months [##REF##32110103##25##], Millot et al. found that its three risk groups differed significantly from each other with respect to progression-free survival in 350 children with imatinib as first-line treatment—despite only 23 events (progression or death) [##REF##28838993##26##]. In both studies, the authors concluded that the ELTS score outperformed all other scores [##REF##32110103##25##, ##REF##28838993##26##]. However, instead of the conventional Sokal score, Millot et al. considered the Sokal score for younger patients (≤45 years) [##REF##28838993##26##, ##REF##3904870##27##].</p>", "<p id=\"Par40\">In 202 Italian patients ≥65 years treated with imatinib or nilotinib, in contrast to the Sokal score, the ELTS score provided significant discrimination of the three risk groups regarding major (BCR-ABL1 ≤ 0.1%, international scale, IS) and deep molecular remission (BCR-ABL1 ≤ 0.01%, IS) and the probabilities of leukemia-related deaths [##UREF##4##28##]. The ELTS score also worked best when applied to 258 patients diagnosed in advanced phase [##REF##31456269##29##]. Lauseker et al. concluded that the ELTS score could be applied to distinguish long-term survival between high-risk and non-high-risk patients until a better model developed in patients with accelerated phase and/or blast crisis is introduced [##REF##31456269##29##].</p>", "<p id=\"Par41\">The ELTS score has been validated several times for its ability to significantly discriminate risk groups regarding long-term survival outcome but mainly in patients first-line treated with imatinib [##REF##26416462##6##, ##REF##31079264##8##, ##REF##27568522##15##, ##REF##29743721##24##–##REF##28838993##26##, ##UREF##4##28##]. Despite significantly faster achievement of molecular reponses with second generation TKIs [##REF##26837842##10##, ##REF##29091516##13##, ##REF##20525993##30##–##REF##31869412##33##], first-line treatment with imatinib and its generics is still widespread. Most physicians continue to see room for first-line treatment with imatinib depending on age, comorbidities, kinase domain mutations, treatment goal, costs, and availability of generic imatinib [##REF##32127639##1##, ##REF##31869412##33##–##REF##30587493##37##]. In prognostic support of first-line treatment selection, the ELTS score offers the most appropriate risk group classification. This is also of interest as imatinib has fewer side effects than second generation TKIs, and it is perceived that a statistically significant overall superiority in long-term efficacy over imatinib has not yet been shown for another TKI [##REF##32127639##1##, ##REF##31869412##33##, ##UREF##6##36##, ##REF##30587493##37##]. There is indication that the ELTS score would also discriminate risk groups with respect to long-term survival if a second generation TKI were chosen as first-line treatment [##REF##29743721##24##]. More evidence is needed. A large patient sample would be necessary to recognize significant differences in long-term survival between TKIs within a certain risk group.</p>", "<p id=\"Par42\">Regarding risk group discrimination, the ELTS score outperformed the Sokal score, the Euro, and the EUTOS score. Due to our large patient sample, it was possible to show, for the first time with statistical significance, that the Sokal score is much more likely to provide an incorrect risk group classification. The mechanism behind the superiority of the ELTS score is its development in imatinib-treated patients and its different weighting of the four prognostic factors, together with a more adequate patient distribution into risk groups (about 60%/30%/10%) than the Sokal score (about 40%/40%/20%) in times when patients have much better survival prospects due to TKIs.</p>", "<p id=\"Par43\">In the most recently published ELN recommendations, the panel recommend the use of the ELTS score as the preferred method to assess baseline CML risk. Through our work, we back the ELN recommendation with statistical evidence. A valid score and its common application support comparative assessment of efficacy and safety. The ELTS score can be calculated via the “Hematology app” or the website: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.leukemia-net.org/content/leukemias/cml/elts_score\">https://www.leukemia-net.org/content/leukemias/cml/elts_score</ext-link>.</p>" ]
[]
[ "<p id=\"Par1\">Prognostic scores support clinicians in selecting risk-adjusted treatments and in comparatively assessing different results. For patients with chronic-phase chronic myeloid leukemia (CML), four baseline prognostic scores are commonly used. Our aim was to compare the prognostic performance of the scores and to arrive at an evidence-based score recommendation. In 2949 patients not involved in any score development, higher hazard ratios and concordance indices in any comparison demonstrated the best discrimination of long-term survival with the ELTS score. In a second step, of 5154 patients analyzed to investigate risk group classification differences, 23% (<italic>n</italic> = 1197) were allocated to high-risk by the Sokal score. Of the 1197 Sokal high-risk patients, 56% were non-high-risk according to the ELTS score and had a significantly more favorable long-term survival prognosis than the 526 high-risk patients according to both scores. The Sokal score identified too many patients as high-risk and relatively few (40%) as low-risk (versus 60% with the ELTS score). Inappropriate risk classification jeopardizes optimal treatment selection. The ELTS score outperformed the Sokal score, the Euro, and the EUTOS score regarding risk group discrimination. The recent recommendation of the European LeukemiaNet for preferred use of the ELTS score was supported with significant statistical evidence.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0931-9) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>Open access funding was provided by Projekt DEAL. The valuable assistance of Eva Spaeh is gratefully appreciated. The EUTOS was a joint project of the European LeukemiaNet and Novartis Oncology Europe. Novartis Oncology Europe provided financial support for the EUTOS registry project from 2007 to 2015.</p>", "<title>Data availability</title>", "<p>For original data, please contact [email protected]. Deidentified individual participant data are available upon request and agreement of the scientific committee and the data security officer of our faculty.</p>", "<title>Code availability</title>", "<p>Most analyses were undertaken using SAS (version 9.4). The truncated concordance index was calculated using the function <italic>pec</italic> implemented in the programming software R (version 3.4.3) [##REF##25317082##38##]. Estimates of the competing risk and the progressive illness-death model were obtained from the R function <italic>etm</italic> and the association between risk group and transition probabilities was assessed using the R function <italic>mstate</italic> [##UREF##3##23##, ##UREF##7##39##].</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par44\">The authors declare the following potential conflicts of interest: REC, Research support: Novartis, Bristol Myers Squibb. Honoraria: Novartis, Bristol Myers Squibb, Pfizer. MB, Research support: Ariad. Honoraria: Novartis, Teva, Pfizer, Juno, Astellas, Ambit. Honoraria: Novartis, BMS, Pfizer, Incyte, Ariad, Takeda, Fusion Pharma. Logistic support: Novartis, BMS, Pfizer, Incyte, Ariad. SS, Research support: Bristol Myers Squibb, Incyte, Novartis. Honoraria: Bristol Myers Squibb, Incyte, Novartis, Pfizer. EF, Research support: Angelini, Bristol Myers Squibb, Novartis. Speaker: Angelini, Bristol Myers Squibb, Novartis. Consultation: Angelini, Bristol Myers Squibb, Novartis. Travel Grants: Angelini, Bristol Myers Squibb, Novartis, Pfizer, Terumo. AT, Consultation: Novartis, Pfizer, Fusion Pharma. Honoraria: Novartis, Pfizer. AZ, Research support: Cellgene, Janssen. TS, Honoraria: Novartis, Bristol-Myers Squibb, Pfizer, Angelini. Speakers’ bureau: Novartis, Bristol-Myers-Squibb, Pfizer, Angelini. DZ, Advisory board: Novartis, Incyte. Speakers’ bureau: Bristol Myers Squibb, Angelini. Consultation: Angelini. Travel grant: Birstol Myers Squibb. FC, Advisory board: Pfizer, Bristol Myers Squibb. Speakers’ bureau: Incyte, Pfizer. AB, Honoraria: Novartis, Pfizer. Travel grant: Takeda. F Castagnetti, Consultation: Novartis, Incyte, Pfizer, Bristol Myers Squibb. Honoraria: Novartis, Incyte, Pfizer, Bristol Myers Squibb. AH, Research support: Novartis, BMS, Pfizer, Incyte. MP, WP, FG, ML, SH, RH, GO, MH, LG, UOS, HE, PK, BL, AC, IZ, JG, JH, and VSH declare no potential competing interests.</p>", "<title>Ethics statement</title>", "<p id=\"Par45\">All studies complied with the Declaration of Helsinki. They were approved by the local human investigations committee and performed in accordance with the legal requirements of the corresponding country. Informed consent was obtained from all patients.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Cumulative incidence probabilities of dying because of CML in 2949 patients from the combined out-study and population-based registry sections.</title><p><bold>a</bold> Stratified for the risk groups according to the Sokal score and <bold>b</bold> Stratified for the risk groups according to the ELTS score. At 3, 6, and 9 years, horizontal crossbars indicate the upper and lower limit of the 95% confidence interval (CI) for the estimated probability. <bold>a</bold> The high-risk group of the Sokal score, though not the intermediate-risk group, had significantly higher probabilities of dying because of CML than the low-risk group, <italic>P</italic> &lt; 0.0001 and <italic>P</italic> = 0.0835, respectively. The corresponding SHRs were 3.559 (95% CI: 2.030–6.240) and 1.668 (95% CI: 0.934–2.978). The concordance indices at 1, 5, and 10 years were 59.7, 62.4, and 63.3, respectively. <bold>b</bold> The intermediate- and high-risk groups of the ELTS score had significantly higher probabilities of dying due to CML than the low-risk group with <italic>P</italic> = 0.0031 and <italic>P</italic> &lt; 0.0001, respectively. The corresponding hazard ratios were 2.203 (95% CI: 1.306–3.718) and 5.646 (95% CI: 3.397–9.387). The concordance indices at 1, 5, and 10 years were 68.0, 66.0, and 68.1, respectively.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Overall survival probabilities in 2949 patients from the combined out-study and population-based registry sections.</title><p><bold>a</bold> Stratified for the risk groups according to the Sokal score and <bold>b</bold> Stratified for the risk groups according to the ELTS score. At 3, 6, and 9 years, horizontal crossbars indicate the upper and lower limit of the 95%-confidence interval (CI) for the estimated probability. <bold>a</bold> The intermediate- and high-risk groups of the Sokal score had significantly lower survival probabilities than the low-risk group with both <italic>P</italic> &lt; 0.0001. The corresponding hazard ratios were 2.256 (95% CI: 1.590–3.201) and 3.384 (95% CI: 2.359–4.852). The concordance indices at 1, 5, and 10 years were 62.9, 62.0, and 61.3, respectively. <bold>b</bold> The intermediate- and high-risk groups of the ELTS score had significantly lower survival probabilities than the low-risk group with both <italic>P</italic> &lt; 0.0001. The corresponding hazard ratios were 2.479 (95% CI: 1.836–3.345) and 4.012 (95% CI: 2.884–5.582). The concordance indices at 1, 5, and 10 years were 65.6, 64.0, and 64.0, respectively.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Cumulative incidence probabilities of dying due to CML in 5154 patients from the combined in-study, out-study, and population-based registry sections.</title><p><bold>a</bold> Stratified for the risk groups according to the Sokal score and <bold>b</bold> with the 1197 high-risk patients according to the Sokal score stratified for non-high-risk and high-risk according to the ELTS Score. At 3, 6, and 9 years, horizontal crossbars indicate the upper and lower limit of the 95% confidence interval (CI) for the estimated probability. <bold>a</bold> The intermediate- and high-risk groups of the Sokal score had significantly higher probabilities of dying due to CML than the low-risk group with <italic>P</italic> = 0.0088 and <italic>P</italic> &lt; 0.0001, respectively. The corresponding hazard ratios were 1.695 (95% CI: 1.142–2.515) and 3.161 (95% CI: 2.146–4.655). The concordance indices at 1, 5, and 10 years were 58.8, 62.1, and 62.2, respectively. <bold>b</bold> The high-risk group according to both scores had significantly higher probabilities of dying due to CML than the non-high-risk group identified by the ELTS score, <italic>P</italic> = 0.0003. The corresponding hazard ratio was 0.415 (95% CI: 0.256–0.671). The concordance indices at 1, 5, and 10 years were 63.3, 60.8, and 59.9, respectively.</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Cumulative incidence probabilities of dying due to CML in 5154 patients from the combined in-study, out-study, and population-based registry sections.</title><p><bold>a</bold> Stratified for the risk groups according to the ELTS score and <bold>b</bold> with the 3037 low-risk patients according to the ELTS score stratified for low-risk and non-low-risk according to the Sokal Score. At 3, 6, and 9 years, horizontal crossbars indicate the upper and lower limit of the 95% confidence interval (CI) for the estimated probability. <bold>a</bold> The intermediate- and high-risk groups of the ELTS score had significantly higher probabilities of dying due to CML than the low-risk group with both <italic>P</italic> &lt; 0.0001. The corresponding hazard ratios were 2.584 (95% CI: 1.795–3.721) and 5.667 (95% CI: 3.912–8.209). The concordance indices at 1, 5, and 10 years were 69.6, 66.8, and 67.3, respectively. <bold>b</bold> The non-low-risk group identified by the Sokal score had no significantly different probabilities of dying due to CML than the low-risk group according to both scores, <italic>P</italic> = 0.6635. The corresponding hazard ratio was 1.129 (95% CI: 0.653–1.951). The concordance indices at 1, 5, and 10 years were not analysable, 47.6, and 47.7, respectively.</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Overall survival probabilities in 5154 patients from the combined in-study, out-study, and population-based registry sections.</title><p><bold>a</bold> Stratified for the risk groups according to the Sokal score and <bold>b</bold> with the 1197 high-risk patients according to the Sokal score stratified for non-high-risk and high-risk according to the ELTS Score. At 3, 6, and 9 years, horizontal crossbars indicate the upper and lower limit of the 95%-confidence interval (CI) for the estimated probability. <bold>a</bold> The intermediate- and high-risk groups of the Sokal score had significantly lower survival probabilities than the low-risk group with both <italic>P</italic> &lt; 0.0001. The corresponding hazard ratios were 2.049 (95% CI: 1.607–2.611) and 2.596 (95% CI: 2.009–3.355). The concordance indices at 1, 5, and 10 years were 61.2, 60.6, and 59.7, respectively. <bold>b</bold> The high-risk group according to both scores had significantly lower survival probabilities than the non-high-risk group identified by the ELTS score, <italic>P</italic> = 0.0041. The hazard ratio for non-high- to high-risk patients was 0.615 (95% CI: 0.442–0.857). The concordance indices at 1, 5, and 10 years were 56.5, 55.6, and 55.4, respectively.</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Overall survival probabilities in 5154 patients from the combined in-study, out-study, and population-based registry sections.</title><p><bold>a</bold> Stratified for the risk groups according to the ELTS score and <bold>b</bold> with the 3037 low-risk patients according to the ELTS score stratified for low-risk and non-low-risk according to the Sokal Score. At 3, 6, and 9 years, horizontal crossbars indicate the upper and lower limit of the 95% confidence interval (CI) for the estimated probability. <bold>a</bold> The intermediate- and high-risk groups of the ELTS score had significantly lower survival probabilities than the low-risk group with both <italic>P</italic> &lt; 0.0001. The corresponding hazard ratios were 2.631 (95% CI: 2.116–3.273) and 3.675 (95% CI: 2.861–4.720). The concordance indices at 1, 5, and 10 years were 66.6, 63.8, and 63.7, respectively. <bold>b</bold> The non-low-risk group identified by the Sokal score had significantly lower survival probabilities than the low-risk group according to both scores, <italic>P</italic> = 0.0147. The corresponding hazard ratio was 1.490 (95% CI: 1.082–2.053). The concordance indices at 1, 5, and 10 years were 49.8, 54.4, and 54.4, respectively.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
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[ "<media xlink:href=\"41375_2020_931_MOESM1_ESM.docx\"><caption><p>Supplementary Information</p></caption></media>" ]
[{"label": ["17."], "surname": ["Therneau", "Grambsch"], "given-names": ["TM", "PM"], "source": ["Modeling Survival Data: Extending the Cox Model"], "year": ["2000"], "publisher-loc": ["New York, USA"], "publisher-name": ["Springer"]}, {"label": ["18."], "surname": ["Kalbfleisch", "Prentice"], "given-names": ["JD", "RL"], "source": ["The Statistical Analysis of Failure Time Data"], "year": ["1980"], "publisher-loc": ["New York, USA"], "publisher-name": ["Wiley"]}, {"label": ["20."], "surname": ["Fine", "Gray"], "given-names": ["JP", "RJ"], "article-title": ["A proportional hazards model for the subdistribution of a competing risk"], "source": ["J Am Stat Assoc"], "year": ["1999"], "volume": ["94"], "fpage": ["496"], "lpage": ["509"], "pub-id": ["10.1080/01621459.1999.10474144"]}, {"label": ["23."], "surname": ["de Wreede", "Fiocco", "Putter"], "given-names": ["LC", "M", "H"], "article-title": ["The mstate package for estimation and prediction in non- and semi-parametric multi-state and competing risks models"], "source": ["Comput Meth Prog Bio"], "year": ["2010"], "volume": ["99"], "fpage": ["261"], "lpage": ["74"], "pub-id": ["10.1016/j.cmpb.2010.01.001"]}, {"label": ["28."], "surname": ["Castagnetti", "Gugliotta", "Breccia", "Stagno", "Specchia", "Levato"], "given-names": ["F", "G", "M", "F", "G", "L"], "article-title": ["The use of EUTOS long-term survival score instead of sokal score is strongly advised in elderly chronic myeloid leukemia patients"], "source": ["Blood."], "year": ["2018"], "volume": ["132"], "fpage": ["44"], "pub-id": ["10.1182/blood-2018-99-117409"]}, {"label": ["34."], "surname": ["Radich", "Deininger", "Abboud", "Altman", "Berman", "Bhatia"], "given-names": ["JP", "M", "CN", "JK", "E", "R"], "article-title": ["Chronic myeloid leukemia, version 1.2019, NCCN clinical practice guidelines in oncology"], "source": ["J Natl Compr Cancer Netw"], "year": ["2018"], "volume": ["16"], "fpage": ["1108"], "lpage": ["35"], "pub-id": ["10.6004/jnccn.2018.0071"]}, {"label": ["36."], "surname": ["Claudiani", "Apperley"], "given-names": ["S", "JF"], "article-title": ["The argument for using imatinib in CML"], "source": ["Hematol Am Soc Hematol Educ Program"], "year": ["2018"], "volume": ["2018"], "fpage": ["161"], "lpage": ["7"], "pub-id": ["10.1182/asheducation-2018.1.161"]}, {"label": ["39."], "surname": ["Allignol", "Schumacher", "Beyersmann"], "given-names": ["A", "M", "J"], "article-title": ["Empirical transition matrix of multi-state models: The etm package"], "source": ["J Stat Softw"], "year": ["2011"], "volume": ["38"], "fpage": ["1"], "lpage": ["15"], "pub-id": ["10.18637/jss.v038.i04"]}]
{ "acronym": [], "definition": [] }
39
CC BY
no
2024-01-13 23:35:07
Leukemia. 2020 Jun 29; 34(8):2138-2149
oa_package/07/0f/PMC7387299.tar.gz
PMC7387306
32457354
[ "<title>Introduction</title>", "<p id=\"Par2\">Chronic myeloid leukemia (CML) is a myeloproliferative disorder that is most commonly characterized by the presence of a Philadelphia chromosome, caused by the genetic translocation t(9;22)(q34;q11). This genetic abnormality juxtaposes two genes (BCR and ABL1), whose fusion codes for the constitutively active tyrosine kinase is BCR-ABL1. Targeting this protein with tyrosine kinase inhibitors (TKIs) such as imatinib mesylate revolutionized treatment of this disorder [##UREF##0##1##]. The use of TKIs has fundamentally improved survival rates for the majority of patients with CML and many patients can expect a near normal life expectancy [##REF##21422402##2##–##REF##26088952##5##]. However, patients can experience TKI-related adverse side effects [##REF##21750313##6##–##REF##17151364##9##], toxicities [##REF##24180494##7##, ##REF##25676422##10##, ##REF##23459449##11##], impact on quality of life (QoL) [##REF##21750313##6##], and for some, the price of the drugs [##UREF##1##12##, ##UREF##2##13##]. Furthermore, TKIs are not recommended for female patients who are trying to conceive, during pregnancy or are breastfeeding [##UREF##3##14##].</p>", "<p id=\"Par3\">In recent years, a number of studies have been conducted to demonstrate the possibility and safety of TKI cessation in well responding patients. The results of such studies have seen born the concept of treatment-free remission (TFR). Following a pilot study [##REF##16973963##15##], two pioneer studies confirmed that treatment discontinuation was feasible and safe [##REF##20965785##16##, ##REF##23704092##17##], and numerous clinical trials and observational studies have been reported, not only after imatinib treatment [##REF##22180435##18##–##REF##31292142##31##], but also after second generation TKI (2GTKI) whether used in 1st or 2nd line [##REF##29735299##23##–##REF##30880635##27##, ##REF##27932374##32##–##REF##29976734##37##]. Recently, second attempts to discontinue TKI in patients who relapsed have been reported [##REF##27932374##32##, ##REF##22936742##38##, ##REF##28743166##39##]. Recommendations on selection of patients with a higher probability of successful discontinuation have been proposed by global experts [##UREF##7##40##–##REF##32127639##43##]. However, the point of view of the patients undergoing discontinuation of treatment is less frequently reported on.</p>", "<p id=\"Par4\">The CML Advocates Network (CMLAN) is an active international network for leaders of Chronic Myeloid Leukemia (CML) patient groups. Its aim is to facilitate and support best practice sharing among patient advocates across the world. CMLAN’s objectives in this research were to inform the development of a range of guides for patients and healthcare professionals, that consider the patient experience of discontinuing treatment. The research did not seek to replicate the formal collection of scientific data such as prognosis, or clinical requirements for stopping treatment, as this has been expertly collected through existing medical studies and clinical trials. The CMLAN study was explicitly designed to focus on the patient perspective. Therefore, the areas of investigation include subjects less frequently covered in existing scientific research, but have importance to patients. These include, but are not limited to: the patient’s reasons for discontinuing treatment, the emotional impact within the TFR journey, and what information and support was provided. Through analysis of the survey data, it was identified that there were areas within the TFR journey where opportunities exist to improve patient care and experience. This manuscript summarizes and evidences these findings.</p>" ]
[ "<title>Methods</title>", "<title>Questionnaire design</title>", "<p id=\"Par5\">The questionnaire was designed by an expert panel of eight CML patients and went through two rounds of testing. Information was collected across five sections: about you (demographics); considerations around stopping treatment; experiences during the first six months of stopping treatment; restarting treatment; and, ongoing long-term TFR. Patients only completed the sections that were relevant to their experiences of stopping treatment. The demographic section collected data on relevant CML patient characteristics, including gender, age and country of residence. Consideration of stopping treatment was classified as “Phase I” of the stopping treatment journey. The questions collected information about the considerations respondents took before attempting, or rejecting TFR. Patients’ experiences during the first 6 months of discontinuing treatment were classified as “Phase II”. Most molecular recurrences (relapses) happen within the first 6 months after stopping treatment, therefore this stage of the stopping process is also known as the probation phase. This section collected information on respondents’ experiences during the probation phase. The restarting treatment section was classified as “Phase IIIA” and collected information from the respondents who had stopped treatment, but subsequently had to restart. The experiences of those in long-term remission beyond the 6-month probation phase were classified as “Phase IIIB”. Questions here included information on the concerns respondents still have and what they would change about their experience of discontinuing treatment.</p>", "<p id=\"Par6\">Across all sections of the questionnaire, the aim was to ask questions that allowed insight and understanding into what the patient had experienced, rather than the clinical perspective. Due to this focus on patient perspective, it was the authors’ opinion that the views of patients discontinuing treatment within the context of a clinical trial, were no more or less important than patients who discontinue in “real life”. Consequently, information around if respondents had participated in TFR clinical trials was not collected as part of this study. It is not believed that the conclusions are biased or weaker due to the lack of this data.</p>", "<title>Administration</title>", "<p id=\"Par7\">Administration of the survey was through a web-based questionnaire, between 14th March 2018 and 1st August 2018. The questionnaire was made available in 11 languages: Arabic, Danish, English, Finnish, French, German, Hebrew, Italian, Japanese, Russian and Spanish. The survey was promoted by the CMLAN, across 12 CML patient organization websites; CMLAN social media channels (Facebook, Twitter, Instagram, LinkedIn,); 64 CMLAN members’ Facebook pages; six emails to CMLAN members; three newsletters to CMLAN members; at the European Hematology Association Congress 2018; and, at the CML Horizons 2018 Conference. The project elicited 1016 responses from patients across 68 countries.</p>", "<p id=\"Par8\">Anecdotal evidence from other survey programmes suggests that patients who are already engaged in wider discussions about treatment options (for example, through patient advocacy organizations), are more likely to complete questionnaires; and that because of this engagement (for which we can find no scientific evidence) they may report either better (because they are better informed) or worse (because poor treatment led them to seek help) experiences. In the survey design for this study, there is an explicit assumption that the experience of patients stopping treatment is not statistically associated with their involvement with patient advocacy organizations, or their desire to engage in completing a questionnaire—and therefore that there is no inherent bias in the methodology or results.</p>", "<title>Analysis of responses</title>", "<p id=\"Par9\">For all questions (except for those asked in the form of “tick all that apply”) the percentage responses are calculated after excluding those respondents that did not answer that question. All percentages are rounded to the nearest whole number. When added together, the answers to a particular question may not total 100% because of this rounding. Figures have been calculated excluding responses where the question was not applicable to the respondent’s circumstances, or they felt unable to give a definite answer. The base size for questions that have been asked as “tick all that apply” is determined by the number of eligible respondents. As such, the missing count for a “tick all that apply” response option represents any eligible respondents who have chosen not to select that particular option.</p>", "<title>Limitations</title>", "<p id=\"Par10\">The survey has several limitations. The survey was only available online. Whilst there were benefits to this methodology, it will have introduced limitations to accessibility by factors such as region and socioeconomic status. Respondents were self-selected, participated on a voluntary basis and were recruited through patient organizations, therefore cannot reflect the perspectives of all CML patients.</p>" ]
[ "<title>Results</title>", "<title>Demographics</title>", "<p id=\"Par11\">1016 responses were collected across 68 countries. Fifty-five percent (<italic>n</italic> = 550) of respondents were female, the median age was 53 (with a range of 16–92); education levels were varied. Respondents were grouped into the designated World Health Organization (WHO) regions; 56% (<italic>n</italic> = 563) were from countries assigned to the European region. Sixty percent (<italic>n</italic> = 608) of respondents reported that their main place of treatment was a hospital, and 39% (<italic>n</italic> = 389) of respondents had been living with CML for 10 years or more. Forty-nine percent (<italic>n</italic> = 494) of respondents proceeded to stop treatment. Full demographics of respondents at each phase are shown in Table ##TAB##0##1##. Fifty-one percent (<italic>n</italic> = 242) of respondents who stopped treatment reported that the medication they were taking before stopping was imatinib. Full range of responses are shown in Table ##TAB##1##2##.</p>", "<title>Key findings in Phase I—the consideration phase</title>", "<p id=\"Par12\">All 1016 respondents were asked to complete the Phase I section of the questionnaire.</p>", "<p id=\"Par13\">Most respondents reported first hearing about the possibility of stopping treatment through a healthcare professional 49% (<italic>n</italic> = 476), followed by 21% (<italic>n</italic> = 209) who heard through a patient organization. When asked to select the main reasons that made them consider attempting TFR, 51% (<italic>n</italic> = 516) said they wanted to get rid of current treatment side effects, and 48% (<italic>n</italic> = 488) wanted to see if they could be free of CML without therapy. Seventeen percent (<italic>n</italic> = 168) reported they considered stopping because their doctor proposed they join a “stopping treatment” clinical trial. The full range of responses is shown in Table ##TAB##2##3##.</p>", "<p id=\"Par14\">Respondents were asked which topics they discussed with their doctor whilst making their decision to try stopping treatment: 60% (<italic>n</italic> = 606) of respondents discussed risks of stopping; 50% (<italic>n</italic> = 513) discussed the requirements to be met in order to stop; 48% (<italic>n</italic> = 490) discussed the benefits of stopping; 34% (<italic>n</italic> = 346) discussed the timing of when to stop; 21% (<italic>n</italic> = 215) discussed the drug withdrawal symptoms. Fourteen percent (<italic>n</italic> = 143) did not have a discussion with their doctor. Following the discussion with their doctor, 55% (<italic>n</italic> = 555) of respondents said they still had concerns about TFR being unsuccessful, and their disease reoccurring.</p>", "<p id=\"Par15\">Respondents were asked to indicate the reasons that made them worry about stopping treatment. Fifty-seven percent (<italic>n</italic> = 579) reported recurrence of CML; 20% (<italic>n </italic>= 203) said they would not feel safe going off treatment; 16% (<italic>n</italic> = 158) did not have enough information about stopping treatment; 12% (<italic>n</italic> = 120) had a fear of withdrawal symptoms; 7% (<italic>n</italic> = 70) said there is a lack of proper quality PCR monitoring. Twenty-six percent (<italic>n</italic> = 261) reported that they were not worried about stopping treatment.</p>", "<p id=\"Par16\">Seventy-three percent (<italic>n</italic> = 591) of respondents said their doctor supported their decision to try stopping treatment. Sixty-one percent (<italic>n</italic> = 624) of respondents reported they received support and information about stopping treatment from their doctor or another healthcare professional; 32% (<italic>n</italic> = 327) said they received it from a patient organization; 30% (<italic>n</italic> = 303) from other CML patients who have stopped treatment; and, 29% (<italic>n</italic> = 299) from the internet.</p>", "<p id=\"Par17\">When asked what information about stopping treatment and TFR they would have liked to have received: 52% (<italic>n</italic> = 533) of respondents would have liked information on results from clinical trials; 49% (<italic>n</italic> = 499) general information on all the steps of the stopping treatment journey; 44% (<italic>n</italic> = 452) the expectations of the risks and opportunities of stopping treatment; 40% (<italic>n</italic> = 407) the withdrawal effects after stopping; 36% (<italic>n</italic> = 368) the side effects on restarting therapy; 32% (<italic>n</italic> = 330) the required PCR monitoring; and, 22% (<italic>n</italic> = 227) the psychological effects.</p>", "<title>Key findings in Phase II—the probation phase</title>", "<p id=\"Par18\">At the time of the study, no (published) consensus concerning the minimal requirements for TFR had been reached. Subsequent recommendations published by European LeukemiaNet [##REF##32127639##43##] advise minimal requirements for TFR that include duration of TKI therapy &gt;5 years (&gt;4 years for 2GTKI) and duration of deep molecular response (DMR) (MR<sup>4</sup> or better) &gt;2 years. Seventy-seven percent (<italic>n</italic> = 359) of respondents reported that they had been on CML medication for ≥5 years, the median was 7 years (with a range of 1–27). Seventy-eight percent (<italic>n</italic> = 382) said they had been in DMR of at least MR<sup>4</sup>, or BCR-ABL transcript levels below 0.01%, for over 2 years before stopping. Table ##TAB##3##4## illustrates the full range of responses.</p>", "<p id=\"Par19\">Fifty-six percent (<italic>n</italic> = 266) of respondents said that they “completely” had all the information they wanted when they stopped treatment. When asked what topics they discussed with their doctor during Phase II, 39% (<italic>n</italic> = 168) of respondents reported having a discussion on how to deal with withdrawal symptoms. Forty-seven percent (<italic>n</italic> = 222) reported that their doctor or another healthcare professional asked them if they were experiencing any physical withdrawal symptoms whilst stopping treatment; 27% (<italic>n</italic> = 128) were not asked, but would have liked to have been; and 26% (<italic>n</italic> = 121) were not asked but did not feel it was necessary. Sixty percent (<italic>n</italic> = 288) of respondents reported experiencing withdrawal symptoms; of these, 32% (<italic>n</italic> = 91) said they continued for a few months after stopping. Of those experiencing withdrawal symptoms: 89% (<italic>n</italic> = 257) reported withdrawal pain in muscles, joints or bones; 51% (<italic>n</italic> = 147) tiredness; 26% (<italic>n</italic> = 76) depressive episodes, fear or bad mood; 26% (<italic>n</italic> = 74) sweating or skin problems; and, 11% (<italic>n</italic> = 31) weight loss. Forty percent (<italic>n</italic> = 112) of the respondents who experienced side effects, reported that their doctor did not support them in managing all their physical withdrawal effects during discontinuation of treatment.</p>", "<p id=\"Par20\">When asked what topics they discussed with their doctor during the stopping phase, 18% (<italic>n</italic> = 69) of respondents reported having a discussion on how to deal with psychological aspects. Seventy-four percent (<italic>n</italic> = 268) reported that one of the benefits of stopping treatment was that it had a positive impact on their emotional wellbeing, however, 56% (<italic>n</italic> = 268) of respondents said that they felt fear or anxiety at some point during the stopping phase, and 55% (<italic>n</italic> = 148) of these reported this happened around the time of their PCR monitoring tests. Figure ##FIG##0##1## illustrates the full range of responses.</p>", "<p id=\"Par21\">Seven percent (<italic>n</italic> = 32) of respondents reported that their doctor asked them if they needed psychological support whilst stopping treatment; 26% (<italic>n</italic> = 120) were not asked but would have liked to have been; 67% (<italic>n</italic> = 312) were not asked, but did not feel it was necessary. Twenty percent (<italic>n</italic> = 93) of respondents reported that they did receive psychological and/or emotional support during discontinuation of treatment; 23% (<italic>n</italic> = 110) did not receive any, but would have liked to; and 57% (<italic>n</italic> = 269) did not receive any, but felt it was not necessary. Of the respondents who said they had received psychological and/or emotional support: 71% (<italic>n</italic> = 66) reported receiving support from friends and family; 26% (<italic>n</italic> = 24) from patient organization/s; 25% (<italic>n</italic> = 23) from a social media group; and, 20% (<italic>n</italic> = 19) received counseling.</p>", "<p id=\"Par22\">During Phase II, 92% (<italic>n</italic> = 426) of respondents reported having a discussion on how often to monitor their BCR-ABL transcript levels, 76% (<italic>n</italic> = 339) discussed response levels and when/if to restart treatment and 70% (<italic>n</italic> = 294) discussed the time taken to receive the results of the last PCR test. Sixty-four percent (<italic>n</italic> = 308) of respondents reported that on average they were monitored monthly by their doctor through PCR tests during the probation phase, and 34% (<italic>n</italic> = 165) were monitored less frequently.</p>", "<title>Key findings in Phase IIIA—the restarting treatment phase</title>", "<p id=\"Par23\">Of the 494 respondents who reported having stopped treatment, 32% (<italic>n</italic> = 159) reported that their disease had reoccurred, and treatment had to restart. Seventy-seven percent (<italic>n</italic> = 119) of patients reported that their disease reoccurred within the first 6 months of stopping treatment.</p>", "<p id=\"Par24\">When they were first told their disease had reoccurred: 59% (<italic>n</italic> = 81) of respondents strongly agreed/agreed that they felt scared/anxious; 91% (<italic>n</italic> = 138) strongly agreed/agreed that they felt disappointed; 56% (<italic>n</italic> = 74) strongly agreed/agreed that they felt depressed; 20% (<italic>n</italic> = 25) strongly agreed/agreed that they felt confused; and, 4% (<italic>n</italic> = 5) strongly agreed/agreed that they felt relieved.</p>", "<p id=\"Par25\">When asked how they felt emotionally in the first few weeks after restarting treatment, 35% (<italic>n</italic> = 55) of respondents reported feeling emotionally worse at this point compared with before stopping, and 30% (<italic>n</italic> = 48) reported feeling emotionally worse than during stopping treatment.</p>", "<p id=\"Par26\">Respondents were asked to rate side effects by the extent they affected their everyday life in the weeks before stopping treatment, and then again when they restarted treatment. A score of 1 indicated that the side effect mildly affected their everyday life, and 5 indicated that the side effect completely affected their everyday life. No response indicated they did not experience the side effect. A mean score was calculated for each side effect. The side effects that showed the biggest mean score increase in impact between before stopping and restarting were feeling sad, feeling distressed, and anxiety (Table ##TAB##4##5##).</p>", "<p id=\"Par27\">Twenty-six percent (<italic>n</italic> = 40) of respondents reported that they did receive psychological and/or emotional support during the restarting of treatment; 25% (<italic>n</italic> = 39) did not receive any, but would have liked to; and, 48% (<italic>n</italic> = 74) did not receive any, but felt it was not necessary. Of the respondents who said they had received psychological and/or emotional support: 85% (<italic>n</italic> = 34) reported receiving support from friends and family; 25% (<italic>n</italic> = 10) received counseling; 20% (<italic>n</italic> = 8) reported receiving support from a social media group; and, 18% (<italic>n</italic> = 7) received support from patient organization/s.</p>", "<title>Key findings in Phase IIIB—the long-term remission phase</title>", "<p id=\"Par28\">Sixty-seven percent (<italic>n</italic> = 132) of respondents reported feeling that overall, they receive adequate care in the long-term TFR phase; 30% (<italic>n</italic> = 60) reported that this happens to some extent; and, 3% (<italic>n</italic> = 6) said that they do not receive adequate care.</p>", "<p id=\"Par29\">When asked how they felt their doctor could improve their experience of stopping treatment, 47% (<italic>n</italic> = 95) of respondents reported they felt no improvement was necessary; 32% (<italic>n</italic> = 65) wanted more information on current data in easy to understand language; 11% (<italic>n</italic> = 23) wanted better PCR monitoring; 10% (<italic>n</italic> = 21) wanted a better doctor/patient relationship; and, 10% (<italic>n</italic> = 21) wanted better psychological support.</p>", "<p id=\"Par30\">Respondents reported the major concerns they had while in Phase IIIB. Fifty-eight percent (<italic>n</italic> = 118) reported that late reoccurrence of CML was a major concern; 34% (<italic>n</italic> = 69) had uncertainty about the future in terms of CML; 29% (<italic>n</italic> = 58) were concerned over the misunderstanding of people thinking that they are cured; 26% (<italic>n</italic> = 52) had concerns over late detection of a reoccurrence; and, 4% (<italic>n</italic> = 9) were concerned that they have more frequent PCR tests than before stopping. Twenty-eight percent (<italic>n</italic> = 57) reported not having any concerns.</p>", "<p id=\"Par31\">Twenty-five percent (<italic>n</italic> = 49) of respondents reported that they do receive psychological and/or emotional support during the long-term remission phase; 16% (<italic>n</italic> = 32) do not receive any, but would like to; and, 59% (<italic>n</italic> = 116) do not receive any, but feel it is not necessary.</p>", "<p id=\"Par32\">Of the respondents who said they receive psychological and/or emotional support: 76% (<italic>n</italic> = 37) reported receiving support from friends and family; 33% (<italic>n</italic> = 16) from a social media group; 31% (<italic>n</italic> = 15) from patient organization/s; and, 16% (<italic>n</italic> = 8) receive counseling.</p>", "<title>Advice from patients</title>", "<p id=\"Par33\">All patients (<italic>n</italic> = 494) who attempted stopping treatment (whether successful or not) were asked what their advice would be to other CML patients who are considering stopping treatment. Seventy-nine percent (<italic>n</italic> = 392) advised to always be well informed about your PCR results and treatment options; 69% (<italic>n</italic> = 342) advised to look for the best doctor with experience in stopping treatment; 59% (<italic>n</italic> = 290) advised to talk with other patients who have stopped, or are considering stopping; 51% (<italic>n</italic> = 254) advised to receive information from patient organizations about stopping treatment; 50% (<italic>n</italic> = 248) advised to look for simple and good information about each step of stopping treatment; 33% (<italic>n</italic> = 162) advised to get emotional support; and, 25% (<italic>n</italic> = 125) advised to get psychological support.</p>" ]
[ "<title>Discussion and conclusion</title>", "<p id=\"Par34\">To our knowledge this is the largest study conducted to gain knowledge about undertaking TFR that is taken purely from the perspective of the patient. There is existing research on patient perceptions of TFR [##UREF##8##44##–##UREF##10##47##], however, these focus on motivation and concerns about undertaking TFR. We believe this study is one of a limited number that investigates the experience of patients throughout all stages, and in particular looks at the support and management of the process beyond that of the management of PCR monitoring.</p>", "<title>Psychological and emotional impact of TFR</title>", "<p id=\"Par35\">Our results evidence that respondents in the probation phase particularly felt fear or anxiety around the time of their PCR monitoring tests, an occurrence that is often dubbed “PCR-itis” (or scanxiety, in solid tumors) and is known to affect patients with cancer [##UREF##11##48##]. The implications of test results for TFR patients are perhaps magnified when they are not on treatment, as not only is there the potential to “fail” at TFR, but to “lose” a level of major molecular response that had previously been maintained for many years. With recommendations stating that monitoring should take place monthly in this phase [##REF##27013442##41##–##REF##32127639##43##], there is the potential for fear and anxiety to be frequent at this point in the TFR journey. However, only a small proportion of respondents said they discussed how to deal with psychological aspects with their doctor, and even fewer said that their doctor asked them if they needed psychological support during this phase.</p>", "<p id=\"Par36\">The data also suggests that the requirement to restart treatment can have a strong negative psychological impact on patients, and that the desire for psychological support will be greater in the restarting treatment phase. In Phase IIIA, when they were first told they had molecular reoccurrence, many respondents reported feeling scared/anxious, and depressed. In addition, there were respondents who reported feeling emotionally worse during this phase than before discontinuation, and during discontinuation of treatment. There was also an increase in the reported severity of the impact of the emotional side effects of anxiety, feeling distressed and feeling upset in this phase, compared with before discontinuation of treatment. These results echo those of Sogawa et al. [##UREF##12##49##], who reported significantly higher anxiety and depression on the Hospital Anxiety and Depression Scale (HADS), at reintroduction of TKI than at the point of TKI discontinuation.</p>", "<p id=\"Par37\">Despite the reported negative impact on psychological and emotional wellbeing, the data shows there were patients who wanted emotional support during the probation and restarting phases but did not receive it.</p>", "<p id=\"Par38\">Whilst psychological support seems to be less of an issue in Phase IIIB (long-term TFR, ongoing past the probation period), late reoccurrence was reported as a major concern by a majority of respondents.</p>", "<p id=\"Par39\">The survey did not collect information on what specifically caused concerns during the probation and restarting phases, and further investigation into this would be beneficial for the healthcare and wider CML communities.</p>", "<p id=\"Par40\">This study’s findings support existing recommendations [##REF##29753691##50##] that psychological wellbeing of CML patients attempting TFR should be a consideration of healthcare professionals and form part of routine monitoring.</p>", "<title>TKI withdrawal</title>", "<p id=\"Par41\">Many of the respondents who stopped treatment reported that they experienced withdrawal symptoms. However, results indicate that patients did not always discuss the possibility of drug withdrawal symptoms with their doctor before making their decision to stop treatment. During the probation phase there were patients who did not have a discussion with their doctor about how to deal with withdrawal symptoms. Furthermore, during the discontinuation of treatment not all patients were asked if they were experiencing withdrawal effects, and many of these would have liked to have been. Where respondents experienced physical withdrawal symptoms during the discontinuation of treatment, results indicate there are instances where there could be further doctor support in the management of these. With clinical study data suggesting that up to 30% of patients who stop treatment will experience withdrawal symptoms [##REF##29468438##35##, ##REF##25071107##51##], there is a strong indication that importance needs to be placed on the issues of support and management of TKI withdrawal.</p>" ]
[ "<title>Discussion and conclusion</title>", "<p id=\"Par34\">To our knowledge this is the largest study conducted to gain knowledge about undertaking TFR that is taken purely from the perspective of the patient. There is existing research on patient perceptions of TFR [##UREF##8##44##–##UREF##10##47##], however, these focus on motivation and concerns about undertaking TFR. We believe this study is one of a limited number that investigates the experience of patients throughout all stages, and in particular looks at the support and management of the process beyond that of the management of PCR monitoring.</p>", "<title>Psychological and emotional impact of TFR</title>", "<p id=\"Par35\">Our results evidence that respondents in the probation phase particularly felt fear or anxiety around the time of their PCR monitoring tests, an occurrence that is often dubbed “PCR-itis” (or scanxiety, in solid tumors) and is known to affect patients with cancer [##UREF##11##48##]. The implications of test results for TFR patients are perhaps magnified when they are not on treatment, as not only is there the potential to “fail” at TFR, but to “lose” a level of major molecular response that had previously been maintained for many years. With recommendations stating that monitoring should take place monthly in this phase [##REF##27013442##41##–##REF##32127639##43##], there is the potential for fear and anxiety to be frequent at this point in the TFR journey. However, only a small proportion of respondents said they discussed how to deal with psychological aspects with their doctor, and even fewer said that their doctor asked them if they needed psychological support during this phase.</p>", "<p id=\"Par36\">The data also suggests that the requirement to restart treatment can have a strong negative psychological impact on patients, and that the desire for psychological support will be greater in the restarting treatment phase. In Phase IIIA, when they were first told they had molecular reoccurrence, many respondents reported feeling scared/anxious, and depressed. In addition, there were respondents who reported feeling emotionally worse during this phase than before discontinuation, and during discontinuation of treatment. There was also an increase in the reported severity of the impact of the emotional side effects of anxiety, feeling distressed and feeling upset in this phase, compared with before discontinuation of treatment. These results echo those of Sogawa et al. [##UREF##12##49##], who reported significantly higher anxiety and depression on the Hospital Anxiety and Depression Scale (HADS), at reintroduction of TKI than at the point of TKI discontinuation.</p>", "<p id=\"Par37\">Despite the reported negative impact on psychological and emotional wellbeing, the data shows there were patients who wanted emotional support during the probation and restarting phases but did not receive it.</p>", "<p id=\"Par38\">Whilst psychological support seems to be less of an issue in Phase IIIB (long-term TFR, ongoing past the probation period), late reoccurrence was reported as a major concern by a majority of respondents.</p>", "<p id=\"Par39\">The survey did not collect information on what specifically caused concerns during the probation and restarting phases, and further investigation into this would be beneficial for the healthcare and wider CML communities.</p>", "<p id=\"Par40\">This study’s findings support existing recommendations [##REF##29753691##50##] that psychological wellbeing of CML patients attempting TFR should be a consideration of healthcare professionals and form part of routine monitoring.</p>", "<title>TKI withdrawal</title>", "<p id=\"Par41\">Many of the respondents who stopped treatment reported that they experienced withdrawal symptoms. However, results indicate that patients did not always discuss the possibility of drug withdrawal symptoms with their doctor before making their decision to stop treatment. During the probation phase there were patients who did not have a discussion with their doctor about how to deal with withdrawal symptoms. Furthermore, during the discontinuation of treatment not all patients were asked if they were experiencing withdrawal effects, and many of these would have liked to have been. Where respondents experienced physical withdrawal symptoms during the discontinuation of treatment, results indicate there are instances where there could be further doctor support in the management of these. With clinical study data suggesting that up to 30% of patients who stop treatment will experience withdrawal symptoms [##REF##29468438##35##, ##REF##25071107##51##], there is a strong indication that importance needs to be placed on the issues of support and management of TKI withdrawal.</p>" ]
[ "<p id=\"Par1\">In CML, treatment-free remission (TFR) refers to having a stable deep molecular response without the need for ongoing tyrosine kinase inhibitor treatment. Whilst recommendations exist about the technical management of stopping and re-starting therapy, much is still unknown about the experiences of those considering and undertaking TFR. This study sought to obtain the patient perspective, identify areas of unmet needs and create recommendations for improvements. Fifty-six percent of patients reported fear or anxiety during treatment discontinuation, whereas only 7% of patients were asked if they needed psychological support during this period. Where patients re-initiated treatment; 59% felt scared or anxious, and 56% felt depressed. Twenty-six percent of re-initiated patients received psychological and/or emotional support at this time. Sixty percent of patients experienced withdrawal symptoms whilst discontinuing treatment, however, 40% of patients who experienced withdrawal symptoms reported that they were not fully supported by their doctor in managing all the symptoms. Healthcare professionals should further consider how they monitor the psychological well-being of patients who are discontinuing or re-initiating treatment, and review what support is offered in response to identified concerns. Surveillance of withdrawal symptoms should be a priority during treatment discontinuation, along with how healthcare professionals assist in the management of these.</p>", "<title>Subject terms</title>" ]
[ "<title>Conclusion</title>", "<p id=\"Par42\">Our research indicates that there are points in the TFR journey where patients do not always get all the advice, information or support they want around psychological issues and TKI withdrawal.</p>", "<p id=\"Par43\">Results show that patients in the probation and restarting phases are likely to experience a negative impact on their mental well-being in the form of fear, worry, depression or anxiety. However, doctors do not always discuss the potential impact discontinuing treatment may have on a patient’s mental well-being, or ask them if they need psychological support. It is our recommendation that doctors should discuss mental health with patients early in the consideration and/or probation phases, so they are aware of and prepared for changes. Doctors should decide with the patient if it is appropriate to monitor their mental health, particularly during the probation period and treatment re-initiation. Further investigation into how mental health can be monitored during the TFR journey should be a consideration of future studies.</p>", "<p id=\"Par44\">In addition, it is our recommendation that doctors should discuss what psychological support may be suitable and/or available for patients. While formal medical psychological intervention may not be necessary, signposting to other sources of support should be considered.</p>", "<p id=\"Par45\">Research shows that TKI withdrawal is a possibility for patients who discontinue treatment. However, our results show that this is not always discussed when patients are making their decision to attempt TFR. In addition, once patients have discontinued treatment they are not always asked if they are experiencing withdrawal symptoms or offered support to manage them. It is our recommendation that healthcare professionals should address this by informing patients of the potential for withdrawal symptoms early in the TFR decision process. Once treatment has been discontinued, doctors should monitor for known withdrawal symptoms, encourage patients to report any symptoms they experience, and offer advice and support to on how to manage these.</p>", "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0867-0) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>The authors would like to thank all member organizations of the CML Advocates Network for their assistance in promoting the survey, and the CML patients who participated in the research.</p>", "<title>Data availability</title>", "<p>The data that support the findings of this study are available from the corresponding author on request.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par46\">GS reports: consultancy for Leukemia Patient Advocates Foundation, Israeli CML patient organization, Novartis, Incyte, AbbVie, Takeda, Bristol-Myers Squibb, Celgene and Pfizer; advisory board membership for CML Advocates Network, Leukemia Patient Advocates Foundation and Israeli CML patient organization; organizational grant funding from Novartis, Incyte, Amgen, Bristol-Myers Squibb, Celgene, AOP Orphan, CTI, Medison, Roche, Janssen, Pfizer, Takeda and Gilead. CM reports: employment for Leukemia Patient Advocates Foundation; consultancy for Psoriasis en Red and AbbVie; advisory board membership for Psoriasis en Red and FFpaciente; organizational grant funding from AbbVie, Leo Pharma, UCB, Celgene, Pfizer, Novartis Oncology, Incyte, Takeda, Bristol-Myers Squibb, The Leukemia &amp; Lymphoma Society and International CML Foundation. JB reports employment for Quality Health Ltd. ZPW reports: employment for Leukemia Care; equity ownership of Patient Evidence; consultancy for Acute Leukemia Advocates Network, Amgen, Bristol-Myers Squibb, Celgene, Incyte, Janssen, Novartis and Pfizer; advisory board membership for Acute Leukemia Advocates Network and CML Advocates Network; speakers bureau for Amgen, Bristol-Myers Squibb, Gilead, Jazz, Novartis and Pfizer; organizational grant funding from AbbVie, Amgen, Bristol-Myers Squibb, Celgene, Daiichi Sankyo, Gilead, Incyte, Jazz, Janssen, Kyowa Kirin, Novartis and Takeda. FB reports: consultancy for Abbvie, Bristol-Myers Squibb, Celgene, Jazz, Novartis, Takeda; board membership of AIL Torino onlus, CML Advocates Network, CLL Advocates Network, MPN Advocates Network; organizational grant funding from Abbvie, Bristol-Myers Squibb, Celgene, Daiichi Sankyo, Gilead, Janssen, Novartis, Pfizer, Roche, Takeda. ROC reports: consultancy for LYLE (patient organization for Lymphoma, Leukemia &amp; MDS), Autisme 4700 DK, Bristol-Myers Squibb, Incyte, Janssen, Novartis Nordic, Roche and Takeda; board membership of LYLE and Autisme 4700 DK; organizational grant funding from Abbvie, Gilead, Incyte, Janssen, Novartis Nordic, Pfizer, Roche and Takeda. BG reports: consultancy for Bristol-Myers Squibb, Incyte, Novartis, Takeda and Pfizer; board membership of AMAL (Association des malades atteints de leucémies); organizational grant funding from Novartis. ND reports Honoraria from Novartis. MD reports no competing interests. JG reports: consultancy for AMGEN, Bristol-Myers Squibb, Bayer, Biomarin, Grünenthal, Janssen, Novartis, Pfizer, Roche, Servier, Takeda and UCB; board membership of CML Advocates Network, Leukemia Patient Advocates Foundation and LeukaNET; contributions to IMI-funded consortium projects HARMONY and EUPATI by EFPIA companies; organizational grant funding from Incyte, Novartis, Pfizer, Bristol-Myers Squibb and Takeda.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Frequency of fear or anxiety during Phase II.</title><p>This chart illustrates how patients responded when asked how frequently they experienced fear or anxiety during Phase II.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Demographics of the respondents.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Variables</th><th>Phase I</th><th>Phase II</th><th>Phase IIIA</th><th>Phase IIIB</th></tr><tr><th><italic>N</italic> %</th><th><italic>N</italic> %</th><th><italic>N</italic> %</th><th><italic>N</italic> %</th></tr></thead><tbody><tr><td colspan=\"5\">Gender</td></tr><tr><td> Male</td><td>453 (45)</td><td>196 (40)</td><td>51 (32)</td><td>98 (49)</td></tr><tr><td> Female</td><td>550 (55)</td><td>293 (60)</td><td>106 (68)</td><td>103 (51)</td></tr><tr><td colspan=\"5\">Age (at the time when the survey was completed)</td></tr><tr><td> Median, range (years)</td><td>53 (16–92)</td><td>56 (21–92)</td><td>56 (26–82)</td><td>57 (21–85)</td></tr><tr><td colspan=\"5\">Education</td></tr><tr><td> No formal qualifications</td><td>50 (5)</td><td>19 (4)</td><td>10 (6)</td><td>2 (1)</td></tr><tr><td> High school qualifications or High school diploma</td><td>228 (23)</td><td>107 (22)</td><td>30 (19)</td><td>48 (24)</td></tr><tr><td> University—Bachelors or Undergraduate degree</td><td>361 (36)</td><td>160 (33)</td><td>44 (28)</td><td>62 (31)</td></tr><tr><td> University—Masters or PHD</td><td>208 (21)</td><td>112 (23)</td><td>41 (26)</td><td>45 (22)</td></tr><tr><td> Career or technical qualifications</td><td>160 (16)</td><td>92 (19)</td><td>32 (20)</td><td>44 (22)</td></tr><tr><td colspan=\"5\">WHO Region (grouped from country of residence when the survey was completed)</td></tr><tr><td> Africa (AFRO)</td><td>13 (1)</td><td>2 (0)</td><td>0 (0)</td><td>1 (0)</td></tr><tr><td> Eastern Mediterranean (EMRO)</td><td>27 (3)</td><td>9 (2)</td><td>3 (2)</td><td>3 (1)</td></tr><tr><td> Europe (EURO)</td><td>563 (56)</td><td>330 (67)</td><td>111 (70)</td><td>132 (66)</td></tr><tr><td> Pan Americas (PAHO)</td><td>171 (17)</td><td>79 (16)</td><td>23 (15)</td><td>36 (18)</td></tr><tr><td> South-East Asia (SEARO)</td><td>33 (3)</td><td>3 (1)</td><td>0 (0)</td><td>2 (1)</td></tr><tr><td> Western Pacific (WPRO)</td><td>198 (20)</td><td>67 (14)</td><td>21 (13)</td><td>27 (13)</td></tr><tr><td colspan=\"5\">Place of treatment (tick all that apply)</td></tr><tr><td> Community/family doctor</td><td>52 (5)</td><td>26 (5)</td><td>6 (4)</td><td>15 (7)</td></tr><tr><td> Hospital</td><td>608 (60)</td><td>255 (52)</td><td>85 (53)</td><td>87 (43)</td></tr><tr><td> Specialist all cancers center</td><td>221 (22)</td><td>120 (24)</td><td>43 (27)</td><td>49 (24)</td></tr><tr><td> CML centre of excellence</td><td>214 (21)</td><td>135 (27)</td><td>43 (27)</td><td>65 (32)</td></tr><tr><td> Other</td><td>53 (5)</td><td>26 (5)</td><td>8 (5)</td><td>12 (6)</td></tr><tr><td colspan=\"5\">Time after diagnosis of CML</td></tr><tr><td> Less than 1 year</td><td>34 (3)</td><td>3 (1)</td><td>1 (1)</td><td>1 (1)</td></tr><tr><td> 1–3 years</td><td>120 (12)</td><td>8 (3)</td><td>2 (1)</td><td>2 (1)</td></tr><tr><td> 3–5 years</td><td>140 (14)</td><td>49 (10)</td><td>14 (9)</td><td>13 (7)</td></tr><tr><td> 5–10 years</td><td>308 (31)</td><td>183 (38)</td><td>66 (43)</td><td>62 (32)</td></tr><tr><td> 10 years or more</td><td>389 (39)</td><td>237 (49)</td><td>72 (46)</td><td>115 (60)</td></tr><tr><td colspan=\"5\">Discontinued treatment</td></tr><tr><td> Yes</td><td>494 (49)</td><td/><td/><td/></tr><tr><td> No</td><td>522 (51)</td><td/><td/><td/></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>CML treatment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Variables</th><th>Not discontinued</th><th>Discontinued</th></tr><tr><th><italic>N</italic> %</th><th><italic>N</italic> %</th></tr></thead><tbody><tr><td colspan=\"3\">Medication</td></tr><tr><td> Imatinib (Glivec/Gleevec or generic Imatinib)</td><td>263 (56)</td><td>242 (51)</td></tr><tr><td> Nilotinib (Tasigna)</td><td>100 (21)</td><td>141 (29)</td></tr><tr><td> Dasatinib (Sprycel)</td><td>88 (19)</td><td>75 (16)</td></tr><tr><td> Bosutinib (Bosulif)</td><td>9 (2)</td><td>5 (1)</td></tr><tr><td> Ponatinib (Iclusig)</td><td>3 (1)</td><td>1 (0)</td></tr><tr><td> Interferon Alpha</td><td>0 (0)</td><td>14 (3)</td></tr><tr><td> ABL001/Asciminib</td><td>0 (0)</td><td>0 (0)</td></tr><tr><td> Other</td><td>9 (2)</td><td>1 (0)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Motivation for discontinuing treatment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Variables</th><th><italic>N</italic> %</th></tr></thead><tbody><tr><td colspan=\"2\">Where first heard about the possibility of stopping treatment</td></tr><tr><td> Healthcare professional</td><td>476 (49)</td></tr><tr><td> Patient organization</td><td>209 (21)</td></tr><tr><td> Family</td><td>4 (0)</td></tr><tr><td> Printed materials (e.g. brochures or leaflet)</td><td>7 (1)</td></tr><tr><td> Media (e.g. scientific articles or lay press)</td><td>28 (3)</td></tr><tr><td> Internet</td><td>120 (12)</td></tr><tr><td> Social media (e.g. Facebook or web based group)</td><td>81 (8)</td></tr><tr><td> Other</td><td>51 (5)</td></tr><tr><td colspan=\"2\"> Main reasons for considering stopping treatment (tick all that apply)</td></tr><tr><td> To get rid of current treatment side effects</td><td>516 (51)</td></tr><tr><td> The fear of side effects caused by long-term treatment</td><td>429 (42)</td></tr><tr><td> Not needing to take medication everyday</td><td>378 (37)</td></tr><tr><td> To see if can be free of CML without therapy</td><td>488 (48)</td></tr><tr><td> Doctor proposed joining a “stopping treatment” clinical trial</td><td>168 (17)</td></tr><tr><td> Financial reasons—reduction of costs</td><td>98 (10)</td></tr><tr><td> Planned or unplanned pregnancy</td><td>105 (10)</td></tr><tr><td> Other</td><td>72 (7)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Duration of treatment and deep molecular response reported by respondents to Phase II.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Variables</th><th><italic>N</italic> %</th></tr></thead><tbody><tr><td colspan=\"2\"><bold>Time on CML treatment (years)</bold></td></tr><tr><td> 1</td><td>8 (2)</td></tr><tr><td> 2</td><td>15 (3)</td></tr><tr><td> 3</td><td>44 (9)</td></tr><tr><td> 4</td><td>40 (9)</td></tr><tr><td> 5</td><td>61 (13)</td></tr><tr><td> 6</td><td>33 (7)</td></tr><tr><td> 7</td><td>42 (9)</td></tr><tr><td> 8</td><td>47 (10)</td></tr><tr><td> 9</td><td>35 (8)</td></tr><tr><td> 10</td><td>34 (7)</td></tr><tr><td> 11</td><td>19 (4)</td></tr><tr><td> 12</td><td>25 (5)</td></tr><tr><td> 13</td><td>22 (5)</td></tr><tr><td> 14</td><td>21 (5)</td></tr><tr><td> 15</td><td>9 (2)</td></tr><tr><td> 16</td><td>2 (0.4)</td></tr><tr><td> 17</td><td>2 (0.4)</td></tr><tr><td> 18</td><td>1 (0.2)</td></tr><tr><td> 19</td><td>1 (0.2)</td></tr><tr><td> 24</td><td>2 (0.4)</td></tr><tr><td> 25</td><td>1 (0.2)</td></tr><tr><td> 26</td><td>1 (0.2)</td></tr><tr><td> 27</td><td>1 (0.2)</td></tr><tr><td colspan=\"2\"><bold>Time in deep molecular response (DMR) in years</bold></td></tr><tr><td colspan=\"2\"><italic>DMR defined as at least MR</italic><sup><italic>4.0</italic></sup></td></tr><tr><td> Not in DMR</td><td>7 (2)</td></tr><tr><td> &lt; 1</td><td>16 (3)</td></tr><tr><td> 1–2</td><td>55 (12)</td></tr><tr><td> 2–3</td><td>90 (20)</td></tr><tr><td> 3–4</td><td>84 (18)</td></tr><tr><td> 4–8</td><td>124 (27)</td></tr><tr><td> &gt;8</td><td>84 (18)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Severity of side effects reported by Phase IIIA respondents, before stopping and after restarting treatment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Variables</th><th>N experienced before stopping treatment</th><th>% experienced before stopping treatment</th><th> severity score before stopping treatment</th><th>N experienced after restarting treatment</th><th>% experienced after restarting treatment</th><th> severity score after restarting treatment</th><th>Difference of severity score after restarting treatment</th><th>% difference of severity score after restarting treatment</th></tr></thead><tbody><tr><td colspan=\"9\"><bold>Side effect</bold></td></tr><tr><td> Feeling sad</td><td>85</td><td>(53)</td><td>2.14</td><td>86</td><td>(54)</td><td>2.73</td><td>0.59</td><td>(28)</td></tr><tr><td> Feeling distressed (upset)</td><td>83</td><td>(52)</td><td>2.34</td><td>84</td><td>(53)</td><td>2.90</td><td>0.57</td><td>(24)</td></tr><tr><td> Anxiety</td><td>84</td><td>(53)</td><td>2.31</td><td>91</td><td>(57)</td><td>2.80</td><td>0.49</td><td>(21)</td></tr><tr><td> Headaches</td><td>81</td><td>(51)</td><td>2.14</td><td>77</td><td>(48)</td><td>2.48</td><td>0.34</td><td>(16)</td></tr><tr><td> Lack of appetite</td><td>70</td><td>(44)</td><td>1.53</td><td>66</td><td>(42)</td><td>1.77</td><td>0.24</td><td>(16)</td></tr><tr><td> Disturbed sleep</td><td>98</td><td>(62)</td><td>2.80</td><td>96</td><td>(60)</td><td>3.03</td><td>0.24</td><td>(08)</td></tr><tr><td> Diarrhoea</td><td>89</td><td>(56)</td><td>2.12</td><td>81</td><td>(51)</td><td>2.31</td><td>0.19</td><td>(09)</td></tr><tr><td> Feeling of malaise (not feeling well)</td><td>88</td><td>(55)</td><td>2.63</td><td>86</td><td>(54)</td><td>2.79</td><td>0.17</td><td>(06)</td></tr><tr><td> Hair loss</td><td>89</td><td>(56)</td><td>2.31</td><td>76</td><td>(48)</td><td>2.46</td><td>0.15</td><td>(06)</td></tr><tr><td> Bruising</td><td>77</td><td>(48)</td><td>1.57</td><td>74</td><td>(47)</td><td>1.69</td><td>0.12</td><td>(07)</td></tr><tr><td> Difficulty thinking clearly</td><td>89</td><td>(56)</td><td>2.44</td><td>92</td><td>(58)</td><td>2.53</td><td>0.09</td><td>(04)</td></tr><tr><td> Vomiting</td><td>73</td><td>(46)</td><td>1.48</td><td>63</td><td>(40)</td><td>1.57</td><td>0.09</td><td>(06)</td></tr><tr><td> Nausea</td><td>88</td><td>(55)</td><td>2.38</td><td>82</td><td>(52)</td><td>2.46</td><td>0.09</td><td>(04)</td></tr><tr><td> Menstrual cycle issues</td><td>69</td><td>(43)</td><td>1.83</td><td>55</td><td>(35)</td><td>1.85</td><td>0.03</td><td>(02)</td></tr><tr><td> Shortness of breath</td><td>95</td><td>(60)</td><td>2.44</td><td>80</td><td>(50)</td><td>2.46</td><td>0.02</td><td>(01)</td></tr><tr><td> Pain</td><td>89</td><td>(56)</td><td>2.72</td><td>79</td><td>(50)</td><td>2.72</td><td>0.00</td><td>(0)</td></tr><tr><td> Fatigue (tiredness)</td><td>125</td><td>(79)</td><td>3.30</td><td>121</td><td>(76)</td><td>3.31</td><td>0.00</td><td>(0)</td></tr><tr><td> Dry mouth</td><td>81</td><td>(51)</td><td>2.22</td><td>78</td><td>(49)</td><td>2.22</td><td>0.00</td><td>(0)</td></tr><tr><td> Rash or skin change</td><td>95</td><td>(60)</td><td>2.69</td><td>82</td><td>(52)</td><td>2.67</td><td>−0.02</td><td>−(01)</td></tr><tr><td> Numbness or tingling</td><td>90</td><td>(57)</td><td>2.34</td><td>74</td><td>(47)</td><td>2.31</td><td>−0.03</td><td>−(01)</td></tr><tr><td> Remembering things</td><td>94</td><td>(59)</td><td>2.54</td><td>83</td><td>(52)</td><td>2.48</td><td>−0.06</td><td>−(02)</td></tr><tr><td> Eye bleeds</td><td>90</td><td>(57)</td><td>1.86</td><td>77</td><td>(48)</td><td>1.77</td><td>−0.09</td><td>−(05)</td></tr><tr><td> Swelling of hands, feet, abdomen, and around eyes</td><td>101</td><td>(64)</td><td>2.86</td><td>91</td><td>(57)</td><td>2.65</td><td>−0.21</td><td>−(07)</td></tr><tr><td> Skin pigment changes</td><td>84</td><td>(53)</td><td>2.52</td><td>70</td><td>(44)</td><td>2.29</td><td>−0.24</td><td>−(09)</td></tr><tr><td> Muscle soreness or cramping</td><td>113</td><td>(71)</td><td>3.25</td><td>103</td><td>(65)</td><td>2.97</td><td>−0.28</td><td>−(09)</td></tr><tr><td> Other</td><td>16</td><td>(10)</td><td>2.63</td><td>14</td><td>(09)</td><td>1.50</td><td>−1.13</td><td>−(43)</td></tr></tbody></table></table-wrap>" ]
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[ "<media xlink:href=\"41375_2020_867_MOESM1_ESM.pdf\"><caption><p>TFR Patient Experience Questionnaire - English</p></caption></media>" ]
[{"label": ["1."], "mixed-citation": ["Deininger M, Buchdunger E, Druker BJ. The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood. 2005;105:2640\u201353."]}, {"label": ["12."], "surname": ["Hochhaus"], "given-names": ["A"], "article-title": ["Educational session: managing chronic myeloid leukemia as a chronic disease"], "source": ["Hematol Am Soc Hematol Educ Program"], "year": ["2011"], "volume": ["2011"], "fpage": ["128"], "lpage": ["35"]}, {"label": ["13."], "surname": ["Kanavos", "Vandoros", "Garcia-Gonzale"], "given-names": ["P", "S", "P"], "article-title": ["Benefits of global partnerships to facilitate access to medicines in developing countries: a multi-country analysis of patients and patient outcomes in GIPAP"], "source": ["Glob Health"], "year": ["2009"], "volume": ["5"], "fpage": ["19"]}, {"label": ["14."], "mixed-citation": ["National Comprehensive Cancer Network. Chronic Myelogenous Leukemia. NCCN Clinical Practice Guidelines in Oncology Version 2.2020. National Comprehensive Cancer Network, 2020:45\u20136."]}, {"label": ["19."], "surname": ["Rousselot", "Charbonnier", "Cony-Makhoul", "Agape", "Nicolini", "Varet"], "given-names": ["P", "A", "P", "P", "FE", "BR"], "article-title": ["Loss of major molecular response as a trigger for restarting tyrosine kinase inhibitor therapy in patients with chronic-phase chronic myelogenous leukemia who have stopped imatinib after durable undetectable disease"], "source": ["Blood."], "year": ["2013"], "volume": ["122"], "fpage": ["381"]}, {"label": ["29."], "surname": ["Chamoun", "Kantarjian", "Atallah", "Gonzalez", "Issa", "Rios"], "given-names": ["K", "H", "R", "GN", "GC", "MB"], "article-title": ["Tyrosine kinase inhibitor discontinuation in patients with chronic myeloid leukemia: a single-institution experience"], "source": ["J Hematol Onco"], "year": ["2019"], "volume": ["12"], "fpage": ["1"]}, {"label": ["30."], "surname": ["Fujisawa", "Ueda", "Usuki", "Kobayashi", "Kondo", "Doki"], "given-names": ["S", "Y", "K", "H", "E", "N"], "article-title": ["Feasibility of the imatinib stop study in the Japanese clinical setting: delightedly overcome CML expert stop TKI trial (DOMEST Trial)"], "source": ["Int J Clin Onco"], "year": ["2019"], "volume": ["24"], "fpage": ["445"], "lpage": ["53"]}, {"label": ["40."], "surname": ["Saussele", "Richter", "Guilhot", "Hjorth-Hansen", "Medina de Almeida", "Janssen"], "given-names": ["S", "J", "J", "H", "A", "JJWM"], "article-title": ["\u201cDuration of deep molecular response\u201d has most impact on the success of cessation of tyrosine kinase inhibitor treatment in chronic myeloid leukemia - results from the EURO-SKI trial"], "source": ["Blood"], "year": ["2017"], "volume": ["130"], "fpage": ["313"], "lpage": ["313"]}, {"label": ["44."], "surname": ["Sanford", "Kyle", "Lazo-Langner", "Xenocostas", "Chin-Yee", "Howson-Jan"], "given-names": ["D", "R", "A", "A", "I", "K"], "article-title": ["Patient preferences for stopping tyrosine kinase inhibitors in chronic myeloid leukemia"], "source": ["Curr Onco"], "year": ["2014"], "volume": ["21"], "fpage": ["e241"], "lpage": ["e249"]}, {"label": ["45."], "surname": ["Boquimpani", "Szczudlo", "Mendelson", "Benjamin", "Masszi"], "given-names": ["CM", "T", "E", "K", "T"], "article-title": ["Attitudes and perceptions of patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP) toward treatment-free remission (TFR)"], "source": ["Blood"], "year": ["2014"], "volume": ["124"], "fpage": ["4547"], "lpage": ["4547"]}, {"label": ["47."], "surname": ["Goldberg", "Hamarman"], "given-names": ["S", "S"], "article-title": ["Patients with chronic myelogenous leukemia may not want to discontinue tyrosine kinase inhibitor therapy"], "source": ["Blood"], "year": ["2015"], "volume": ["126"], "fpage": ["1584"], "lpage": ["1584"]}, {"label": ["48."], "surname": ["Cho", "Kang", "Park", "Jeong Eon", "Seok Jin", "Hak Min"], "given-names": ["J", "D", "S", "L", "N", "L"], "article-title": ["Scanxiety and quality of life among breast cancer survivors"], "source": ["J Clin Oncol"], "year": ["2017"], "volume": ["33"], "fpage": ["e20569"], "lpage": ["e20569"]}, {"label": ["49."], "surname": ["Sogawa", "Kimura", "Yakabe", "Mizokami", "Tasaki", "Sueoka-Aragane"], "given-names": ["R", "S", "R", "Y", "M", "N"], "article-title": ["Anxiety and depression associated with tyrosine kinase inhibitor discontinuation in patients with chronic myeloid leukemia"], "source": ["Int J Clin Onco"], "year": ["2018"], "volume": ["23"], "fpage": ["974"], "lpage": ["9"]}]
{ "acronym": [], "definition": [] }
51
CC BY
no
2024-01-13 23:35:08
Leukemia. 2020 May 26; 34(8):2102-2112
oa_package/14/c9/PMC7387306.tar.gz
PMC7387307
32047238
[]
[]
[]
[]
[]
[ "<title>Subject terms</title>" ]
[ "<title>To the Editor:</title>", "<p id=\"Par1\">Polycythemia vera (PV) is a chronic myeloproliferative neoplasm (cMPN) characterized by stem cell-derived clonal myeloproliferation resulting in panmyelosis with persistently raised hematocrit, increased risk of thrombotic complications, and predisposition to evolve to myelofibrosis or leukemia [##REF##30478826##1##]. Therapy is currently based on phlebotomy to normalize hematocrit, and aspirin. Hydroxyurea is used as first line when cytoreduction is necessary [##REF##30478826##1##], although toxicity can result in inadequate disease management [##REF##30919072##2##]. Recently, ropeginterferon α-2b was approved by European Medicinal Agency as first line for patients without symptomatic splenomegaly [##UREF##0##3##]. Ruxolitinib is second-line for patients who are refractory and/or intolerant to hydroxyurea [##REF##29200417##4##]; other treatments include busulfan, pipobroman [##REF##12764352##5##], and nonpegylated and pegylated interferons (off-label) [##REF##30478826##1##, ##REF##18843285##6##, ##REF##31515250##7##], but use is limited by side effects and safety concerns. Additional, targeted therapies are therefore needed.</p>", "<p id=\"Par2\">Up to 98% of patients with PV bear the <italic>JAK2</italic><sup>V617F</sup> gene mutation, which activates erythropoietin receptor signaling pathways. Givinostat is a histone-deacetylase (HDAC) inhibitor that selectively targets <italic>JAK2</italic><sup>V617F</sup> cell growth, reducing hematopoietic cell proliferation [##REF##18079739##8##]. The efficacy and safety of givinostat alone or with hydroxyurea has previously been evaluated in two studies in <italic>JAK2</italic><sup>V617F</sup> positive PV [##REF##20560970##9##, ##REF##23573950##10##]. Although these studies confirmed the positive risk-benefit of givinostat, they did not provide comprehensive efficacy evidence for givinostat monotherapy, and did not identify the most appropriate dose. The current study was therefore conducted to support givinostat monotherapy development in PV, aiming to determine the maximum tolerated dose (MTD), and to assess safety and efficacy of this dose.</p>", "<p id=\"Par3\">This multinational, open-label, nonrandomized study was conducted in two parts. <italic>Part A</italic> (Phase Ib) was dose escalation, with the first 4-week cycle determining the MTD. <italic>Part B</italic> (Phase II), the proof of concept phase, then evaluated efficacy and safety at this MTD. Full details of the methods are in the supplement. Both parts had 24-week treatment periods, with patients receiving six four-week cycles of givinostat. In <italic>Part A</italic>, since givinostat 50 mg twice daily (BID) was previously well tolerated, the first cohort of three patients received 100 mg BID, with the dose to be escalated by 50 mg BID in each subsequent cohort according to a 3 + 3 design, adopting a modified Fibonacci escalation scheme, although only after the third patient had been followed for a minimum of one cycle, and tolerability data had been evaluated by the Safety Review Team (Supplementary Table ##SUPPL##0##1##). For <italic>Part B</italic>, patients initially received givinostat at the MTD, with modification permitted to achieve an optimized dose, balancing tolerability, and response.</p>", "<p id=\"Par4\">Eligible patients were aged ≥18 years, with a confirmed PV diagnosis, <italic>JAK2</italic><sup>V617F</sup> positivity assessed by centralized quantitative real-time polymerase chain reaction, and active/not controlled disease, defined as: (1) hematocrit ≥45% or &lt;45% with phlebotomy, and (2) platelet count &gt;400 × 10<sup>9</sup>/l, and (3) white blood cell count &gt;10 × 10<sup>9</sup>/l. Main exclusion criteria were: absolute neutrophil count &lt;1.2 × 10<sup>9</sup>/l; prior <italic>JAK2</italic> or HDAC inhibitor treatment; systemic treatment for cMPNs other than aspirin; hydroxyurea, interferon alpha, or anagrelide within 28, 14, or 7 days before enrollment, respectively. All patients provided informed consent. Study registration: ClinicalTrials.gov (NCT01901432).</p>", "<p id=\"Par5\">The primary objectives of <italic>Part A</italic> were to determine givinostat’s MTD, and to characterize safety and tolerability in terms of treatment-related adverse events (AEs). Secondary endpoints were to evaluate overall response after three and six cycles (using the clinico-hematological European LeukemiaNet (ELN) response criteria [##REF##19468275##11##]), and to characterize pharmacokinetics. For <italic>Part B</italic>, primary objectives were to evaluate overall response, safety and tolerability after three cycles. Secondary endpoints were to evaluate overall response, safety and tolerability after six cycles, and to characterize pharmacokinetics. Exploratory endpoints are in the supplement.</p>", "<p id=\"Par6\">Twelve patients were studied in <italic>Part A</italic>, with 35 in <italic>Part B</italic> (Supplementary Fig. ##SUPPL##0##1##, Supplementary Tables ##SUPPL##0##2## and ##SUPPL##0##3##). In <italic>Part A</italic>, during the first cycle one patient receiving givinostat 100 mg BID experienced dose-limiting toxicity: Grade 3 dyspepsia, drug related, resolving with sequelae after treatment. Three additional patients therefore received 100 mg BID, none of whom had dose-limiting toxicity during Cycle 1. Although escalation to higher doses was permitted, the Safety Review Team agreed the MTD was 100 mg BID, given: (a) thrombocytopenia is a known side effect of HDAC inhibitors; (b) a platelet count decrease was observed in subsequent cycles; (c) as givinostat is a chronic treatment, it was preferable to not expose patients to higher doses that could be poorly tolerated during chronic treatment. To more accurately define givinostat’s MTD, three additional patients received an intermediate dose (150 mg daily). Finally, three patients received 50 mg BID, to investigate safety, pharmacokinetics, and pharmacodynamics of this dose. A total of 66.7% of patients experienced at least one drug-related AE, mainly Grade 1 or 2 (Supplementary Table ##SUPPL##0##4##), most commonly thrombocytopenia (33.3% of patients). Two patients (16.7%) experienced a serious AE (thrombophlebitis and myocardial infarction), neither drug-related; no patient died. Two patients withdrew due to drug-related AEs (dyspepsia [Grade 3] and thrombocytopenia [Grade 4]), both with 100 mg BID.</p>", "<p id=\"Par7\">The overall response rate in <italic>Part A</italic> was above 70% (Fig. ##FIG##0##1##). One patient achieved complete response after three cycles, and one after six cycles. Median givinostat <italic>T</italic><sub>max</sub> was 1.5–4 h (Supplementary Table ##SUPPL##0##5##), with steady-state reached by day 28 of Cycle 1 (the first repeat-dose pharmacokinetic evaluation). After three cycles, givinostat normalized hematological parameters in 45.5–54.5% of patients (Supplementary Table ##SUPPL##0##6##), normalized spleen volume in 54.5%, resolved disease-related symptoms in 63.6% (Supplementary Table ##SUPPL##0##7##), and reduced pruritus and <italic>JAK2</italic>-mutated allele burden (Supplementary Table ##SUPPL##0##8##).</p>", "<p id=\"Par8\">At the end of Cycles 3 and 6 of <italic>Part B</italic>, 80.6% of patients were responders (Fig. ##FIG##0##1##), with three achieving complete response after three cycles and one after six. Overall, 94.3% of patients had at least one drug-related AE, the majority Grade 1 or 2 and none Grade 4 or 5, with most occurring during the first three cycles of treatment (152 out of 190 events). The most common were diarrhea (51.4% of patients), thrombocytopenia (45.7%), and increased blood creatinine (37.1%) (Table ##TAB##0##1##). Two patients experienced a serious AE, both during the first three cycles, one study drug-related (Grade 3 diarrhea resolving in 7 days without therapy, with study drug temporarily discontinued). Three patients withdrew, one due to study drug-related AEs (Grade 3 neutropenia and Grade 2 thrombocytopenia, both resolving). The other two were withdrawn by their investigators (Supplementary Fig. ##SUPPL##0##1##). No patient died, and there were no clinically relevant vital signs or ECG values.</p>", "<p id=\"Par9\">Overall, <italic>Part B</italic> pharmacokinetics was similar to <italic>Part A</italic> at comparable doses (Supplementary Tables ##SUPPL##0##9## and ##SUPPL##0##10##). Improvements were seen in all individual response criteria (Supplementary Fig. ##SUPPL##0##2##; Supplementary Table ##SUPPL##0##11##), with white blood cell and platelet counts normalized in 90.3% and 74.2% of patients after three cycles, respectively, and hematocrit in 77.4% and 48.4% after three and six cycles, respectively. Improvements were observed in disease-related symptoms assessed by Myeloproliferative Neoplasm Symptom Assessment Form quality of life (QoL) questionnaire, especially during Cycle 6 (Supplementary Table ##SUPPL##0##12##), with a reduction in the proportion with severe pruritus (Score 7–10; Supplementary Fig. ##SUPPL##0##3##). Approximately 50% had no headache (Supplementary Table ##SUPPL##0##12##), and no patients had severe headache (Supplementary Fig. ##SUPPL##0##4##). The proportion of patients without microvascular symptoms improved from baseline (38.7%) to Cycle 6 (51.6%; Supplementary Table ##SUPPL##0##12##), with a low proportion having severe symptoms (6.5–12.9%; Supplementary Fig. ##SUPPL##0##5##). A total of 19.4% had a spleen volumetric index reduction of at least 35% during treatment, with total spleen normalization in two and three patients after three and six cycles, respectively, and a moderate reduction in <italic>JAK2</italic><sup>V617F</sup> allele burden (Supplementary Table ##SUPPL##0##12##). Finally, differential gene expression was observed (Supplementary Fig. ##SUPPL##0##6##), with upregulation for <italic>GLRX</italic>, <italic>STAT4</italic> and <italic>HDAC3</italic>, and downregulation for <italic>MYC</italic>.</p>", "<p id=\"Par10\">The study aims were achieved, with the MTD, 100 mg BID, determined in <italic>Part A</italic>, and this dose effective in <italic>Part B</italic>. In addition to the high overall response rate, givinostat had a positive impact on individual clinico-hematological ELN criteria, both hematological parameters and disease-related symptoms. The three hematological parameters, all abnormal at study entry, were normalized in the majority of patients, and givinostat improved key disease-related symptoms, notably pruritus with complete resolution in many patients, with an associated positive impact on QoL. A reduction in JAK2-mutated allele burden was observed in both parts of the study, and <italic>Part B</italic> provided clear evidence of differential gene expression with givinostat, consistent with disease pathway regulation. Overall, givinostat was well tolerated with no new safety concerns. Unlike previous studies, the recruited population had active or not controlled disease, and were both high- and low-risk, making comparisons difficult. However, the observed response was greater than for other PV therapies [##UREF##1##12##–##REF##23758082##15##]. For example, in a study comparing interferon to hydroxyurea, 45% of patients had a hematologic response to either therapy [##UREF##1##12##], whereas in a second study, 40% of patients had a response of any type to ruxolitinib [##REF##30997748##13##], and in a third the overall response to the HDAC inhibitor vorinostat was 35%, with significant side effects resulting in a high rate of study withdrawal [##REF##23758082##15##].</p>", "<p id=\"Par11\">In conclusion, these data support givinostat monotherapy development in the defined PV target population.</p>", "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0735-y) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>This study was funded by Italfarmaco S.p.A. David Young of Young Medical Communications and Consulting Ltd, a medical writer supported by funding from Italfarmaco S.p.A., provided drafts and editorial assistance to the authors during preparation of this paper. The authors would like to thank the investigators who recruited patients, including Dr Andrzej Hellmann, and the patients at the investigative sites for their support of this study.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par12\">AR has received honoraria for consultancy, and travel support from Italfarmaco, Gilead, Amgen, Novartis, Pfizer, Celgene, Sanofi, Astellas and Roche. AI has received speaker honoraria from Novartis, Pfizer and Incyte. AMV has received honoraria for advisory board participation from Novartis, Celgene, Incyte, CTI and Italfarmaco, and for lectures from Novartis, and CTI. N.v.B. received research funding from Novartis. MFM has received honoraria and has attended advisory boards with Novartis, and has received honoraria from Celgene. RAM has received honoraria as a consultant to Novartis, Sierra Oncology, and La Jolla Oncology, and research support from Incyte, Genentech, Celgene, CTI, and Abbvie. RT has advised and received honoraria from Italfarmaco. PB, SM, and SDT are employees of Italfarmaco SpA, the sponsor of the study. RN, AG, BM, AP, GC, MDM, SL, NC, J-PM, APancrazzi, FG have no conflicts to disclose.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Parts A and B: therapeutic response evaluation (intention-to-treat population).</title><p>Data are from 11 patients in Part A and 31 patients in Part B.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Part B: patients with study drug-related treatment-emergent AEs, overall and by system organ class and preferred term (including only preferred terms reported by one or more patient with Grade 3 events) (safety population).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">System organ class preferred term</th><th colspan=\"2\">Grade 3</th><th colspan=\"2\">Any grade</th></tr><tr><th><italic>N</italic></th><th>%</th><th><italic>N</italic></th><th>%</th></tr><tr><th>Patients with any drug-related AE</th><th>10</th><th>28.6</th><th>33</th><th>94.3</th></tr></thead><tbody><tr><td>Blood and lymphatic system disorders</td><td>3</td><td>8.6</td><td>18</td><td>51.4</td></tr><tr><td> Anemia</td><td>2</td><td>5.7</td><td>6</td><td>17.1</td></tr><tr><td> Neutropenia</td><td>1</td><td>2.9</td><td>2</td><td>5.7</td></tr><tr><td> Thrombocytopenia</td><td>1</td><td>2.9</td><td>16</td><td>45.7</td></tr><tr><td>Cardiac disorders</td><td>0<sup>a</sup></td><td>0</td><td>1</td><td>2.9</td></tr><tr><td>Gastrointestinal disorders</td><td>4</td><td>11.4</td><td>26</td><td>74.3</td></tr><tr><td> Diarrhea</td><td>4</td><td>11.4</td><td>18</td><td>51.4</td></tr><tr><td>General disorders and administration site conditions</td><td>2</td><td>5.7</td><td>9</td><td>25.7</td></tr><tr><td> Asthenia</td><td>2</td><td>5.7</td><td>8</td><td>22.9</td></tr><tr><td>Investigations</td><td>0<sup>a</sup></td><td>0</td><td>19</td><td>54.3</td></tr><tr><td>Metabolism and nutrition disorders</td><td>1</td><td>2.9</td><td>8</td><td>22.9</td></tr><tr><td> Hypocalcemia</td><td>1</td><td>2.9</td><td>4</td><td>11.4</td></tr><tr><td>Nervous system disorders</td><td>0<sup>a</sup></td><td>0</td><td>5</td><td>14.3</td></tr><tr><td>Renal and urinary disorders</td><td>0<sup>a</sup></td><td>0</td><td>2</td><td>5.7</td></tr><tr><td>Respiratory, thoracic and mediastinal disorders</td><td>0<sup>a</sup></td><td>0</td><td>1</td><td>2.9</td></tr><tr><td>Skin and subcutaneous tissue disorders</td><td>1</td><td>2.9</td><td>6</td><td>17.1</td></tr><tr><td> Rash</td><td>1</td><td>2.9</td><td>1</td><td>2.9</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p>Data are from 35 patients. Grades are based on the National Cancer Institute Common Terminology Criteria for Adverse Events Version 4.03, where Grade 1 are mild events, Grade 2 are moderate, Grade 3 are severe, Grade 4 are life-threatening, and Grade 5 events result in death. There were no Grade 4 or 5 events in Part B of the study.</p><p><italic>AE</italic> adverse event.</p><p><sup>a</sup>There were no Grade 3 events for these system organ classes.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_735_Fig1_HTML\" id=\"d32e589\"/>" ]
[ "<media xlink:href=\"41375_2020_735_MOESM1_ESM.pdf\"><caption><p>Supplemental material</p></caption></media>" ]
[{"label": ["3."], "surname": ["Kiladjian", "Cassinat", "Soret-Dulphy", "Verger", "Roy", "Rey"], "given-names": ["J-J", "B", "J", "E", "L", "J"], "article-title": ["Molecular response to hydroxyurea and ropeginterferon alfa-2B in the PROUD-PV randomized Phase 3 trial"], "source": ["Haematologica"], "year": ["2017"], "volume": ["102"], "fpage": ["S787"]}, {"label": ["12."], "surname": ["Gisslinger", "Klade", "Georgiev", "Skotnicki", "Gercheva-Kyuchukova", "Egyed"], "given-names": ["H", "C", "P", "A", "L", "M"], "article-title": ["Final results from PROUD-PV a randomized controlled Phase 3 trial comparing ropeginterferon alfa-2b to hydroxyurea in polycythemia vera patients"], "source": ["Blood"], "year": ["2016"], "volume": ["128"], "fpage": ["475"], "pub-id": ["10.1182/blood.V128.22.475.475"]}]
{ "acronym": [], "definition": [] }
15
CC BY
no
2024-01-13 23:35:09
Leukemia. 2020 Feb 11; 34(8):2234-2237
oa_package/b2/b4/PMC7387307.tar.gz
PMC7387308
32066865
[]
[]
[]
[]
[]
[ "<title>Subject terms</title>" ]
[ "<title>To the Editor:</title>", "<p id=\"Par1\">Extranodal natural killer T-cell lymphoma (ENKTL) is a distinct entity in the World Health Organization (WHO) classification. Patients suffering from this type of disease have poor survival outcomes [##REF##18385201##1##–##REF##25687842##7##]. However, using the Ann Arbor staging system (AASS), a routine lymphoma staging system, most ENKTL patients are categorized as early stage, which is inconsistent with their poor survival [##REF##18385201##1##, ##REF##12182990##8##–##REF##25697894##12##]. Since the AASS has limited utility in the prognostication and treatment decision making for patients with ENKTL, this study aimed to develop a new staging system specific for ENKTL that can effectively identify patients with poor prognosis and provide information for personalized therapy.</p>", "<p id=\"Par2\">There were three components of this study: a training cohort consisting of two stages and a validation cohort. In the training cohort, we first conducted a retrospective study of ENKTL patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) or CHOP-like regimens or radiotherapy (RT) alone between Jan 1999 and Jun 2008 in 19 hospitals in China, with the aim to identify high-risk factors for proposing the new staging system: the Chinese Southwest Oncology Group and Asia Lymphoma Study Group ENKTL (CA) system. Second, we conducted a prospective study of patients treated with asparaginase-based regimens or RT between Jul 2008 and Dec 2012 in the same 19 hospitals to determine whether the CA system was suitable in the era of asparaginase-based treatment. Based on the results of retrospective study and prospective study, the final form of the CA system was established. To validate the results from the training cohort, we performed an independent validation cohort study between Jan 2010 and Dec 2017 with the same inclusion and exclusion criteria using data obtained from Samsung Medical Center in South Korea, the National Cancer Center in Singapore and five hospitals in China, that were not included in the training cohort. The inclusion criteria were: (1) central pathologically confirmed diagnosis of ENKTL according to the 2008 WHO classification of lymphomas; (2) treatment with chemotherapy with or without radiotherapy or with RT alone with curative intent; and (3) availability of all clinical data required for staging and survival analyses. Local invasiveness was defined as invasion of the bone or perforation or invasion of the skin or paranasal extension as previously reported [##REF##16109779##9##]. Regional lymph node involvement was defined as the invasion of lymph nodes corresponding to N1, N2, or N3 of the primary lesion in accordance with 2002 TNM classification of the American Joint Committee on Cancer. The nasal and nonnasal types were defined based on the involvement of the nasal area, as reported previously [##REF##19850638##5##]. The Institutional Review Board of Sun Yat-sen University Cancer Center (Guangzhou, China) reviewed and approved all aspects of this study. The statistical methods are summarized in Supplementary Appendix.</p>", "<p id=\"Par3\">The CONSORT flow of the study was shown in Supplementary Fig. ##SUPPL##3##1##. The training cohort included 1168 patients. Table ##TAB##0##1## summarizes the characteristics of the patients. In the retrospective study, patients with lesions confined to the nasal cavity or nasopharynx without local tumor invasiveness showed a superior 5-year overall survival (OS) rate than patients with lesions complicated by local tumor invasiveness (59.7% vs. 48.1%, <italic>P</italic> = 0.01). Those with nonnasal-type disease without lymph node involvement had a lower 5-year OS rate than those with nasal-type disease without lymph node involvement (56.7% vs. 40.1%, <italic>P</italic> &lt; 0.01). Thus, patients with nonnasal-type disease or lesions confined to the nasal cavity or nasopharynx complicated by local tumor invasiveness were classified as stage II. Patients harboring lesions with regional lymph node involvement exhibited a lower 5-year OS rate than those without regional lymph node involvement (34.7% vs. 52.3%, <italic>P</italic> &lt; 0.01). Thus, patients harboring lesions with regional lymph node involvement were categorized as stage III. Further, patients with non-regional lymph node involvement or lymph node involvement on both sides of the diaphragm or disseminated disease did not show 5-year OS rate difference (26.5% vs. 27.1% vs. 25.7%, <italic>P</italic> = 0.342), these patients were classified as stage IV. Thus, we propose that the CA be stratified as follows: stage I, lesions confined to the nasal cavity or nasopharynx without local invasiveness and lymph node involvement; stage II, nonnasal-type disease or lesions confined to the nasal cavity or nasopharynx with local invasiveness without lymph node involvement; stage III, lesions with regional lymph node involvement; and stage IV, involvement of nonregional lymph node or lymph nodes on both sides of the diaphragm or disseminated disease. In the retrospective study involving patients who received CHOP-like treatment, the CA effectively discriminated survival; we then performed the prospective study and found that CA also effectively in discriminating the survival of patients who received asparaginase-based treatment.</p>", "<p id=\"Par4\">According to the AASS, the patient distribution in the training cohort from stages I through IV was 61.8%, 20.4%, 5.7%, and 12.1%, respectively. However, according to the CA system, the distribution was 27.4%, 35.2%, 18.7%, and 18.7%, respectively, from stages I through IV (Supplementary Table ##SUPPL##1##1##).</p>", "<p id=\"Par5\">The 5-year OS rate for the training cohort was 52.4% (95% confidence interval (CI) 48.9–55.9), and the 5-year progression-free survival (PFS) rate was 49.0% (95% CI 45.5–52.5). In the training cohort, the CA system exhibited good patient stratification, with 5-year OS rates of 70.8%, 53.1%, 38.6%, and 29.9% for stages I through IV (<italic>P</italic> &lt; 0.001), respectively, and 5-year PFS rates of 67.5%, 52.6%, 35.6%, and 21.1% (<italic>P</italic> &lt; 0.001), respectively. Alternatively, the 5-year OS rates were 60.7%, 42.9%, 17.5%, and 32.1% for AASS stages I through IV (<italic>P</italic> &lt; 0.001), respectively, and the 5-year PFS rates were 59.1%, 38.3%, 10.4%, and 22.4% (<italic>P</italic> &lt; 0.001), respectively (Fig. ##FIG##0##1a–d##). For patients receiving CHOP-like treatment who were diagnosed with CA stages I through IV, the 5-year OS rates also showed reasonable declines; however, when diagnosed using the AASS, the survival of staging IV was better than that of staging III (Supplementary Fig. ##SUPPL##3##2a, b##). This result was similar for patients receiving asparaginase-based treatment (Supplementary Fig. ##SUPPL##3##2c, d##).</p>", "<p id=\"Par6\">Patients from Singapore (<italic>n</italic> = 114), South Korea (<italic>n</italic> = 102), and China (<italic>n</italic> = 769) were included in the independent validation cohort, the 5-year OS rate was 50.4% (95% CI 46.1–54.7), and the 5-year PFS rate was 46.0% (95% CI 41.7–50.3) in this cohort. The CA system effectively stratified the OS and PFS for all 985 patients. (Fig. ##FIG##0##1e–h##).</p>", "<p id=\"Par7\">In the receiver operating characteristic (ROC) analysis, the CA system better discriminated survival than the AASS in the training cohort (area under the curve (AUC), 0.68 vs. 0.60, <italic>P</italic> = 0.013) and the validation cohort (AUC, 0.70 vs. 0.61, <italic>P</italic> = 0.032). For all 842 patients who received asparaginase-based treatment in the training cohort and validation cohort, the prognostic index of natural killer lymphoma (PINK) [##REF##26873565##13##] could stratify the survival according to different risk groups (Supplementary Fig. ##SUPPL##3##3##). However, the ROC analysis of CA system was superior than PINK (AUC, 0.71 vs. 0.64, <italic>P</italic> = 0.031). For the 842 asparaginase-based treatment patients, for CA stage I, the 5-year OS rates for RT and chemotherapy combined with radiotherapy were similar (81.6% vs. 85.8%, <italic>P</italic> = 0.248). For CA stage II, RT resulted in the lowest 5-year OS rate of 70.1%, while concurrent chemoradiotherapy (CCRT), induction chemotherapy followed by radiotherapy, or concurrent chemotherapy (CT + CCRT/RT) and CCRT followed by adjuvant chemotherapy (CCRT + CT) showed similar 5-year OS rates (75.2%, 82.3%, and 76.7%, respectively, <italic>P</italic> = 0.754). Patients with CA stage III receiving CT + CCRT/RT exhibited the highest 5-year OS rate of 73.5%, CCRT + CT and CCRT had moderate 5-year OS rates of 67.0% and 55.3%, respectively, and those receiving RT had the lowest 5-year OS of 32.3% (<italic>P</italic> &lt; 0.001). For CA stage IV, patients receiving autologous transplantation after chemotherapy did not show superior survival than those who did not (57.1% vs. 23.5%, <italic>P</italic> = 0.174) (Supplementary Fig. ##SUPPL##3##4##).</p>", "<p id=\"Par8\">The AASS could not reasonably stratify the survival of ENKTL patients, since the survival of patients with stage IV was better than that of stage III. Less than 10% of patients were classified as stage III by the AASS, the highly unbalanced distribution may produce unavoidable survival bias. Yan et al. [##REF##26098892##14##] recently suggested a TNM staging system for ENKTL. However, that study was performed at a single-center, focused only on nasal patients, and the majority of patients enrolled received anthracycline chemotherapy, thus limiting the generalizability of that staging system. Currently, PINK is used to predict prognosis, but factors including the stage and lymph node involvement in this index are traditionally classified as part of the staging system. Thus, the application of PINK should depending on patient’s general characteristics and staging factors. The CA system was established based on anatomic factors and can efficiently classify patients into different stages. The anatomic factors can be conveniently examined through imaging examinations. Moreover, the ROC analysis suggested that CA staging system is superior to AASS and PINK.</p>", "<p id=\"Par9\">In terms of guiding contemporary asparaginase-based treatment, our study recommended RT for stage I; chemotherapy combined with radiotherapy for stage II; CT + CCRT/RT for stage III; and intensive chemotherapy for stage IV. However, the treatment regimens in our study were varied, validation in a second data set is needed, most preferable in a prospective manner.</p>", "<p id=\"Par10\">The CA system demonstrated better survival discrimination than the AASS, and might add prognostic value and inform treatment decisions for ENKTL. It is crucial to accurately identify high-risk patients to improve outcomes in this subset of lymphoma.</p>", "<title>Supplementary information</title>", "<p>\n\n\n\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0740-1) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>This work is supported by Guangdong Science and Technology Department (grant numbers 2017B020227002 and 2017A020215030). We thank all the patients, their families, and the institutions for supporting this study. We thank the committee on malignant lymphoma from the Chinese Southwest Oncology Group and the Asia Lymphoma Study Group.</p>", "<title>Author contributions</title>", "<p>TYL designed the research. TYL and HMH collected and analyzed the data. HMH, CYL, and HH, wrote the article. All authors provided study materials or patients and approved the article.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par11\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>The OS and PFS of the training cohort and the validation cohort.</title><p><bold>a</bold> OS staging using the AASS for the training cohort. <bold>b</bold> OS staging using the CA system for the training cohort. <bold>c</bold> PFS staging using the AASS for the training cohort. <bold>d</bold> PFS staging using the CA system for the training cohort. <bold>e</bold> OS staging using the AASS for the validation cohort. <bold>f</bold> OS staging using the CA system for the validation cohort. <bold>g</bold> PFS staging using the AASS for the validation cohort. <bold>h</bold> PFS staging using the CA system for the validation cohort.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of characteristics between the validation and training cohorts.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Characteristic</th><th>Training cohort <italic>n</italic> = 1168, no. (%)</th><th>Validation cohort <italic>n</italic> = 985, no. (%)</th><th><italic>P</italic></th></tr></thead><tbody><tr><td>Age, years</td><td/><td/><td>0.719</td></tr><tr><td> ≤60</td><td>985 (84.3)</td><td>837 (85.0)</td><td/></tr><tr><td> &gt;60</td><td>183 (15.7)</td><td>148 (15.0)</td><td/></tr><tr><td>Sex</td><td/><td/><td>0.166</td></tr><tr><td> Male</td><td>804 (68.8)</td><td>650 (66.0)</td><td/></tr><tr><td> Female</td><td>364 (31.2)</td><td>335 (34.0)</td><td/></tr><tr><td>ECOG PS</td><td/><td/><td>0.237</td></tr><tr><td> 0–1</td><td>1061 (90.8)</td><td>896 (91.0)</td><td/></tr><tr><td> 2–4</td><td>87 (9.2)</td><td>89 (9.0)</td><td/></tr><tr><td>“B” symptoms</td><td/><td/><td>0.242</td></tr><tr><td> Absent</td><td>570 (48.8)</td><td>455 (46.2)</td><td/></tr><tr><td> Present</td><td>598 (51.2)</td><td>530 (53.8)</td><td/></tr><tr><td>Serum LDH level</td><td/><td/><td>0.510</td></tr><tr><td> Normal</td><td>822 (70.5)</td><td>680 (69.0)</td><td/></tr><tr><td> Elevated</td><td>346 (29.6)</td><td>305 (31.0)</td><td/></tr><tr><td>IPI</td><td/><td/><td>0.377</td></tr><tr><td> 0–1</td><td>869 (74.4)</td><td>716 (72.7)</td><td/></tr><tr><td> 2–5</td><td>299 (25.6)</td><td>269 (27.3)</td><td/></tr><tr><td>NK prognostic index</td><td/><td/><td>0.189</td></tr><tr><td> 1–2</td><td>690 (59.1)</td><td>554 (56.2)</td><td/></tr><tr><td> 3–4</td><td>478 (40.9)</td><td>431 (43.8)</td><td/></tr><tr><td>Local invasiveness</td><td/><td/><td>0.405</td></tr><tr><td> Absent</td><td>688 (58.9)</td><td>562 (57.1)</td><td/></tr><tr><td> Present</td><td>480 (41.1)</td><td>423(42.9)</td><td/></tr><tr><td>Nonnasal type</td><td/><td/><td>0.498</td></tr><tr><td> Yes</td><td>162 (13.9)</td><td>147 (14.9)</td><td/></tr><tr><td> No</td><td>1006 (86.1)</td><td>838 (85.1)</td><td/></tr><tr><td>Regional lymph-node involvement</td><td/><td/><td>0.500</td></tr><tr><td> Absent</td><td>950 (81.3)</td><td>813 (82.5)</td><td/></tr><tr><td> Present</td><td>218 (18.7)</td><td>172 (17.5)</td><td/></tr><tr><td>Bone marrow involvement</td><td/><td/><td>0.441</td></tr><tr><td> Absent</td><td>1093 (93.6)</td><td>913 (92.7)</td><td/></tr><tr><td> Present</td><td>75 (6.4)</td><td>72 (7.3)</td><td/></tr><tr><td>Ann Arbor staging system stage</td><td/><td/><td>0.492</td></tr><tr><td> I–II</td><td>961 (82.3)</td><td>822 (83.5)</td><td/></tr><tr><td> III–IV</td><td>207 (17.7)</td><td>163 (16.5)</td><td/></tr><tr><td>WBC count</td><td/><td/><td>0.232</td></tr><tr><td> &gt;4000 per mm<sup>3</sup></td><td>954 (81.7)</td><td>824 (83.7)</td><td/></tr><tr><td> &lt;4000 per mm<sup>3</sup></td><td>214 (18.3)</td><td>161 (16.3)</td><td/></tr><tr><td>Hb level</td><td/><td/><td>0.869</td></tr><tr><td> &gt;110 g/L</td><td>945 (80.9)</td><td>800 (81.2)</td><td/></tr><tr><td> &lt;110 g/L</td><td>223 (19.1)</td><td>185 (18.8)</td><td/></tr><tr><td>Platelet count</td><td/><td/><td>0.877</td></tr><tr><td> &gt;100,000 per mm<sup>3</sup></td><td>1070 (91.6)</td><td>900 (91.4)</td><td/></tr><tr><td> &lt;100,000 per mm<sup>3</sup></td><td>98 (8.4)</td><td>85 (8.6)</td><td/></tr><tr><td>Absolute lymphocyte count</td><td/><td/><td>0.165</td></tr><tr><td> &gt;1000 per mm<sup>3</sup></td><td>808 (69.2)</td><td>653 (66.3)</td><td/></tr><tr><td> &lt;1000 per mm<sup>3</sup></td><td>360 (30.8)</td><td>332 (33.7)</td><td/></tr><tr><td>Serum albumin level</td><td/><td/><td>0.240</td></tr><tr><td> &gt;35 g/L</td><td>965 (82.6)</td><td>794 (80.6)</td><td/></tr><tr><td> &lt;35 g/L</td><td>203 (17.4)</td><td>191 (19.4)</td><td/></tr><tr><td>Treatment regimens</td><td/><td/><td/></tr><tr><td>Anthracycline-based</td><td/><td/><td>&lt;0.001</td></tr><tr><td> RT alone</td><td>200 (23.1)</td><td>20 (4.5)</td><td/></tr><tr><td> CHOP</td><td>502 (58.1)</td><td>318 (71.1)</td><td/></tr><tr><td> CHOP-like</td><td>162 (18.8)</td><td>109 (24.4)</td><td/></tr><tr><td>Asparaginase-based</td><td/><td/><td>0.070</td></tr><tr><td> RT alone</td><td>82 (30.0)</td><td>186 (34.6)</td><td/></tr><tr><td> SMILE-like</td><td>49 (16.1)</td><td>73 (13.6)</td><td/></tr><tr><td> Platinum containing</td><td>173 (56.9)</td><td>279 (51.8)</td><td/></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM4\"></supplementary-material>" ]
[ "<table-wrap-foot><p>Local invasiveness was defined in the text.</p><p><italic>ECOG PS</italic> eastern cooperative oncology group performance status, <italic>LDH</italic> lactate dehydrogenase, <italic>IPI</italic> International Prognostic Index, <italic>NK</italic> natural killer, <italic>WBC</italic> white blood cell, <italic>Hb</italic> hemoglobin, <italic>RT</italic> radiotherapy, <italic>CHOP</italic> cyclophosphamide, doxorubicin, vincristine, and prednisolone, <italic>SMILE</italic> dexamethasone, methotrexate, ifosfamide, <sc>l</sc>-asparaginase, and etoposide.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Huangming Hong, Yexiong Li, Soon Thye Lim, Chaoyong Liang, He Huang</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_740_Fig1_HTML\" id=\"d32e1266\"/>" ]
[ "<media xlink:href=\"41375_2020_740_MOESM1_ESM.docx\"><caption><p>Statistical analysis</p></caption></media>", "<media xlink:href=\"41375_2020_740_MOESM2_ESM.docx\"><caption><p>Patient distribution according to the different staging systems</p></caption></media>", "<media xlink:href=\"41375_2020_740_MOESM3_ESM.docx\"><caption><p>Supplementary Figures Legends</p></caption></media>", "<media xlink:href=\"41375_2020_740_MOESM4_ESM.pptx\"><caption><p>Supplementary Figure 1–4</p></caption></media>" ]
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{ "acronym": [], "definition": [] }
14
CC BY
no
2024-01-13 23:35:09
Leukemia. 2020 Feb 17; 34(8):2243-2248
oa_package/99/62/PMC7387308.tar.gz
PMC7387317
32439895
[ "<title>Introduction</title>", "<p id=\"Par2\">Resistance to cancer treatments may be due to cancer cell-intrinsic mechanisms, such as alteration of the drug target, deregulation of (anti-) apoptotic pathways, drug inactivation or shuttling of the drug out of the cancer cell [##REF##15803154##1##–##REF##16014887##3##]. Cancer cell-extrinsic mechanisms of chemoresistance may be mediated by alterations of drug binding to plasma proteins or their catabolism or the tumor microenvironment [##REF##24048067##4##]. However, whether leukemia cell-intrinsic mutations conferring resistance to therapy lead to differential interactions with the bone marrow (BM) microenvironment (BMM), a complex arrangement of various cell types, extracellular matrix (ECM) proteins, and other factors [##REF##26521296##5##], has not been shown.</p>", "<p id=\"Par3\">The <italic>BCR-ABL1</italic> oncogene, generated by the translocation between chromosomes 9 and 22, results in deregulated activity of the BCR-ABL1 tyrosine kinase driving chronic myeloid leukemia (CML) at early disease stages [##REF##29455643##6##]. BCR-ABL1 is targeted by tyrosine kinase inhibitors (TKIs) [##REF##8548747##7##]. However, resistance to TKIs such as imatinib may occur due to the <italic>BCR-ABL1</italic><sup><italic>T315I</italic></sup> and other mutations [##REF##11423618##8##, ##REF##12204532##9##]. <italic>BCR-ABL1</italic><sup><italic>T315I</italic></sup> arises in the ABL kinase domain interfering with binding to TKIs like imatinib and others [##REF##11423618##8##, ##REF##12204532##9##] accounting for 15–20% of mutations found in CML patients [##REF##24131888##10##, ##REF##16642048##11##]. Patients with imatinib resistance due to BCR-ABL1<sup>T315I</sup> have a rapid clinical course and poor prognosis [##REF##16642048##11##–##REF##23716543##13##], although other mechanisms may be contributory [##REF##24847658##14##]. In the case of BCR-ABL1<sup>T315I</sup> kinase activity does not correlate with increased transformation potency [##REF##17164333##15##, ##REF##16880519##16##]. Global phosphoproteome analysis of BCR-ABL1<sup>T315I+</sup> cells identified a unique signature of phosphosubstrates compared with cells positive for native BCR-ABL1 or other imatinib-resistance conferring mutations leading to altered biological properties [##REF##17164333##15##]. However, exactly how these leukemia cell-intrinsic alterations might influence disease outcome, for instance via altered interactions with the BMM, has not been demonstrated.</p>", "<p id=\"Par4\">Niche occupation in the BMM by normal hematopoietic stem and progenitor cells (HSPC) [##REF##19052546##17##] or leukemic stem cells (LSC) in acute myeloid leukemia (AML) [##REF##21765021##18##] depends on their maturation stage or the state of disease progression, respectively. We hypothesized that differences in clinical outcome of patients with CML due to imatinib-resistant mutations in <italic>BCR-ABL1</italic> may correlate with LSC location in the BMM and specific interactions with the BMM. Indeed, here we show that interactions of leukemic murine and human cells with the BMM via the fibronectin/integrin β3/integrin-linked kinase (ILK)-mediated signaling pathway influence leukemia progression and clinical outcome in BCR-ABL1<sup>T315I+</sup> imatinib-resistant CML in vivo. Targeting these interactions may offer a beneficial, innovative, additive treatment strategy for patients with BCR-ABL1<sup>T315I+</sup> CML, and possibly other leukemias.</p>" ]
[ "<title>Materials and methods</title>", "<title>Statistical analysis</title>", "<p id=\"Par5\">All statistical analyses were performed using GraphPad Prism software. Survival curves were analyzed by Kaplan–Meier-style curves and Log-rank (Mantel–Cox) or Gehan–Breslow–Wilcoxon tests. Differences between groups were assessed by student’s <italic>t</italic>-test. When multiple hypotheses were tested, one-way ANOVA and a Tukey test were used as post-hoc test. The data were presented as mean ± s.d. <italic>P</italic> values ≤ 0.05 were considered significant.</p>" ]
[ "<title>Results</title>", "<title>BCR-ABL1<sup>T315I+</sup> differ from BCR-ABL1<sup>+</sup> cells with regards to diverse biological functions</title>", "<p id=\"Par6\">To test the location of CML-initiating cells (LIC) in the BMM, we performed in vivo confocal 2-photon microscopy of the murine calvarium. Measuring the shortest three-dimensional distance to the endosteum [##REF##19052546##17##], we demonstrated that transplanted BCR-ABL1<sup>+</sup> Lin<sup>−</sup> c-Kit<sup>+</sup> Sca-1<sup>+</sup> (LKS) cells, which harbor the LSC [##REF##17077147##19##] in the retroviral transduction/transplantation model of CML [##REF##24162813##20##], and, particularly, LKS CD150<sup>+</sup> CD48<sup>−</sup> (SLAM) cells, were located significantly further away from the endosteum than control cells (<italic>P</italic> = 0.0029 and <italic>P</italic> = 0.0035, respectively, Figs. ##FIG##0##1a##, ##SUPPL##0##S1A## and Supplementary Table ##SUPPL##0##1##). Prior in vitro treatment of BCR-ABL1<sup>+</sup> LKS cells with imatinib led to closer localization of imatinib-treated LKS cells to the endosteum (<italic>P</italic> = 0.003, Figs. ##FIG##0##1b## and ##SUPPL##0##S1B##). However, BCR-ABL1<sup>T315I+</sup> LIC, which are resistant to all TKIs apart from ponatinib and the allosteric inhibitor of the ABL1 kinase, asciminib [##REF##28329763##21##], localized closer to osteoblastic cells than BCR-ABL1<sup>+</sup> LKS cells (<italic>P</italic> &lt; 0.0001, Fig. ##FIG##0##1c##). We tested whether murine recipients of BM transduced with BCR-ABL1, BCR-ABL1<sup>T315I</sup>, BCR-ABL1<sup>M351T</sup>, BCR-ABL1<sup>Y253F</sup>, or BCR-ABL1<sup>E255K</sup> (the latter both P-loop mutations) [##REF##24162813##20##, ##REF##10224280##22##] may recapitulate the accelerated disease course in patients. Indeed, the survival of untreated recipients of BCR-ABL1<sup>T315I</sup>− or BCR-ABL1<sup>Y253F</sup>− transduced BM was significantly shortened compared with recipients of BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>M351T+</sup> BM (Fig. ##FIG##0##1d##). Consistent with increased engraftment of BCR-ABL1<sup>T315I+</sup> proviral clones and more aggressive disease of BCR-ABL1<sup>T315I+</sup> CML [##REF##24162813##20##, ##REF##16998483##23##], the disease clonality was significantly higher in recipients of BCR-ABL1<sup>T315I+</sup> than BCR-ABL1<sup>+</sup> BM (<italic>P</italic> = 0.035, Fig. ##FIG##0##1e, f##). CML was detectable 8 days after transplantation (Fig. ##SUPPL##0##S1C##–##SUPPL##0##D##), and the leukocyte count was higher in recipients of BCR-ABL1<sup>T315I+</sup> than BCR-ABL1<sup>+</sup> BM (<italic>P</italic> = 0.0335, Fig. ##FIG##0##1g##). Similar to human imatinib-resistant patients with BCR-ABL1<sup>T315I+</sup> disease, blasts were increased in the peripheral blood (Fig. ##SUPPL##0##S1E##) and the BM of mice with BCR-ABL1<sup>T315I+</sup> CML (Fig. ##FIG##0##1h, i##). GFP<sup>+</sup> (BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup>) Gr-1<sup>+</sup> (Fig. ##FIG##0##1j##) and in particular GFP<sup>+</sup> CD11b<sup>medium+</sup> (Figs. ##FIG##0##1k## and ##SUPPL##0##S1F##) myeloid cells, which likely represent the blasts (Fig. ##FIG##0##1l##), were higher in mice with BCR-ABL1<sup>T315I+</sup> than BCR-ABL1<sup>+</sup> CML. BCR-ABL1<sup>T315I+</sup> CD11b<sup>medium+</sup> cells expressed the highest levels of c-Kit (<italic>P</italic> &lt; 0.0001, Fig. ##SUPPL##0##S1G##), but lower levels of myeloperoxidase (<italic>P</italic> = 0.0121, Fig. ##SUPPL##0##S1H##–##SUPPL##0##I##). Overall, a decrease of the percentage of GFP<sup>+</sup> c-Kit<sup>+</sup> cells (<italic>P</italic> = 0.0095, Fig. ##FIG##0##1m##) and an increase of GFP<sup>+</sup> CD34<sup>+</sup> (<italic>P</italic> = 0.0003, Fig. ##SUPPL##0##S2A##) and GFP<sup>+</sup> CD13<sup>+</sup> (<italic>P</italic> = 0.0019, Fig. ##SUPPL##0##S2B##) cells were observed in mice with BCR-ABL1<sup>T315I+</sup> CML. The frequency of BCR-ABL1<sup>T315I+</sup> (GFP<sup>+</sup>) LKS cells (<italic>P</italic> = 0.0045, Fig. ##SUPPL##0##S2C##) and myeloid progenitor cells (Fig. ##SUPPL##0##S2D##) in the BM were reduced. However, the percentage of BCR-ABL1<sup>T315I+</sup> (GFP<sup>+</sup>) LKS SLAM did not differ compared with BCR-ABL1<sup>+</sup> CML (Fig. ##SUPPL##0##S2E##). Concordant with decreased myeloid maturation expression of the myeloid transcription factor <italic>Cebpa</italic> in total BM cells of mice with BCR-ABL1<sup>T315I+</sup> CML was significantly decreased (<italic>P</italic> = 0.03, Fig. ##FIG##0##1n## and Supplementary Table ##SUPPL##0##2##). There was a trend toward reduced expression of <italic>Spi1</italic> (PU.1), another myeloid transcription factor, in BCR-ABL1<sup>T315I+</sup> cells (Fig. ##SUPPL##0##S2F##). The migration of BCR-ABL1<sup>T315I+</sup> BA/F3 cells (<italic>P</italic> = 0.0267, Fig. ##FIG##0##1o##), a frequently used in vitro model [##REF##22184410##24##, ##REF##19171873##25##], and the adhesion of BCR-ABL1<sup>T315I+</sup> CD11b<sup>+</sup> cells to the stroma cell line MS-5 in vitro (<italic>P</italic> = 0.0063, Fig. ##FIG##0##1p##) were significantly increased compared with BCR-ABL1<sup>+</sup> BA/F3 cells. In summary, these findings suggest that BCR-ABL1<sup>+</sup> and BCR-ABL1<sup>T315I+</sup> leukemia cells differ with respect to homing localization in the BMM, migration, adhesion, disease aggressiveness and (immuno-) phenotype.</p>", "<title>The actin cytoskeleton and expression and function of focal adhesion kinase differ between BCR-ABL1<sup>T315I+</sup> and BCR-ABL1<sup>+</sup> cells</title>", "<p id=\"Par7\">We hypothesized that differences in the actin cytoskeleton and/or focal adhesion kinase (FAK) [##REF##12124845##26##], which is phosphorylated by BCR-ABL1 [##REF##7556524##27##], underlie the increased migration (Fig. ##FIG##0##1o##) and adhesion (Fig. ##FIG##0##1p##) of BCR-ABL1<sup>T315I+</sup> cells. Indeed, immunofluorescence staining of 3T3 fibroblasts, frequently used as model system to visualize the actin cytoskeleton in BCR-ABL1<sup>+</sup> cells [##REF##2670246##28##, ##REF##9622054##29##], revealed that the evenly distributed and polymerized actin cytoskeleton in empty vector-transduced 3T3 cells was less finely arranged in BCR-ABL1<sup>+</sup> and decreased in BCR-ABL1<sup>T315+</sup> 3T3 cells (Figs. ##FIG##1##2a## and ##SUPPL##0##S3A##). Staining of FAK, a focal adhesion protein involved in actin polymerization [##REF##27402964##30##] and cytoskeletal stability [##REF##22067150##31##], which lies downstream of the integrin receptors, in 3T3 cells transduced with empty vector-expressing retrovirus, was punctate, possibly consistent with an intact focal adhesosome. However, the punctae were reduced in BCR-ABL1<sup>+</sup> and completely dispersed and granular in BCR-ABL1<sup>T315I+</sup> 3T3 cells (<italic>P</italic> &lt; 0.0001; Fig. ##FIG##1##2b, c##). Phosphorylation at phosphotyrosine pY397 in FAK, an autophosphorylation site [##REF##12640026##32##], was higher in BCR-ABL1<sup>+</sup> compared with BCR-ABL1<sup>T315I+</sup> BA/F3 (Figs. ##FIG##1##2d, e## and ##SUPPL##0##S3B##) and Lin<sup>−</sup> cells (Fig. ##SUPPL##0##S3C##), while phosphorylation at pY925 was similar. shRNA-mediated knockdown of <italic>Ptk2</italic> (FAK) (Fig. ##SUPPL##0##S3D##) or the gene of another adapter protein at focal adhesion sites, paxillin (<italic>Pxn</italic>), (Fig. ##SUPPL##0##S3E##) in BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> donor BM did not lead to survival prolongation in recipient wildtype mice. Taken together, these data suggest that the cytoskeleton differs between BCR-ABL1<sup>+</sup> versus BCR-ABL1<sup>T315I+</sup> cells. Possibly due to differing phosphorylation of FAK by BCR-ABL1 versus BCR-ABL1<sup>T315I</sup> focal adhesions may be dysfunctional in BCR-ABL1<sup>T315I+</sup> cells.</p>", "<title>Integrin β3 expression on BCR-ABL1<sup>T315I+</sup> cells influences CML progression</title>", "<p id=\"Par8\">Given our above observations, the known involvement of pY397 of FAK for FAK-mediated cell migration and the phosphorylation of FAK at pY397 upon clustering of integrins [##REF##12640026##32##], we assessed expression of various integrins. While there was no difference in mean fluorescence intensity of integrin α5 (CD49e) (Fig. ##SUPPL##0##S4A##), integrin α IIb (CD41), which forms a heterodimer with integrin β3 [##REF##15906242##33##], was less expressed on BCR-ABL1<sup>T315I+</sup> compared with BCR-ABL1<sup>+</sup> cells (<italic>P</italic> = 0.0103, Fig. ##SUPPL##0##S4B##). Further, integrin β3, previously implicated in the development and progression of AML [##REF##23770013##34##], was more highly expressed on BCR-ABL1<sup>T315I+</sup> versus BCR-ABL1<sup>+</sup> or empty vector-transduced BA/F3 cells by immunoblotting (Figs. ##FIG##2##3a## and ##SUPPL##0##S4C##) and super-resolution microscopy [##REF##28287710##35##] (<italic>P</italic> = 0.0228, Figs. ##FIG##2##3b## and ##SUPPL##0##S4D##). Primary Gr-1<sup>+</sup> BCR-ABL1<sup>T315I+</sup> myeloid cells (<italic>P</italic> = 0.0306, Fig. ##FIG##2##3c##) and total GFP<sup>+</sup> BM cells (<italic>P</italic> = 0.0021, Fig. ##FIG##2##3d##) from CML mice also revealed significantly increased expression of integrin β3. This was also independently confirmed using stable isotope labeling with amino acids in cell culture-based quantitative mass spectrometry (Fig. ##SUPPL##0##S4E##, Supplementary Table ##SUPPL##1##3##). Consistently, coimmunoprecipitation of BA/F3 cells transduced with BCR-ABL1 or BCR-ABL1<sup>T315I</sup> with an antibody to integrin β3 revealed increased binding of FAK to integrin β3 in BCR-ABL1<sup>T315I+</sup> cells (Fig. ##SUPPL##0##S4F##). Exogenous overexpression of integrin β3 on BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> donor BM by retroviral cotransduction [##REF##24162813##20##] (Fig. ##SUPPL##0##S3G##), led to significantly reduced leukocyte counts in peripheral blood (<italic>P</italic> = 0.0031 for BCR-ABL1<sup>+</sup> and <italic>P</italic> &lt; 0.0001 for BCR-ABL1<sup>T315I+</sup>, Fig. ##FIG##2##3e##) and prolonged survival (<italic>P</italic> = 0.0018 for BCR-ABL1<sup>+</sup> and <italic>P</italic> = 0.0025 for BCR-ABL1<sup>T315I+</sup>, Fig. ##FIG##2##3f##) in most recipients of integrin β3<sup>+</sup> compared with empty vector-transduced BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> donor BM. Homing of empty vector- or integrin β3-overexpressing LIC did not differ (Fig. ##SUPPL##0##S4H##–##SUPPL##0##I##). In contrast, knockdown of integrin β3 on BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> donor BM did not alter survival (Fig. ##SUPPL##0##S4J##–##SUPPL##0##K##). Testing phagocytic activity, characteristic of mature myeloid cells, we demonstrated that` phagocytosis was significantly reduced in BCR-ABL1<sup>T315I+</sup> versus BCR-ABL1<sup>+</sup> myeloid cells (<italic>P</italic> = 0.0256, Fig. ##FIG##2##3g##). However, overexpression of integrin β3 on BCR-ABL1<sup>T315I+</sup> myeloid cells ‘rescued’ or restored the phagocytosis of bacterial particles (<italic>P</italic> = 0.0418, Fig. ##FIG##2##3g##). In summary, these data suggested that integrin β3 plays a role in the outcome of, particularly, BCR-ABL1<sup>T315I+</sup> CML, while also influencing the differentiation of BCR-ABL1<sup>+</sup> and BCR-ABL1<sup>T315I+</sup> myeloid cells.</p>", "<title>Fibronectin is decreased in the BMM of mice with BCR-ABL1<sup>T315I+</sup> CML</title>", "<p id=\"Par9\">Next, we tested the adhesion of empty vector-, BCR-ABL1- or BCR-ABL1<sup>T315I+</sup> primary BM cells to the ECM protein fibronectin, one of the ligands of integrin β3. This revealed increased adhesion of BCR-ABL1<sup>T315I+</sup> cells to fibronectin compared with empty vector- or BCR-ABL1-transduced cells (<italic>P</italic> &lt; 0.0001, Fig. ##FIG##3##4a##). We hypothesized that BCR-ABL1<sup>T315I+</sup> CML cells, similar to other cancers [##REF##9622054##29##], may deposit less fibronectin. Indeed, 3T3 fibroblasts transduced with BCR-ABL1<sup>T315I</sup> deposited significantly less fibronectin than BCR-ABL1<sup>+</sup> fibroblasts (Fig. ##SUPPL##0##S5A##). Deposition of fibronectin was kinase-dependent, as treatment of BCR-ABL1<sup>+</sup>, BCR-ABL1<sup>Y253F+</sup>, or BCR-ABL1<sup>T315I+</sup> 3T3 fibroblasts with the TKI ponatinib significantly increased fibronectin deposition, while imatinib had no effect on imatinib-resistant BCR-ABL1 mutants (Fig. ##FIG##3##4b##). Less fibronectin was also found in the BMM of mice with CML due to BCR-ABL1<sup>T315I</sup>, BCR-ABL1<sup>E255K</sup>, or BCR-ABL1<sup>Y253F</sup>, but not BCR-ABL1<sup>M351T</sup> compared with control mice (Figs. ##FIG##3##4c## and ##SUPPL##0##S5B##). In order to test whether the generation of fibronectin by leukemia cells influences leukemia progression and accelerates BCR-ABL1<sup>+</sup> CML similar to BCR-ABL1<sup>T315I+</sup> CML, we transduced the BM of fibronectin flox/flox × Mx1-Cre (FN Mx1-Cre) mice with BCR-ABL1 or BCR-ABL1<sup>T315I</sup>, before transplantation into wildtype recipients and subsequent administration of poly I:C to induce Cre (Fig. ##SUPPL##0##S5C##). This led to a significant increase of the number of leukocytes in the peripheral blood of mice that had received BCR-ABL1<sup>+</sup> FN Mx1-Cre BM (<italic>P</italic> = 0.0403, Fig. ##FIG##3##4d##) and significant shortening of survival of those mice which received BCR-ABL1<sup>+</sup> (<italic>P</italic> = 0.0162, Fig. ##FIG##3##4e##) or BCR-ABL1<sup>T315I+</sup> FN Mx1-Cre BM compared with their respective controls (<italic>P</italic> = 0.0018, Fig. ##SUPPL##0##S5D##). All mice succumbed to CML-like disease. Later deletion of fibronectin in LIC had the same effect (Fig. ##FIG##3##4f, g##). In contrast, no effect on survival was observed when BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> wildtype BM was transplanted into fibronectin flox/flox × Col1a2-Cre (FN Col1a2-Cre) mice (Fig. ##SUPPL##0##S5E##–##SUPPL##0##F##), characterized by lack of production of fibronectin by fibroblasts [##REF##22140539##36##]. In summary, these data suggest that BCR-ABL1<sup>T315I+</sup> CML cells produce less fibronectin than BCR-ABL1<sup>+</sup> cells and that fibronectin production by leukemia cells influences CML progression.</p>", "<title>Integrin-linked kinase is involved in fibronectin deposition and influences survival in BCR-ABL1<sup>T315I+</sup> CML</title>", "<p id=\"Par10\">Hypothesizing that BCR-ABL1<sup>T315I+</sup> leukemia cells deposit less fibronectin than BCR-ABL1<sup>+</sup> leukemia cells due to differences in signaling pathways, we focused on ILK, a pseudokinase belonging to the family of RAF-like kinases involved in integrin-mediated signal transduction [##REF##23863169##37##] and fibronectin deposition [##REF##12060675##38##, ##REF##19885839##39##]. ILK is linked to the cytoplasmic domains of integrins β1 and β3 [##REF##22637643##40##]. We demonstrated that levels of total ILK and ILK phosphorylated at its autophosphorylation site S246 (ILK pS246) were increased in BCR-ABL1<sup>T315I+</sup> compared with BCR-ABL1<sup>+</sup> BA/F3 (Figs. ##FIG##4##5a## and ##SUPPL##0##S6A##–##SUPPL##0##B##) or Lin<sup>−</sup> cells (Fig. ##SUPPL##0##S6C##–##SUPPL##0##D##). Treatment with ponatinib reduced levels of ILK, and ILK pS246 in BCR-ABL1<sup>+</sup> and BCR-ABL1<sup>T315I+</sup> BA/F3 cells, suggesting that protein levels of ILK and possibly phosphorylation of ILK may be BCR-ABL1-dependent (Figs. ##FIG##4##5b## and ##SUPPL##0##S6E##–##SUPPL##0##F##), though not directly mediated by the tyrosine kinase BCR-ABL1. ILK and integrin β3 colocalized in BCR-ABL1<sup>+</sup> and BCR-ABL1<sup>T315I+</sup> BA/F3 cells, but the staining pattern for ILK and integrin β3 was more diffuse and less punctate in BCR-ABL1<sup>T315I+</sup> compared with BCR-ABL1<sup>+</sup> cells (Figs. ##FIG##4##5c## and ##SUPPL##0##S6G##). Consistently, in spite of the overexpression of integrin β3, binding between ILK and integrin β3 was reduced in BCR-ABL1<sup>T315I+</sup> compared with BCR-ABL1<sup>+</sup> cells (Figs. ##FIG##4##5d## and ##SUPPL##0##S6H##). Cotransduction of donor BM with BCR-ABL1- versus BCR-ABL1<sup>T315I</sup>-expressing retrovirus and <italic>scrambled</italic>- or <italic>Ilk</italic> shRNA-expressing lentivirus (<italic>P</italic> = 0.0133, Fig. ##SUPPL##0##S6I##) led to a significant reduction of leukocyte counts (<italic>P</italic> = 0.0018, Fig. ##FIG##4##5e##) and significant prolongation of survival (<italic>P</italic> = 0.0476, Figs. ##FIG##4##5f## and  ##SUPPL##0##S6I##) in recipients of BCR-ABL1<sup>T315I+</sup> sh <italic>Ilk</italic><sup><italic>+</italic></sup> donor BM compared with controls, also when knockdown of <italic>Ilk</italic> was induced at a later timepoint after transplantation (<italic>P</italic> = 0.0028, Figs. ##FIG##4##5g## and ##SUPPL##0##S6J##). Knockdown of <italic>Ilk</italic> also led to an increase in fibronectin deposition by BCR-ABL1<sup>T315I+</sup> sh <italic>Ilk</italic><sup><italic>+</italic></sup> 3T3 fibroblasts (Fig. ##FIG##4##5h##) and in the BMM of recipients of BCR-ABL1<sup>T315I+</sup> sh <italic>Ilk</italic><sup><italic>+</italic></sup> donor BM compared with controls (Figs. ##FIG##4##5i## and ##SUPPL##0##S6K##). As BCR-ABL1 only phosphorylates tyrosine and not serine residues, we tested the phosphorylation of ILK at pS246 after treatment of BCR-ABL1<sup>T315I+</sup> BA/F3 cells with vehicle, the ILK inhibitor Cpd22, ponatinib or the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin, as PI3K is activated by BCR-ABL1 [##REF##18704194##41##]. This revealed that levels of pS246ILK were decreased by Cpd22, ponatinib, and wortmannin, while total ILK was mostly decreased by ponatinib (Figs. ##FIG##4##5j## and ##SUPPL##0##S6L##–##SUPPL##0##M##), suggesting that PI3K may differentially phosphorylate ILK in BCR-ABL1<sup>+</sup> versus BCR-ABL1<sup>T315I+</sup> cells. Taken together, these data suggest that binding between ILK and integrin β3 is impaired in BCR-ABL1<sup>T315I+</sup> cells and that ILK plays an important role in progression of BCR-ABL1<sup>T315I+</sup> CML, at least partly via modulation of fibronectin levels in the BMM.</p>", "<title>Treatment with fibronectin prolongs survival in BCR-ABL1<sup>T315I+</sup> CML</title>", "<p id=\"Par11\">Hypothesizing that replenishing reduced fibronectin levels in mice with BCR-ABL1<sup>T315I+</sup> CML may decelerate leukemia progression, we transplanted LIC, resuspended in vehicle or fibronectin, intrafemorally into mice as proof of principle. This plus two further intrafemoral applications of fibronectin led to a significant reduction of leukocytes in peripheral blood (<italic>P</italic> = 0.0096; Fig. ##FIG##5##6a##) and significant survival prolongation (<italic>P</italic> = 0.0246; Fig. ##FIG##5##6b##) in mice with BCR-ABL1<sup>T315I+</sup> CML compared with controls. Intravenous administration of fibronectin also significantly prolonged the survival of mice with BCR-ABL1<sup>T315I+</sup> CML (<italic>P</italic> = 0.017; Fig. ##FIG##5##6c##) and increased fibronectin levels in the BM (Fig. ##SUPPL##0##S7A##). However, administration of fibronectin to mice with BCR-ABL1<sup>+</sup> B-cell acute lymphoblastic leukemia (Fig. ##SUPPL##0##S7B##) [##REF##16998483##23##] or MLL-AF9<sup>+</sup> AML (Fig. ##SUPPL##0##S7C##) [##REF##24162813##20##, ##REF##16862118##42##] did not lead to significant differences in survival. Taken together, these data suggest that fibronectin may be involved in regulating the progression of BCR-ABL1<sup>T315I+</sup> CML.</p>", "<title>Treatment with the ILK inhibitor Cpd22 and ponatinib prolongs survival in BCR-ABL1<sup>T315I+</sup> CML</title>", "<p id=\"Par12\">In order to test whether inhibition of ILK may have a role for the treatment of BCR-ABL1<sup>T315I+</sup> CML, we treated mice with BCR-ABL1<sup>T315I+</sup> CML with vehicle, ponatinib, the ILK inhibitor Cpd22 [##UREF##0##43##], or the combination of Cpd22 and ponatinib. Cotreatment with the ILK inhibitor Cpd22 and ponatinib led to a modest, but significant prolongation of survival compared with treatment with ponatinib alone (<italic>P</italic> = 0.0036; Fig. ##FIG##6##7a##). Consistent with a presumed role of ILK in fibronectin deposition, treatment with Cpd22 alone, ponatinib alone, or the combination of Cpd22 and ponatinib increased the levels of fibronectin in bone sections (Fig. ##FIG##6##7b##). Treatment with Cpd22 or ponatinib increased the percentage of integrin β3<sup>+</sup> BCR-ABL1<sup>+</sup> (Fig. ##SUPPL##0##S8A##) and BCR-ABL1<sup>T315I+</sup> (Fig. ##SUPPL##0##S8B##) cells. Integrin β3 clusters per area, which fortify the interaction with focal adhesosomes [##REF##21307119##44##], also significantly increased by treatment of BCR-ABL1<sup>T315I+</sup> cells with Cpd22 (<italic>P</italic> = 0.0051, Fig. ##SUPPL##0##S8C##). In summary, inhibition of ILK with Cpd22 in combination with ponatinib prolongs survival in BCR-ABL1<sup>T315I+</sup> CML. The benefit of ILK inhibition may lie in the combination of increasing fibronectin levels in the BMM, as well as in the further increase of integrin β3 on leukemia cells.</p>", "<title>The fibronectin/integrin β3/ILK-axis in human BCR-ABL1<sup>T315I+</sup> CML cells</title>", "<p id=\"Par13\">Validating our results in the human setting, we demonstrated increased levels of integrin β3 (<italic>P</italic> = 0.025, Figs. ##FIG##6##7c## and ##SUPPL##0##S8D##–##SUPPL##0##E##), increased migration (<italic>P</italic> = 0.0439, Fig. ##FIG##6##7d##) and adhesion of K562<sup>T315I</sup> (<italic>P</italic> = 0.0288, Fig. ##FIG##6##7e##) compared with K562 cells [##REF##29967475##45##]. Transplantation of K562 versus K562<sup>T315I</sup> cells into NOD SCID interleukin-2 receptor γ (NSG) knockout mice led to increased percentages of human CD45<sup>+</sup> leukocytes in peripheral blood (<italic>P</italic> = 0.0133, Fig. ##FIG##6##7f##) and BM (<italic>P</italic> = 0.0357, Fig. ##FIG##6##7g##) in recipients of K562<sup>T315I</sup> cells. Similar, but less striking results were observed after transplantation of KCL-22<sup>T315I</sup> compared with KCL-22 cells (Fig. ##SUPPL##0##S8F##–##SUPPL##0##G##). Treatment of NSG mice transplanted with K562<sup>T315I</sup> cells with Cpd22, ponatinib or their combination led to a trend towards reduced engraftment of human CD45<sup>+</sup> leukocytes compared with vehicle (Figs. ##FIG##6##7h## and ##SUPPL##0##S8H##). Furthermore, fibronectin levels were significantly reduced in bone sections (<italic>P</italic> = 0.043; Fig. ##FIG##6##7i, j##) and levels of ILK, ILK pS246 and integrin β3 were higher in leukemia cell samples from most patients with BCR-ABL1<sup>T315I+</sup> compared with BCR-ABL1<sup>+</sup> CML, though inter-patient variability was observed (Figs. ##FIG##6##7k## and ##SUPPL##0##S8I##). Lastly, transplantation of human BCR-ABL1<sup>+</sup> versus BCR-ABL1T315I<sup>+</sup> CML cells into NSG mice treated in pairs with vehicle or fibronectin led to a nonsignificant reduction of the engraftment of human CD45<sup>+</sup> leukocytes in the majority of treated versus untreated mouse pairs (Fig. ##FIG##6##7l##). Treatment with fibronectin also led to a reduction of oncogene transcript levels in 2/4 (50%) recipients of human BCR-ABL1<sup>+</sup> and 4/5 (80%) recipients of BCR-ABL1<sup>T315I+</sup> CML cells (Fig. ##FIG##6##7m##). In summary, our data with human material suggest that the described link between fibronectin/integrin β3/ILK may also be applicable to human BCR-ABL1<sup>T315I+</sup> CML.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par14\">In continuation of previous studies [##REF##17164333##15##, ##REF##16880519##16##] we demonstrate here that the increased oncogenicity of the <italic>BCR-ABL1</italic><sup><italic>T315I</italic></sup> mutation is at least partly due to differences in the interaction with the BMM and its remodeling compared with native <italic>BCR-ABL1</italic>. BCR-ABL1<sup>T315I+</sup> cells differ from BCR-ABL1<sup>+</sup> cells with regards to the actin cytoskeleton, migratory properties, expression of integrin β3, the levels and phosphorylation of FAK and ILK, as well as ILK-dependent deposition of fibronectin in the BMM. Further increased expression of integrin β3 led to increased myeloid maturation and a deceleration of leukemic progression, while a decrease of fibronectin expression in BCR-ABL1<sup>+</sup> cells accelerated the disease similar to BCR-ABL1<sup>T315I+</sup> CML. Administration of fibronectin decelerated BCR-ABL1<sup>T315I+</sup> disease. In summary, our data suggest that interactions with proteins of the ECM influence CML progression and that therapeutic manipulation of the levels of ECM proteins may be beneficial in (resistant) CML.</p>", "<p id=\"Par15\">Our data extend the observation that integrin β3 is essential for leukemogenesis and influences outcome in AML [##REF##23770013##34##] and suggest that integrin β3—possibly via its interaction with ECM proteins such as fibronectin—may be involved in myeloid maturation, as suggested for integrin β1 [##REF##7678511##46##].</p>", "<p id=\"Par16\">Our findings on the differential location of normal HSPC and BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> LIC are consistent with a previous study on the distinct physical engagement of the BMM by MLL-AF9<sup>+</sup> AML cells, which is dependent on the degree of leukemic progression [##REF##21765021##18##]. Our results further suggest that niche location of malignant cells may influence survival, possibly also in response to TKI treatment.</p>", "<p id=\"Par17\">The involvement of ILK in the pathophysiology of BCR-ABL1<sup>T315I+</sup> (and BCR-ABL1<sup>+</sup>) CML, given its linkage to the cytoplasmic domains of integrin β1 and β3, its support of scaffolding proteins [##REF##22637643##40##] and its known role in the deposition of fibronectin by epithelial cells [##REF##9417112##47##]—though opposite from our results in hematopoietic cells—and other ECM proteins [##UREF##1##48##] was not surprising and may be context dependent. The expression and activity of ILK is known to be increased in several epithelial cancers [##REF##15630415##49##], where it may also contribute to chemoresistance via regulation of adhesion to fibronectin [##REF##24452447##50##]. In addition, ILK has been postulated to be a novel target in solid cancers [##REF##15630415##49##] and leukemia [##UREF##2##51##], though a connection to the BMM was not established. In our work, treatment with Cpd22 and ponatinib significantly prolonged survival in BCR-ABL1<sup>T315I+</sup> CML compared with ponatinib alone.</p>", "<p id=\"Par18\">Fibronectin is overexpressed in certain cancers and contributes to the tumorigenic process [##REF##28367247##52##] contrary to our findings in BCR-ABL1<sup>T315I+</sup> CML, where decreased levels of fibronectin exacerbate leukemia progression. In hematopoiesis, fibronectin supports the growth of HSPC [##REF##1861722##53##, ##REF##9558382##54##], whereas fibronectin may inhibit the proliferation of B-ALL cells in vitro [##REF##29895128##55##]. Our data suggest that administration of fibronectin—or an increase of fibronectin levels via inhibition of ILK—may be beneficial in BCR-ABL1<sup>T315I+</sup> CML, for which limited therapies or only treatments with significant side effects exist. Although fibronectin did not lead to a significant survival prolongation in BCR-ABL1<sup>+</sup> B-ALL or MLL-AF9<sup>+</sup> AML, it is likely that this may have been due to suboptimal dosing and timing of the administration of fibronectin, whose high purchasing costs were prohibitive of further exploration in this study.</p>", "<p id=\"Par19\">In conclusion, we have demonstrated that a point mutation in the kinase domain of <italic>BCR-ABL1</italic>, which leads to imatinib resistance, in our models has altered biological activity and is associated with more aggressive disease due to altered interactions with the BMM via the fibronectin-integrin β3-ILK pathway. We confirmed altered, though variable expression levels and targetability of these proteins in human BCR-ABL1<sup>T315I+</sup> CML cells. This may also explain the accelerated phenotype in humans. These altered interactions and specifically decreased levels of fibronectin or ILK in BCR-ABL1<sup>T315I+</sup> CML are valuable targets. It is to be hoped that modification of this pathway will lead to the development of novel therapies for imatinib-resistant CML and, hopefully, other leukemias.</p>" ]
[]
[ "<p id=\"Par1\">Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within <italic>BCR-ABL1</italic>, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene <italic>BCR-ABL1</italic><sup><italic>T315I</italic></sup> differ from <italic>BCR-ABL1</italic> native (<italic>BCR-ABL1</italic>) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1<sup>T315I+</sup> tumor burden and significantly prolonged survival of mice with BCR-ABL1<sup>T315I+</sup> CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1<sup>T315I+</sup> cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0866-1) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>The authors thank Joel A. Spencer, Cristina Lo Celso, and Charles P. Lin for assistance with in vivo microscopy. This work was supported by the LOEWE Center for Cell and Gene Therapy Frankfurt (CGT) and institutional funds of the Georg-Speyer-Haus to DSK. The Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health (BMG) and the Ministry of Higher Education, Research and the Arts of the State of Hessen (HMWK). The LOEWE Center for Cell and Gene Therapy Frankfurt is funded by HMWK, reference number: III L 4-518/17.004 (2010).</p>", "<title>Author contributions</title>", "<p>RK designed and carried out experiments, analyzed the data and reviewed the paper. RSP, CZ, MaMe, and JN assisted with experiments. MeMe and VM designed and carried out experiments and analyzed the data. NR assisted with mouse work. MSD and MH performed and analyzed super-resolution microscopy experiments. FS provided expertise in molecular biology. MT, KA, and TI generated the K562<sup>T315I</sup> cells. TO designed and carried out the mass spectrometry experiments and analyzed the data. FEN and HP contributed patient samples. HMK contributed patient material, stained, and analyzed slides. RVE performed the initial murine survival experiments on imatinib-resistant mutants. DSK designed experiments, supervised the project, analyzed data, and wrote the paper.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par20\">RK, MeMe, and DSK hold patent no. WO2018/046666 for the use of fibronectin and ILK-inhibitors in leukemia.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>BCR-ABL1<sup>T315I+</sup> differ from BCR-ABL1<sup>+</sup> cells with regards to diverse biological functions.</title><p><bold>a</bold>–<bold>c</bold> Measurement of the shortest three-dimensional distance of (<bold>a</bold>) normal (black circles) or BCR-ABL1<sup>+</sup> LKS (open circles) (<italic>P</italic> = 0.0029, <italic>t</italic>-test) or LKS CD150<sup>+</sup> CD48<sup>−</sup> (LKS SLAM) (black (normal) or open (BCR-ABL1<sup>+</sup>) squares) (<italic>P</italic> = 0.0035, <italic>t</italic>-test) cells or (<bold>b</bold>) BCR-ABL1<sup>+</sup> LKS in vitro treated with vehicle (black circles) or 10 μM imatinib [##REF##11756187##56##] for 4 h (open circles) (<italic>P</italic> = 0.003, <italic>t</italic>-test) to bone and (<bold>c</bold>) BCR-ABL1<sup>+</sup> (black circles) versus BCR-ABL1<sup>T315I+</sup> (open circles) LKS cells to osteoblasts (<italic>P</italic> &lt; 0.0001, <italic>t</italic>-test) in μm. Hematopoietic cells were labeled with the lipophilic dye DiD and injected into unirradiated Col2.3 kb GFP mice. Imaging was performed 2 h after injection. The horizontal black line represents the mean. Each symbol represents a distinct cell from three separate experiments. <bold>d</bold> Kaplan–Meier-style survival curve of untreated BALB/c recipient mice transplanted with 2.5 × 10<sup>5</sup> BCR-ABL1-(blue), BCR-ABL1<sup>Y253F</sup>-(red), BCR-ABL1<sup>T315I</sup>-(gray), BCR-ABL1<sup>E255K</sup>-(brown), or BCR-ABL1<sup>M351T</sup>-(black) transduced bone marrow. The difference in survival between BCR-ABL1<sup>+</sup> and BCR-ABL1<sup>T315I+</sup> (<italic>P</italic> = 0.002, Log-rank test) or BCR-ABL1<sup>Y253F+</sup> (<italic>P</italic> = 0.001, Log-rank test) CML is significant (<italic>n</italic> = 8-9). <bold>e</bold>, <bold>f</bold> Southern blot showing distinct proviral integration events (<bold>e</bold>) and disease clonality (<bold>f</bold>) in spleens of BALB/c recipients of BCR-ABL1-(lanes 1–5) or BCR-ABL1<sup>T315I</sup>-(lanes 6–10) transduced bone marrow at the time of death (<italic>P</italic> = 0.035, <italic>t</italic>-test). <bold>g</bold> Leukocyte counts (WBC) × 10<sup>3</sup> per μl in the peripheral blood of BALB/c recipient mice transplanted with BCR-ABL1-(black) or BCR-ABL1<sup>T315I</sup>-(gray) transduced bone marrow on days 8, 12, and 15 after transplantation (<italic>P</italic> = 0.0335; ANOVA, Tukey test, <italic>n</italic> = 4–5). <bold>h</bold> Hematoxylin and eosin stain of bone sections of mice with BCR-ABL1<sup>+</sup> (top) or BCR-ABL1<sup>T315I+</sup> (bottom) CML. The open arrows are pointing toward mature myeloid cells, while the closed arrows are pointing toward blasts. The scale bar depicts 200 μm (<italic>n</italic> = 5). <bold>i</bold> Giemsa stain of the cytospins of total bone marrow of representative BALB/c recipient mice transplanted with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-transduced bone marrow on day 15 after transplantation. A total of 50,000 bone marrow cells had been plated. The open arrows are pointing towards mature myeloid cells, while the closed arrows are pointing towards blasts. The scale bar depicts 100 μm (<italic>n</italic> = 5). <bold>j</bold> Percentage of GFP<sup>+</sup> (BCR-ABL1<sup>+</sup>) Gr-1<sup>+</sup> myeloid cells in peripheral blood of mice with BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) CML on day 15 after transplantation (<italic>P</italic> = 0.0387; <italic>t</italic>-test, <italic>n</italic> = 4–6). <bold>k</bold> Percentage of GFP<sup>+</sup> (BCR-ABL1<sup>+</sup>) CD11b<sup>medium+</sup> myeloid cells in peripheral blood of mice with BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) CML 15 days after transplantation (<italic>P</italic> = 0.0011; <italic>t</italic>-test, <italic>n</italic> = 4–6). <bold>l</bold> Giemsa stain of the cytospins of sorted CD11b medium<sup>+</sup> bone marrow cells from representative BALB/c recipient mice transplanted with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-transduced bone marrow on day 15 after transplantation. A total of 10,000 CD11b medium<sup>+</sup> bone marrow cells had been plated. The open arrows are pointing toward mature myeloid cells, while the closed arrows are pointing towards blasts. The scale bar depicts 100 μm (<italic>n</italic> = 3). <bold>m</bold> Percentage of GFP<sup>+</sup> (BCR-ABL1<sup>+</sup>) c-Kit<sup>+</sup> cells in the bone marrow of mice with BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) CML (<italic>P</italic> = 0.0095; <italic>t</italic>-test, <italic>n</italic> = 4–6) on day 15 after transplantation. <bold>n</bold> Relative expression of <italic>Cebpa</italic> in total bone marrow of murine recipients of empty vector (white)-, BCR-ABL1<sup>+</sup> (black)-, or BCR-ABL1<sup>T315I+</sup> (dark gray)-donor bone marrow 15 days after transplantation (<italic>P</italic> = 0.03; ANOVA, Tukey test, <italic>n</italic> = 5). <bold>o</bold> Percentage of empty vector (white)-, BCR-ABL1<sup>+</sup> (black)-, BCR-ABL1<sup>T315I+</sup> (dark gray)- or BCR-ABL1<sup>Y253F+</sup> (light gray)-BA/F3 cells which migrated to the bottom chamber containing MS-5 stroma cells in medium containing 10% serum in a transwell migration assay after 8 h (<italic>P</italic> = 0.0267; ANOVA, Tukey test, <italic>n</italic> = 3). 10<sup>5</sup> cells had been plated. <bold>p</bold> Percentage of BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) (GFP<sup>+</sup>) myeloid CD11b<sup>+</sup> cells adhering to MS-5 stroma cells in vitro (<italic>P</italic> = 0.0063; <italic>t</italic>-test, <italic>n</italic> = 3). 1.5 × 10<sup>5</sup> cells had been plated and allowed to adhere for 72 h. The data are representative of three independent experiments.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>The actin cytoskeleton and expression of focal adhesion kinase differ between BCR-ABL1<sup>T315I+</sup> and BCR-ABL1<sup>+</sup> cells.</title><p>Immunofluorescence studies on 3T3 fibroblasts transduced with empty vector, BCR-ABL1 or BCR-ABL1<sup>T315I</sup> grown on coverslips and stained with phalloidin (<bold>a</bold>) or an antibody to FAK (<bold>b</bold>). <bold>c</bold> Quantification of the focal adhesions (FA) per 3T3 fibroblast transduced with empty vector (white), BCR-ABL1 (black), or BCR-ABL1<sup>T315I</sup> (gray) from (b) (<italic>P</italic> &lt; 0.0001; ANOVA, Tukey test, <italic>n</italic> = 3). The data in (<bold>a–c</bold>) are from three independent experiments. <bold>d</bold> Immunoblot showing the expression of FAKpY397 (130 kDa), FAKpY925 (130 kDa), FAK (125 kDa), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (38 kDa) in lysates of BA/F3 cells transduced with empty vector, BCR-ABL1 or BCR-ABL1<sup>T315I</sup>. The immunoblot is representative of three experiments. <bold>e</bold> Immunofluorescence studies of BA/F3 cells transduced with empty vector-, BCR-ABL1-, BCR-ABL1<sup>T315I</sup>-, or BCR-ABL1<sup>Y253F</sup>-expressing retrovirus<sup>,</sup> stained with an antibody to FAKpY397 and 4′,6-diamidino-2-phenylindole (DAPI). The data are representative of two experiments.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Integrin β3 is involved in progression of BCR-ABL1<sup>T315I+</sup> CML.</title><p><bold>a</bold> Immunoblot showing the expression of integrin β3 (ITGB3) (92 kDa) or GAPDH (38 kDa) in lysates of BA/F3 cells transduced with empty vector, BCR-ABL1 or BCR-ABL1<sup>T315I</sup>. The immunoblot is representative of three experiments. <bold>b</bold> Results of super-resolution microscopy (dSTORM) for immunolabeled integrin β3 showing enhanced expression of the epitope on the surface of BA/F3 cells transduced with BCR-ABL1<sup>T315I</sup> versus BCR-ABL1 (<italic>P</italic> = 0.0228; <italic>t</italic>-test, <italic>n</italic> = 3). The data are representative of three experiments. <bold>c</bold> Percentage of GFP<sup>+</sup> (BCR-ABL1<sup>+</sup>) Gr-1<sup>+</sup> integrin β3<sup>+</sup> myeloid cells in peripheral blood of mice with BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) CML on day 15 after transplantation (<italic>P</italic> = 0.0306; <italic>t</italic>-test, <italic>n</italic> = 4–6). <bold>d</bold> Mean fluorescence intensity (MFI) of integrin β3 on CD11b<sup>+</sup> cells from bone marrow of mice with BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) CML on day 15 after transplantation (<italic>n</italic> = 5). <bold>e</bold>, <bold>f</bold> Leukocyte counts (WBC) × 10<sup>3</sup> per μl in peripheral blood (<italic>P</italic> &lt; 0.0001 for BCR-ABL1<sup>T315I+</sup>; <italic>t</italic>-test, <italic>n</italic> = 7-8) (<bold>e</bold>) and Kaplan–Meier-style survival curve (<bold>f</bold>) of BALB/c recipient mice transplanted with bone marrow cotransduced with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-expressing retrovirus and integrin β3 (ITGB3)-overexpressing retrovirus (<italic>P</italic> = 0.0025 for BCR-ABL1<sup>T315I</sup>, Log-rank test). <bold>g</bold> Percentage of BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> CD11b<sup>+</sup> myeloid cells cotransduced with empty vector or integrin β3 (GFP<sup>+</sup>) from mice with established disease after incubation for 90 min with pHrodo-phycoerythrin (PE)-labeled <italic>Escherichia coli</italic> particles (<italic>P</italic> = 0.0256; <italic>t</italic>-test, <italic>n</italic> = 5).</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Fibronectin is decreased in the BMM of mice with BCR-ABL1<sup>T315I+</sup> CML.</title><p><bold>a</bold> Optical density read at 570 nm after adhesion of sorted empty vector<sup>+</sup>, BCR-ABL1<sup>+</sup>, or BCR-ABL1<sup>T315I+</sup> (GFP<sup>+</sup>) CD11b<sup>+</sup> splenocytes to fibronectin (FN) in vitro (<italic>P</italic> &lt; 0.0001 for BCR-ABL1 versus BCR-ABL1<sup>T315I</sup>; ANOVA, Tukey test, <italic>n</italic> = 3). 10<sup>5</sup> cells had been plated and allowed to adhere for 72 h. The data are representative of three independent experiments. <bold>b</bold> Immunofluorescence of 3T3 fibroblasts transduced with BCR-ABL1-, BCR-ABL1<sup>T315I</sup>-, or BCR-ABL1<sup>Y253F</sup>-expressing retrovirus, treated with vehicle, 750 nM imatinib or 60 nM ponatinib for 6 h, stained with an antibody to fibronectin (pink). The nuclei are counterstained with DAPI. <bold>c</bold> Immunohistochemistry for fibronectin (detected by immunoperoxidase using yellow–brown horseradish-peroxidase chromogen) on bones of BALB/c recipient mice transplanted with empty vector-, BCR-ABL1-, or BCR-ABL1<sup>T315I</sup>-transduced bone marrow at time of death. The scale bar depicts 200 μm (<italic>n</italic> = 3). Leukocyte counts (WBC) × 10<sup>3</sup> per μl in peripheral blood (<italic>P</italic> = 0.0403 for BCR-ABL1; <italic>t</italic>-test, <italic>n</italic> = 5–6) on day 12 after transplantation (<bold>d</bold>) and Kaplan–Meier-style survival curve (<bold>e</bold>) of C57BL/6 recipient mice transplanted with FN fl/fl Mx1-Cre<sup>−</sup> or FN fl/fl Mx1-Cre<sup>+</sup> bone marrow transduced with BCR-ABL1 (<italic>P</italic> = 0.0162, Log-rank test) (<bold>e</bold>) or BCR-ABL1<sup>T315I</sup> (<bold>d</bold>). 10 mg/kg polyinosinic:polycytidylic acid (poly I:C) per dose was administered on days 1, 2, 3, and 5 after transplantation. <bold>f</bold>, <bold>g</bold> Leukocyte counts (WBC) × 10<sup>3</sup> per μl in peripheral blood on day 12 after transplantation (<italic>P</italic> = 0.009 for BCR-ABL1<sup>+</sup>; <italic>t</italic>-test, <italic>n</italic> = 5–6) (<bold>f</bold>) and Kaplan–Meier-style survival curve (<bold>g</bold>) of C57BL/6 recipient mice transplanted with FN fl/fl Mx1-Cre<sup>−</sup> or FN fl/fl Mx1-Cre<sup>+</sup> bone marrow transduced with BCR-ABL1 (<italic>P</italic> = 0.0003, Log-rank test) or BCR-ABL1<sup>T315I</sup> (<bold>g</bold>). Ten milligram per kilogram polyinosinic:polycytidylic acid (poly I:C) per dose was administered on days 8, 9, and 10 after transplantation.</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Integrin-linked kinase influences fibronectin deposition and survival in BCR-ABL1<sup>T315I+</sup> CML.</title><p><bold>a</bold> Immunoblot showing the expression of ILK pS246 (65 kDa), ILK (51 kDa), or glycerinaldehyde-3-phosphate dehydrogenase (GAPDH) (38 kDa) in lysates of BA/F3 cells transduced with empty vector, BCR-ABL1 or BCR-ABL1<sup>T315I</sup>. The immunoblot is representative of three independent experiments. <bold>b</bold> Immunoblot showing the expression of ILK pS246 (65 kDa), ILK (51 kDa), or GAPDH (38 kDa) in lysates of BA/F3 cells transduced with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-expressing retrovirus treated with vehicle, 60 nM ponatinib or 750 nM imatinib for 4 h. The immunoblot is representative of three independent experiments. <bold>c</bold> Immunofluorescence of BA/F3 cells transduced with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-expressing retrovirus, stained with an antibody to ILK (red) or integrin β3 (green). The nuclei are counterstained with DAPI. The images are representative of three experiments. The scale bar represents 50 μm. <bold>d</bold> Coimmunoprecipitation (IP) of lysates of BA/F3 cells transduced with BCR-ABL1 or BCR-ABL1<sup>T315I</sup> with an anti-integrin β3 (ITGB3) antibody. The immunoblot was performed with an antibody to integrin β3 (92 kDa) and ILK (51 kDa). <bold>e</bold>, <bold>f</bold> Leukocyte counts (WBC) × 10<sup>3</sup> per μl in peripheral blood (<italic>P</italic> = 0.0018 for BCR-ABL1<sup>T315I+</sup>; <italic>t</italic>-test, <italic>n</italic> = 8–10) (<bold>e</bold>) and Kaplan–Meier-style survival curve (<bold>f</bold>) of BALB/c recipient mice transplanted with bone marrow cotransduced with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-expressing retrovirus and <italic>Scrambled</italic> or <italic>Ilk</italic> shRNA-expressing lentivirus (<italic>P</italic> = 0.0476 for BCR-ABL1<sup>T315I+</sup>, Log-rank test, <italic>n</italic> = 10). <bold>g</bold> Kaplan–Meier-style survival curve of BALB/c recipient mice transplanted with bone marrow cotransduced with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-expressing retrovirus and a lentivirus expressing inducible nontarget control or <italic>Ilk</italic> shRNA (<italic>P</italic> = 0.0028 for BCR-ABL1<sup>T315I+</sup> and <italic>P</italic> = 0.0386 for BCR-ABL1, Log-rank test, <italic>n</italic> = 10). 50 mg/kg of doxcyclin to induce shRNA-expression was administered intraperitoneally to recipient mice on days 8, 9, 10, and 12 after transplantation. <bold>h</bold> Immunofluorescence of normal wildtype (WT) 3T3 fibroblasts or 3T3 fibroblasts transduced with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>-expressing retrovirus and <italic>Scrambled</italic> or <italic>Ilk</italic> shRNA-expressing lentivirus. The nuclei are counterstained with DAPI. The images are representative of four independent experiments. <bold>i</bold> Immunohistochemistry for fibronectin (detected by immunoperoxidase using yellow–brown horseradish-peroxidase chromogen) on bones of representative BALB/c recipient mice transplanted with BCR-ABL1- or BCR-ABL1<sup>T315I</sup>- and sh <italic>Scrambled</italic> or sh <italic>Ilk-</italic>cotransduced bone marrow. <bold>j</bold> Western blot showing the expression of ILK pS246 (65 kDa), ILK (51 kDa), AKT pS473 (62 kDa), or GAPDH (38 kDa) as loading control in lysates of BA/F3 cells transduced with BCR-ABL1<sup>T315I</sup> and treated with vehicle, 50 nM Cpd22, 60 nM ponatinib, or 40 nM wortmannin for 6 h.</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Treatment with fibronectin prolongs survival in BCR-ABL1<sup>T315I+</sup> CML.</title><p><bold>a</bold>–<bold>b</bold> Leukocyte counts (WBC) × 10<sup>3</sup> per μl in peripheral blood (<italic>P</italic> = 0.0096 for BCR-ABL1<sup>T315I+</sup>; <italic>t</italic>-test, <italic>n</italic> = 3) (<bold>a</bold>) and Kaplan–Meier-style survival curve (<bold>b</bold>) of BALB/c recipient mice transplanted with bone marrow transduced with BCR-ABL1 (black)- or BCR-ABL1<sup>T315I</sup> (blue)-expressing retrovirus and treated with intrafemoral (i.f.) administrations of vehicle (solid line) or 50 μg fibronectin resuspended in 50 μl PBS (dashed line) per mouse per injection on days 0, 1, and 2 (<italic>P</italic> = 0.0246 for BCR-ABL1<sup>T315I+</sup>, Log-rank test). At the time of transplant (day 0), the leukemia-initiating cells had been resuspended in vehicle or fibronectin. <bold>c</bold> Kaplan–Meier-style survival curve of BALB/c recipient mice transplanted with bone marrow transduced with BCR-ABL1 (black)- or BCR-ABL1<sup>T315I</sup> (blue)-expressing retrovirus and treated with intravenous (i.v.) administrations of vehicle (solid line) or 200 μg fibronectin per mouse per injection (dashed line) on days 9, 10, and 12 (<italic>P</italic> = 0.017 for BCR-ABL1<sup>T315I+</sup>, Log-rank test, <italic>n</italic> = 3–5).</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig. 7</label><caption><title>Levels of fibronectin are decreased and levels of integrin β3 and ILK are increased in human <italic>BCR-ABL1</italic><sup><italic>T315I+</italic></sup> CML cells.</title><p><bold>a</bold> Kaplan–Meier-style survival curve of BALB/c recipient mice transplanted with bone marrow transduced with BCR-ABL1<sup>T315I</sup>-expressing retrovirus and treated with vehicle (solid black line), 20 mg/kg ponatinib (dashed black line), 15 mg/kg Cpd22 (solid blue line), or ponatinib and Cpd22 (blue dashed line) (doses as above) (<italic>P</italic> = 0.0036 for ponatinib versus ponatinib plus Cpd22, Log-rank test, <italic>n</italic> = 5–6). Ponatinib was given daily on days 9–22, while Cpd22 was given daily on days 9–14 and every other day on days 14–22 after transplantation. <bold>b</bold> Immunohistochemistry for fibronectin (detected by immunoperoxidase using yellow–brown horseradish-peroxidase chromogen) on bones of BALB/c recipient mice transplanted with BCR-ABL1<sup>T315I</sup>-transduced bone marrow treated with vehicle, ponatinib, Cpd22, or the combination of ponatinib and Cpd22 as in (<bold>a</bold>). <bold>c</bold> Relative mean fluorescence intensity (MFI) of integrin β3 (ITGB3) on K562 (black) or K562<sup>T315I</sup> (gray) cells (<italic>P</italic> = 0.025; <italic>t</italic>-test, <italic>n</italic> = 3). <bold>d</bold> Percentage of K562 (black) or K562<sup>T315I</sup> (gray) cells which migrated to the bottom chamber containing MS-5 stroma cells in medium containing 10% serum in a transwell migration assay after 8 h (<italic>P</italic> = 0.0439; <italic>t</italic>-test, <italic>n</italic> = 4). In total, 10<sup>5</sup> K562 cells and 30,000 MS-5 cells had been plated. <bold>e</bold> Percentage of K562 (black) or K562<sup>T315I</sup> (gray) cells adhering to MS-5 stroma cells in vitro (<italic>P</italic> = 0.0288; <italic>t</italic>-test, <italic>n</italic> = 4). In total, 10<sup>5</sup> K562 cells were plated on top of 30,000 MS-5 stroma cells. Cells had been allowed to adhere for 12 h. Percentage of human CD45<sup>+</sup> leukocytes in the peripheral blood (PB) (<bold>f</bold>) or bone marrow (BM) (<bold>g</bold>) of NOD SCID interleukin-2 receptor γ knockout (NSG) mice transplanted with 3 × 10<sup>6</sup> K562 (black) or K562<sup>T315I</sup> (gray) cells on day 38 after transplantation. The mice had not been irradiated (<italic>P</italic> = 0.0133 for PB and <italic>P</italic> = 0.0357 for BM; <italic>t</italic>-test, <italic>n</italic> = 3–5). <bold>h</bold> Percentage of human CD45<sup>+</sup> leukocytes in the bone marrow of NOD SCID interleukin-2 receptor γ knockout (NSG) mice transplanted with 3 × 10<sup>6</sup> K562<sup>T315I</sup> cells treated with vehicle, ponatinib, Cpd22 or ponatinib plus Cpd22 as in (<bold>a</bold>) at the time of death. The results are not significant (<italic>n</italic> = 4–7). Immunohistochemistry for fibronectin (detected by immunoperoxidase using yellow–brown horseradish-peroxidase chromogen) (<bold>i</bold>) and quantification of the area of fibronectin (FN) staining (<bold>j</bold>) in bone sections of patients with BCR-ABL1<sup>+</sup> (black) or BCR-ABL1<sup>T315I+</sup> (gray) CML (<italic>n</italic> = 4–11). <bold>k</bold> Immunoblot showing the expression of ILK pS246 (65 kDa), ILK (51 kDa), integrin β3 (92 kDa), or GAPDH (38 kDa) as loading control in lysates of leukocytes from the bone marrow or peripheral blood of patients with BCR-ABL1<sup>+</sup> or BCR-ABL1<sup>T315I+</sup> CML. Percentage of engraftment of human CD45<sup>+</sup> (hCD45<sup>+</sup>) leukocytes (<bold>l</bold>), or relative levels of <italic>BCR-ABL1</italic><sup><italic>+</italic></sup> or <italic>BCR-ABL1</italic><sup><italic>T315I+</italic></sup> engraftment in BM aspirates from individual NSG recipients of primary human CML samples (peripheral blood or BM) on day 28 after transplantation (<italic>n</italic> = 4–5) (<bold>m</bold>). The recipient mice were treated with either saline or 200 μg/dose fibronectin on days 9, 10, 11, 13, and 15 after transplantation. Recipients of peripheral blood or BM grafts from the same human CML sample are indicated by the same individual symbols. Each human patient sample was transplanted into one vehicle- and two to three fibronectin-treated recipient mice.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>" ]
[ "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Raquel S. Pereira, Costanza Zanetti</p></fn></fn-group>" ]
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[ "<media xlink:href=\"41375_2020_866_MOESM1_ESM.pdf\"><caption><p>Supplementary figures, tables, methods</p></caption></media>", "<media xlink:href=\"41375_2020_866_MOESM2_ESM.pdf\"><caption><p>Supplementary table 3</p></caption></media>" ]
[{"label": ["43."], "mixed-citation": ["Ahmed AU, Yim HCH, Alorro M, Ernst M, Williams BRG. Integrin-linked kinase expression in myeloid cells promotes inflammatory signaling during experimental colitis. J Immunol. 2017; pii: ji1700125. 10.4049/jimmunol.1700125. [Epub ahead of print]."]}, {"label": ["48."], "surname": ["Morgner", "Ghatak", "Jakobi", "Dieterich", "Aumailley", "Wickstr\u00f6m"], "given-names": ["J", "S", "T", "C", "M", "SA"], "article-title": ["Integrin-linked kinase regulates the niche of quiescent epidermal stem cells"], "source": ["Nat Comm"], "year": ["2015"], "volume": ["8"], "fpage": ["8198"]}, {"label": ["51."], "mixed-citation": ["de la Puente P, Weisberg E, Muz B, Nonami A, Luderer M, Stone RM, et al. Identification of ILK as a novel therapeutic target for acute and chronic myeloid leukemia. Leuk Res. 2015; pii: S0145-2126(15)30377-5. 10.1016/j.leukres.2015.09.005. [Epub ahead of print]."]}]
{ "acronym": [], "definition": [] }
56
CC BY
no
2024-01-13 23:35:09
Leukemia. 2020 May 21; 34(8):2087-2101
oa_package/a3/71/PMC7387317.tar.gz
PMC7387319
32071431
[ "<title>Introduction</title>", "<p id=\"Par2\">Clinical observation without therapy—defined as “watch and wait” (W&amp;W)—has been the gold standard for the management of early-stage chronic lymphocytic leukemia (CLL). This principle is based on the repeated failure of previous attempts to improve the clinical outcome of CLL patients by early therapeutic intervention [##REF##9593789##1##–##UREF##1##4##]. Moreover, a reasonable subset of patients with CLL experience an indolent disease course with neither compromising morbidity nor an elevated risk of premature death caused by the leukemia. Such patients have a life expectancy comparable with the normal population, and there is no justification to expose these cases to any potentially harmful antileukemic therapy [##REF##7237385##5##–##REF##1139039##7##].</p>", "<p id=\"Par3\">However, there has still been a debate, whether cases with a more aggressive disease course could benefit from earlier treatment, in particular with the recent advent of targeted drugs. To date, reported trials that address the role of immediate therapy at an early disease stage have only tested single-agent chemotherapies (i.e., chlorambucil and fludarabine), but no modern treatment options, such as combined chemoimmunotherapy or novel small-molecule inhibitors.</p>", "<p id=\"Par4\">The study presented here (named “CLL7” trial) was aimed at testing whether chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) would improve the outcome of patients with unfavorable prognosis when administered at an early stage. FCR has been the first regimen to prolong survival of advanced-stage CLL, and represents a standard of care option for first-line treatment of physically fit CLL patients [##REF##15767648##8##–##UREF##2##12##]. We present data of a German–French collaborative phase 3 trial that compared early FCR therapy versus “watch-and-wait” in Binet A patients with the categorized high-risk CLL disease. We implemented an advanced four-parameter risk stratification system, including genetic disease features to prospectively segregate cases with the Binet A high-risk CLL from those with the low-risk disease, and to direct their therapeutic management in a randomized fashion.</p>" ]
[ "<title>Methods</title>", "<title>Trial design and participants</title>", "<p id=\"Par5\">A prospective randomized phase 3 trial (CLL7) was collaboratively conducted by the German CLL study group (GCLLSG) and the French Cooperative Group on CLL (FCGCLL). Patients with early-stage CLL were registered at 69 sites in Germany, Austria, and Switzerland, and 25 sites in France, in case the following main inclusion criteria had been fulfilled (Supplementary Table ##SUPPL##0##1##): diagnosis of CLL according to NCI-working group criteria [##REF##8652811##13##], established not earlier than 12 months prior to registration, Binet stage A disease, no prior treatment, age ≥ 18 years, and Eastern Cooperative Oncology Group performance status 0–2. Patients with clinically evident autoimmune cytopenias, active second malignancies or infections, long-term use of steroids, or other severe medical illnesses or organ dysfunctions were not eligible. All patients provided written informed consent before registration. The trial was conducted according to the Declaration of Helsinki, and approved by ethical review boards responsible for each of the participating centers. It was registered at the US National Institute of Health (NCT00275054) and the EU clinical trial database (EudraCT 2005-003018-14).</p>", "<title>Risk stratification and randomization</title>", "<p id=\"Par6\">After registration, the following risk parameters were assessed in central laboratories of the GCLLSG and FCGCLL according to standard protocols: serum thymidine kinase (TK) levels, the mutation status of the immunoglobulin heavy-chain variable region genes (IGHV), and recurrent chromosomal abnormalities by fluorescence in situ hybridization. The lymphocyte doubling time (LDT) was calculated by regression curve analysis from a minimum of three lymphocyte counts obtained in at least 4-week intervals within 6 months before registration. Risk factor results were collected at the German and French biometry centers, respectively (Institute for Medical Statistics and Epidemiology (IMSE), Technical University of Munich, Germany; Department de Biostatistiques et Informatique Medicale, Hôpital Saint Louis, Paris, France), where for each patient the final risk evaluation and stratification/randomization procedures were performed. Patients with at least two of four adverse prognostic markers present (TK &gt; 10 U/L, LDT &lt; 12 months, IGHV unmutated, or deletion (del) in chromosome 11q or 17p, or trisomy 12) were categorized as high-risk (Hi) patients, while patients with &lt;2 of these markers present were categorized as low risk (Lo). High-risk patients were one-to-one randomized to either receive FCR chemoimmunotherapy (Hi-FCR) or being observed (Hi-W&amp;W) using a previously generated randomization list (IMSE). The randomization was balanced by the use of randomly permuted blocks with a block size of four, and was stratified according to country and number of adverse prognostic markers. Low-risk patients were only assigned to clinical observation (Lo-W&amp;W).</p>", "<title>Patient treatment and procedures</title>", "<p id=\"Par7\">Patients randomized to the Hi-FCR arm were assigned to receive a maximum of six cycles of intravenous FCR, given in 28-day intervals. Fludarabine (25 mg/m<sup>2</sup>) and cyclophosphamide (250 mg/m<sup>2</sup>) were administered on day 1–3 of each cycle. Rituximab was given at 375 mg/m<sup>2</sup> on day 0 of cycle 1, and at 500 mg/m<sup>2</sup> on day 1 of cycles 2–6. According to the protocol, the prophylactic use of growth factors was left to the discretion of the local investigator. In case of grades 3–4 neutropenia with signs of a concurrent infection, the administration of G-CSF was mandatory per protocol. Anti-infective prophylaxis with trimethoprim/sulfmethoxazole was recommended from day 1 until the end of 2 months after the last dose of the last cycle. Additional details on parental drug administration, concomitant medication, and dose reduction rules are described in ##SUPPL##0##Supplementary Methods##.</p>", "<p id=\"Par8\">Baseline disease assessment included physical examination, ECOG performance status, assessment of B symptoms and comorbidity, imaging of disease manifestations via ultrasound or computed tomography (CT), laboratory assessments from peripheral blood (PB) including parameters routinely assessed prior to the administration of cytoreductive therapies, serum beta-2-microglobulin, and lymphocyte immunophenotyping. Patients underwent baseline and follow-up disease assessments at month 4 (interim staging after three cycles of therapy, Hi-FCR only), month 8 (final staging after therapy), and 12, in 6-month intervals between months 12 and 60, and once per year thereafter. Response assessment after FCR therapy included routine clinical and laboratory assessments, radiographic imaging of CLL manifestations (used method at the discretion of the local investigator), and flow cytometry for minimal residual disease (MRD) assessment. The latter was performed using four-color flow cytometry for the German and six-color flow cytometry for the French cohort. For further details refer to ##SUPPL##0##Supplementary Methods## [##REF##17361231##14##, ##REF##19641522##15##]. A uniform threshold was applied to define MRD negativity as less than one detected CLL cell per 10,000 leukocytes analyzed per flow cytometry. After treatment completion, a bone marrow (BM) aspirate/biopsy was recommended per protocol in case the patient achieved a complete remission (CR).</p>", "<title>Outcomes</title>", "<p id=\"Par9\">The primary objective of the study was to compare the efficacy of early versus deferred FCR in Binet stage A patients at high risk for disease progression. The secondary objective was to prospectively validate the prognostic value of the above-mentioned four-parameter risk stratification system for Binet A patients. The primary endpoint was event-free survival (EFS), considering progression, treatment, or death as events. Among the secondary endpoints were overall response rate (ORR), overall survival (OS), progression-free survival (PFS), adverse events related to treatment, molecular response, response duration, and time to (re)treatment (TTT). The toxicity of FCR treatment was determined according to the Common Terminology Criteria (CTC) for Adverse Events version 3.0. The response status after FCR therapy and disease status during follow-up was evaluated according to the NCI-working group criteria [##REF##8652811##13##].</p>", "<title>Statistical analysis</title>", "<p id=\"Par10\">Details on the sample size computation for this study, data responsibilities, and data sharing are described in ##SUPPL##0##Supplementary Methods##. The primary analysis was a two-sided log-rank test that was stratified by country and number of risk factors in a second step to confirm the results. Time-to-event endpoints were estimated according to the Kaplan–Meier method. Survival curves were compared using nonstratified log-rank tests. Hazard ratios (HR), including 95% confidence intervals (CI), were calculated by Cox regression analysis under the assumption of proportional hazards. Exploratory post hoc subgroup analyses were done considering MRD status, IGHV mutational status, and cytogenetic categories. All tests were two sided, and a <italic>p</italic> value &lt; 0.05 was considered significant. Adjustments for multiple testing were not done. Safety analyses were restricted to patients from the intention-to-treat population who received at least one dose of one component of the study treatment (safety population). ORR was calculated based on both the intention-to-treat and on the safety population. Statistical analyses were performed using SPSS v23 (SPSS, Chicago/IL, USA).</p>" ]
[ "<title>Results</title>", "<title>Study population</title>", "<p id=\"Par11\">Between 2005 and 2010, a total of 824 patients were registered for the CLL7 study, 423 in 69 GCLLSG centers in Germany (51.3%), Austria, and Switzerland, and 401 (48.7%) in 25 centers of the FCGCLL in France. After exclusion of patients, who did not fulfill the study requirements, and completion of risk assessment, 800 patients (ITT population), aged 27–81 years, were stratified into 201 high-risk (25.1%) and 599 low-risk (Lo-W&amp;W) patients (74.9%) (Fig. ##FIG##0##1##). The median time from registration to risk stratification was 3 months (0–29.1 months). One hundred and one high-risk patients were randomized to the observation arm (Hi-W&amp;W), while the remaining 100 patients were allocated to receive early FCR (Hi-FCR). Both high-risk arms were well balanced with respect to country, age, comorbidity, ECOG status, white blood count (WBC), IGHV mutation status, trisomy 12, and del(17p) (Table ##TAB##0##1##). There was an imbalance in the prevalence of elevated TK, short LDT, male sex (each more common in Hi-FCR), and del(11q) (more common in Hi-W&amp;W) between the two high-risk cohorts. B symptoms and lymphadenopathy as signs of a more aggressive disease course were more common in high-risk than in low-risk patients.</p>", "<title>Early FCR treatment and safety</title>", "<p id=\"Par12\">Eighty-two percent of 100 Hi-FCR patients received at least one dose of FCR, and were included in the safety analysis (safety population). Eighteen percent patients withdrew their consent for early therapy after randomization and before FCR had been initiated. The median number of administered treatment cycles was 6 (range, 1–6) and 67 patients (81.7%) completed six cycles of study therapy. The documented reasons for discontinued FCR (&lt;6 cycles, 15 patients, 18.3%) were hematotoxicity (6 patients, predominantly neutropenia), fever/infections (2 patients; 1 CMV reactivation, 1 infection of unknown origin), consent withdrawal or allergic exanthema (2 patients each), 1 hospitalization due to rupture of an aortic aneurysm, 1 thrombosis with consecutive pulmonary embolism, and 1 autoimmune hemolytic anemia (AIHA).</p>", "<p id=\"Par13\">In 20 patients (24.4%) at least one study drug was dose reduced &gt;20% in one or more cycles. Most frequently, the doses of fludarabine and cyclophosphamide were reduced (15 cases) due to hematologic toxicity (11 cases with at least one of the following events: 9 leukopenia/neutropenia, 2 thrombocytopenia, 1 cytopenia not further specified, and 1 anemia). Other reasons for dose-reduced FC were febrile infections (two cases), inpatient treatment due to a ruptured aortic aneurysm (one case), a collapse during infusion (one case), or unknown (two cases). Dose-reduced rituximab was given in five patients (twice unintentionally by missing the rituximab dose increase at cycle 2, in two cases for unknown reasons, and in one case due to an event of bradycardia).</p>", "<p id=\"Par14\">Overall, 203 grade 3–5 adverse events in 61 patients (74.4% of safety population) were reported. In addition, there were 18 events documented in five patients, without sufficient information (including missing CTC grade) available. One hundred and twenty-five of those 203 events (86.2%) were categorized as at least possibly related to the study treatment by the local investigator. The three most common categories were hematotoxicity (50 patients, 61.0% of safety population; most frequently leukopenia/neutropenia), infections (18 patients, 22.0%), and metabolic/laboratory events (5 patients, 6.1%; most frequently elevated liver enzymes) (Table ##TAB##1##2##). Recurrent types of infections were respiratory tract infections (seven patients, 8.5% of the safety population), fever/infections of unknown origin (three patients, 3.7%), herpes zoster reactivations (three patients, 3.7%), and catheter-related infections (two patients, 2.4%). Use of growth factor support with G-CSF was documented in 25 out of 82 FCR treated patients (30.5%).</p>", "<p id=\"Par15\">There were two of total four fatal adverse events during follow-up, documented with a potential relationship to administered FCR: one patient succumbed 9.3 months from stratification to a suspected viral encephalitis (clinical/radiologic diagnosis), which was judged as possibly related to the study therapy. Another patient died 9.8 months from stratification due to a persisting AIHA that had occurred under FCR therapy. Two additional deaths were documented at month 41.8 and 53.2 from stratification with no relationship to study therapy. Causes of death were reported as a pulmonary fibrosis and progressive renal failure in the context of a Richter’s transformation, respectively.</p>", "<p id=\"Par16\">In the overall study, we did not detect an elevated risk of disease transformation, second(ary) malignancies, or AIHA related to an early FCR therapy (Supplementay Table ##SUPPL##0##2##). One Hi-FCR patient developed a secondary acute myeloid leukemia (AML) at month 43.4 during treatment with R-CHOP for Richter’s transformation. Two Lo-W&amp;W patients developed an AML at month 10.9 and at month 90.4, respectively. Both patients had received prior FCR for progressive CLL. Ten patients (1.25% of ITT) developed a Richter’s transformation after median 50.1 months (11.6–84.8 months), two in the Hi-FCR arm (2.0%), three in the Hi-W&amp;W arm (3.0%), and five in the Lo-W&amp;W cohort (0.8%).</p>", "<title>Efficacy of early FCR and survival</title>", "<p id=\"Par17\">The overall response to early FCR based on the ITT population according to NCI-working group criteria [##REF##8652811##13##] was 76.0% (76 out of 100 patients allocated to the Hi-FCR arm). Out of 82 patients, who had received at least one dose of FCR (safety population), 76 (92.7%) achieved a remission (Table ##TAB##2##3##). Twenty-seven patients (32.9%) obtained a BM confirmed CR, 34 patients (41.5%) an at least clinical CR without BM evaluation, and 15 patients (18.3%) obtained a partial remission (PR). In three patients, response assessment was missing, but they had received only one or two cycles of the study therapy, respectively. Three patients (3.8%) had stable disease after therapy. In two of those three cases treatment had been stopped prematurely after one or two cycles due to grade 3 neutropenia and grade 3 febrile neutropenia, respectively. The highest CR rates were achieved in patients who underwent at least three cycles of therapy, were IGHV mutated, or carried a del(11q).</p>", "<p id=\"Par18\">Fifty-three and 28 Hi-FCR patients were available for MRD assessment by four-color flow cytometry from PB and BM, respectively. Forty of 53 patients (75.5%) were MRD negative (≤10<sup>−4</sup>) in PB at the time of final response assessment, 13 patients (24.5) were MRD positive (&gt;10<sup>−4</sup>). In BM, 67.9% (19 out of 28 patients) achieved an MRD-negative remission.</p>", "<p id=\"Par19\">The primary endpoint, EFS, was significantly prolonged in high-risk patients treated with an early FCR (Hi-FCR) versus deferred treatment according to the current standard of care (Hi-W&amp;W). After a median follow-up of 55.6 months (range 0–99.2 months), only 36 patients (36.0%) in the Hi-FCR arm had progressed, received new CLL therapy, or died, compared with 83 patients (82.2%) in the Hi-W&amp;W arm (median EFS not reached in Hi-FCR vs. 18.5 months in Hi-W&amp;W, HR 0.22, 95% CI 0.15–0.33, <italic>p</italic> &lt; 0.001 for stratified and nonstratified log-rank test) (Fig. ##FIG##1##2a##). High-risk patients with a MRD-negative response to early FCR in PB significantly benefited from the quality of remission with regard to EFS compared with patients with an MRD-positive response (landmark analysis, median EFS not reached versus 41.2 months, log-rank <italic>p</italic> &lt; 0.001, HR 10.68, 95%CI 3.51–32.55, Fig. ##FIG##2##3a##; for MRD from BM refer to Supplementary Fig. ##SUPPL##0##1##). Twelve Hi-FCR and 11 Hi-W&amp;W patients had died. In both studies, arms major causes of death were infections and progressive disease including Richter’s transformation (Supplementary Table ##SUPPL##0##3##). There was no significant OS benefit for high-risk patients receiving early versus deferred FCR (5-year OS 82.9% in Hi-FCR vs. 79.9% in Hi-W&amp;W, HR 0.93, 95% CI 0.41–0.22, <italic>p</italic> = 0.864, Figs. ##FIG##1##2##b and ##FIG##2##3b##, Supplementary Fig. ##SUPPL##0##2##).</p>", "<p id=\"Par20\">After 5 years from the last dose of study medication, 80.4% of Hi-FCR patients (safety population) had not received any further treatment for CLL (median TTT not reached, Fig. ##FIG##3##4b##), compared with 21.8% of patients in the Hi-W&amp;W arm (Fig. ##FIG##3##4a##).</p>", "<p id=\"Par21\">Our risk stratification system successfully segregated patients with differential prognosis. High-risk patients with or without early FCR therapy (Hi-FCR/Hi-W&amp;W) demonstrated a significantly shorter EFS, PFS, and OS than patients categorized with the low-risk disease (Fig. ##FIG##1##2a, b##, Supplementary Fig. ##SUPPL##0##3##). Briefly, patients assigned to Hi-W&amp;W had a 8.0 times higher risk of progression, treatment or death (HR 8.02, 95%CI 6.04–10.65, <italic>p</italic> &lt; 0.001), while Hi-FCR patients had a 1.8 times higher risk (HR 1.82, 95% CI 1.26–2.63, <italic>p</italic> = 0.002) of progression, treatment or death, compared with the Lo-W&amp;W cohort. Corresponding 5-year EFS rates were 12.6%, 55.2%, and 77.1% in Hi-W&amp;W, Hi-FCR, and Lo-W&amp;W patients, respectively. In total, 87.1% of Lo-W&amp;W patients were without treatment at 5 years from stratification (median time to first-line treatment 27.6 months in Hi-W&amp;W vs. not reached in Lo-W&amp;W, HR 11.62, 95% CI 8.23–16.39, <italic>p</italic> &lt; 0.001). Patients with the low-risk disease demonstrated an excellent survival at 97.2% 5 years from stratification (HR Hi-FCR/W&amp;W vs. Lo-W&amp;W 5.82, 95% CI 2.98–11.36, <italic>p</italic> &lt; 0.001) (Fig. ##FIG##1##2b##).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par22\">We present data of a phase 3 trial (CLL7), which successfully implemented molecular genetic disease characteristics into a risk-tailored treatment allocation strategy for patients with stage Binet A CLL. Twenty-five percent of our ITT study population exhibited a “high risk” disease type according to our four-factor risk assessment, and these patients clearly segregated from the low-risk group with regard to all time-to-event parameters investigated, i.e., illustrated by EFS, PFS (Supplementary Fig. ##SUPPL##0##3##), and OS.</p>", "<p id=\"Par23\">All four risk parameters used for our study design were chosen due to their confirmed value as prognostic factors for PFS/OS in multivariate analyses performed in the first 147 patients registered in the preceding CLL1 trial (phase 3 comparison of fludarabine vs. W&amp;W in Binet A CLL) in 2004 [##REF##28804126##2##]. In particular, we found serum levels of the TK (cutoff 10 U/L) rather than beta-2-microglobulin (3.5 mg/L) as a preferred independent prognostic factor for time-to-event outcome in our test set analysis, and therefore implemented serum TK in our study design [##REF##10029603##16##]. The parameter LDT reflects the disease dynamics, and is recommended by current guidelines to determine the right time a patient requires therapy [##REF##18216293##17##]. Particularly at an early disease stage, an LDT &lt; 12 months has been identified as an independent indicator of an unfavorable prognosis [##REF##3954968##18##–##REF##10397712##20##]. Although easily assessable in clinical practice, the parameter is not commonly documented in large trial datasets, and therefore not considered in the latest CLL scoring systems, such as the CLL-IPI [##REF##24797299##21##–##REF##21969505##23##].</p>", "<p id=\"Par24\">The scientific background to include trisomy 12 as a risk factor in our stratification approach was formed by the hierarchical model, developed by Döhner et al. before this study was designed [##REF##11136261##24##]. Recent long-term follow-up data in FCR studies, however, demonstrated that patients with trisomy 12 have a particularly favorable PFS/OS after FCR, when treated at an advanced disease stage [##REF##20888994##9##, ##REF##26486789##25##, ##REF##26492934##26##]. Thus, in retrospect it might have been specifically difficult to achieve further improvement for this patient population with our early treatment strategy (Supplementary Figs. ##SUPPL##0##4## and ##SUPPL##0##14##).</p>", "<p id=\"Par25\">A comparative analysis of our risk stratification and the CLL-IPI as a current standard risk assessment in CLL is included as Supplementary Table ##SUPPL##0##4##. It indicates that the CLL7 stratification between low-risk and high-risk subsets correponds to a segregation between CLL-IPI low risk versus CLL-IPI intermediate/high/very high risk in the majority of cases.</p>", "<p id=\"Par26\">The data presented here demonstrate that an early application of FCR was able to postpone events of disease progression and the need of therapy in stage Binet A high-risk CLL, but despite this effect, there was no OS benefit in the long run.</p>", "<p id=\"Par27\">FCR was highly effective in reducing the tumor load in treated patients, as demonstrated by a high OR and CR rate. Moreover, while the significance of the MRD data set is limited by a relatively low number of assessments, the frequency of achieved MRD negativity (PB: 75.5%, BM: 67.9%) compares favorably to the respective data from the FCR arm of the CLL8 trial (63% and 44%, respectively) [##REF##22331940##27##]. Patients who achieved a MRD-negative status (at a threshold of 10<sup>−4</sup> in PB) appeared to enjoy a better prognosis (median EFS not reached) than previously reported for MRD-negative patients with active disease treated within the CLL8 trial (median PFS 64 months). These findings underline not only the important ability but also potentially higher likelihood of disease-eradicating activity by treatment regimens applied at an early disease stage.</p>", "<p id=\"Par28\">The shortcomings of this study might be the primary endpoint EFS from stratification may be criticized for not considering the difference in the disease load in early treated versus observed patients, and hence, for implementing an upfront advantage or disadvantage, respectively, in the risk of progression. It should be considered that this study was initiated at a time when the clinical experience with FCR, used at an advanced disease stage, was still limited to make projections on outcome for a study design like ours. We preferred to choose a primary endpoint, which commences at trial outset for all patients, most independent from other dynamic variables, and which allows a study design realistic to be accrued.</p>", "<p id=\"Par29\">Not all patients in the Hi-W&amp;W arm did receive FCR as a deferred frontline therapy. Per protocol, the use of FCR was recommended, in case Hi-W&amp;W patients were in need of therapy. According to collected data on the choice of first-line therapy in the Hi-W&amp;W arm (available in 70 patients, Supplementary Table ##SUPPL##0##5##), the use of anti-CD20 treatment was a common choice made for first-line therapy in the Hi-W&amp;W arm, but also use of less efficacious treatments (i.e., R-CHOP, obinutuzumab + chlorambucil, various monotherapies) were given. In addition, the application of new oral kinase/small-molecule inhibitors at later disease stages in the overall high-risk population might have influenced the survival data as they are.</p>", "<p id=\"Par30\">It could be argued that an elevated risk to die from treatment-related early or late toxicity might have mitigated any survival benefits in the Hi-FCR arm. In comparison to other studies investigating frontline FCR at an advanced disease stage, our study did not clearly detect a significantly higher or unexpected toxicity of FCR, when administered at an early stage. For example, the documented rate of CTC grade 3/4 hematotoxicity after deferred FCR was 56% in the CLL8 trial (phase 3 registration study for FCR versus fludarabine plus cyclophosphamide (FC)) [##REF##20888994##9##]. In the FCR arm of the CLL10 study (phase 3 study on FCR versus BR) [##REF##27216274##28##], grade 3/4 neutropenia occurred in 84% of patients, the overall rate of patients with grade 3 hematological events was 21% and 69% for grade 4, respectively. We observed grade 3/4 infections in 22% of treated patients in the Hi-FCR arm compared with 25% of patients treated with FCR in CLL8, and 35% (grade 3) and 3% (grade 4) of patients treated with FCR in CLL10. The use of growth factors was not generally recommended in all of these protocols and not equally documented for a head-to-head comparison. Further, the causes of death documented in both high-risk arms of our study—predominantly progressive CLL disease and infections—did reveal an increased mortality by late adverse treatment effects. Although a direct comparison of toxicity rates between different trials has to be interpreted with caution, these data allow the conclusion, that the tolerability of FCR in our study was comparable to what has been experienced with its use in advanced-stage CLL. A mandatory use of growth factors like G-CSF might have been adequate to limit the rate of neutropenia and the associated risk of infections.</p>", "<p id=\"Par31\">To rule out a particular hazard of an early FCR in a distinct molecular subset of patients we also compared time-to-event outcome according to the IGHV mutation status, and in cytogenetic subsets [##REF##11136261##24##] (Supplementary Figs. ##SUPPL##0##4##–##SUPPL##0##15##). No particular benefit or disadvantage of early versus late therapy could be detected in these subgroups with respect to EFS and OS. Although not statistically significant due to low patient numbers, there was a particular adverse disease course in three of four early treated patients with del(17p), who died within 12.2 months from stratification (Supplementary Fig. ##SUPPL##0##13##). The causes of death were persisting AIHA, a cerebral stroke, and hemophagocytosis/infectious complications after allogeneic stem cell transplant, respectively.</p>", "<p id=\"Par32\">Molecular genetic studies in advanced CLL have revealed a high level of clonal heterogeneity and ongoing genetic evolution of CLL cells throughout the disease course and in particular under applied treatment pressure [##REF##26466571##29##, ##REF##29463802##30##]. Clinically, clonal evolution might have become evident in the Hi-FCR arm of our trial with lower remission rates or response durations after second-line therapies. These data were not the focus of this trial or analysis. However, those considerations warrant careful monitoring of molecular alterations evolving under ongoing treatment pressure, and their consequences on sequential treatment outcome in future studies in early-stage CLL.</p>", "<p id=\"Par33\">In conclusion, FCR therapy is feasible in Binet A stage CLL and extends EFS and PFS in patients with high-risk disease. As a caveat of early FCR we observed possibly treatment-related deaths in 2.4% of treated patients. In accordance with previous treatment studies in an early-stage CLL, our trial does not provide any evidence that the significant improvement of EFS in this patient population translates into a survival benefit. Therefore, “watch &amp; wait” after diagnosis, until “active disease” criteria [##REF##29540348##31##] are met, remains the standard of care, irrespective of unfavorable prognostic features. Ongoing and future studies may elucidate, whether the immediate use of such targeted and potentially disease-eradicating therapies (i.e., venetoclax combinations), will be able to overcome adverse disease courses (particularly for patients with del(17p)), and to displace the current standard of care “watch &amp; wait” [##REF##26332019##11##].</p>" ]
[]
[ "<p id=\"Par1\">We report a randomized prospective phase 3 study (CLL7), designed to evaluate the efficacy of fludarabine, cyclophosphamide, and rituximab (FCR) in patients with an early-stage high-risk chronic lymphocytic leukemia (CLL). Eight hundred patients with untreated-stage Binet A disease were enrolled as intent-to-treat population and assessed for four prognostic markers: lymphocyte doubling time &lt;12 months, serum thymidine kinase &gt;10 U/L, unmutated IGHV genes, and unfavorable cytogenetics (del(11q)/del(17p)/trisomy 12). Two hundred and one patients with ≥2 risk features were classified as high-risk CLL and 1:1 randomized to receive either immediate therapy with 6xFCR (Hi-FCR, 100 patients), or to be observed according to standard of care (Hi-W&amp;W, 101 patients). The overall response rate after early FCR was 92.7%. Common adverse events were hematological toxicities and infections (61.0%/41.5% of patients, respectively). After median observation time of 55.6 (0–99.2) months, event-free survival was significantly prolonged in Hi-FCR compared with Hi-W&amp;W patients (median not reached vs. 18.5 months, <italic>p</italic> &lt; 0.001). There was no significant overall survival benefit for high-risk patients receiving early FCR therapy (5-year OS 82.9% in Hi-FCR vs. 79.9% in Hi-W&amp;W, <italic>p</italic> = 0.864). In conclusion, although FCR is efficient to induce remissions in the Binet A high-risk CLL, our data do not provide evidence that alters the current standard of care “watch and wait” for these patients.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0747-7) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>We are indebted to the participating centers and investigators, as well as all patients who contributed to the completion of this study. For the GCLLSG we acknowledge Henrik Gerwin, Gabriele Kranz, Silke Schott, and Irene Stodden for their work as project and/or data managers for the study, Sabine Frohs for her activity as safety manager. The database of the study was generated with the support of WiSP (Wissenschaftlicher Service Pharma GmbH, Langenfeld, Germany). Monitoring of participating centers was performed with support of WiSP and the Competence Net Malignant Lymphoma (KML), Germany. This was an investigator initiated trial which was sponsored by the University of Cologne and the FCGCLL. The trial was primarily funded by the German Cancer Aid (106142/107080) and the French National Research Agency (ANR). Additional funding support, as well as the study drug rituximab was provided by Roche Pharma AG, Grenzach, Germany/Boulogne-Billancourt Cedex, France. Subprojects with respect to detailed analyses of infections and molecular profiling of patients included in the study were funded by the German Ministry for Education and Research (01KI0771/01KI101) and the German Research Foundation (KFO286). SS was supported by European Commission/BMBF (“FIRE CLL”, 01KT160) and Deutsche Forschungsgemeinschaft (SFB 1074, project B1, B2). All funders did not have any involvement in the study design, collection, analysis and interpretation of data, or writing of the report.Open access funding provided by Projekt DEAL.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par34\">CDH received research funding and travel support from Roche. PL and KF received travel support from Roche. JB received consulting honoraria and travel support from Roche. MH and CMW report advisory board membership for Roche, and obtained research funding, consulting honoraria, and speaker’s honoraria from Roche. SB reports consulting fees, honoraria, and research funding from Roche. SS declares advisory board membership, consulting honoraria, speaker’s honoraria, research grants and travel support from Roche. All other authors declare no competing financial interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Trial flow diagram illustrating patient assessment and allocation within the CLL7 study.</title><p>AIHA autoimmune hemolytic anemia, EFS event-free survival, FCR fludarabine, cyclophosphamide, and rituximab, PFS progression-free survival, OS overall survival, W&amp;W watch and wait.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Event-free survival (EFS) and overall survival (OS) according to risk stratification/randomization (ITT).</title><p><bold>a</bold> EFS from stratification. <bold>b</bold> OS from stratification. Hi-FCR high-risk CLL treated with early FCR, Hi-W&amp;W high-risk CLL under observation, and Lo-W&amp;W low-risk CLL under observation (watch and wait).</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Event-free survival (EFS) and overall survival (OS) according to MRD status in peripheral blood.</title><p><bold>a</bold> EFS from MRD landmark (final response assessment/MRD evaluation). <bold>b</bold> OS from MRD landmark (final response assessment/MRD evaluation). MRD minimal residual disease. For this calculation, the MRD status at the final restaging was considered. MRD negative &lt; 10<sup>−4</sup>; positive ≥ 10<sup>−4</sup> detected CLL cells per leukocytes, according to MRD-flow cytometry. For MRD-results from bone marrow please refer to Supplementary Figs. ##SUPPL##0##1## and ##SUPPL##0##2##.</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Time to (re)treatment (TTT) according to risk stratification/randomization and treatment status per protocol.</title><p><bold>a</bold> Time to first treatment (TTT) in Hi- and Lo-risk patient categories, considering also Hi-FCR patients, who had withdrawn their consent for early FCR after trial inclusion. <bold>b</bold> Time to re-treatment in Hi-FCR patients, who actually underwent early FCR therapy according to the protocol (SP safety population).</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Baseline characteristics (intention-to-treat population).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Parameter</th><th colspan=\"2\">High risk</th><th>Low risk</th><th rowspan=\"2\">Total<break/><italic>N</italic> (%)</th></tr><tr><th>Hi-FCR<break/><italic>N</italic> (%)</th><th>Hi-W&amp;W<break/><italic>N</italic> (%)</th><th>Lo-W&amp;W<break/><italic>N</italic> (%)</th></tr></thead><tbody><tr><td>All patients</td><td>100</td><td>101</td><td>599</td><td>800</td></tr><tr><td>Germany</td><td>59 (59.0)</td><td>60 (59.4)</td><td>295 (49.2)</td><td>414 (51.8)</td></tr><tr><td>France</td><td>41 (41.0)</td><td>41 (40.6)</td><td>304 (50.8)</td><td>386 (48.3)</td></tr><tr><td>Median age (range)</td><td>58 (33–77)</td><td>60 (40–81)</td><td>59 (27–81)</td><td>59 (27–81)</td></tr><tr><td>Age ≤ 60 years</td><td>58 (58.0)</td><td>54 (53.5)</td><td>335 (55.9)</td><td>447 (55.9)</td></tr><tr><td>Age &gt; 70 years</td><td>10 (10.0)</td><td>15 (14.5)</td><td>59 (9.8)</td><td>84 (10.5)</td></tr><tr><td>Male sex</td><td>69 (69.0)</td><td>78 (77.2)</td><td>366 (61.1)</td><td>513 (64.1)</td></tr><tr><td>≥1 comorbidity (CIRS<sup>a</sup>, <italic>N</italic> = 765)</td><td>54 (54.0)</td><td>61 (60.4)</td><td>354 (62.8)</td><td>469 (61.3)</td></tr><tr><td>&gt;6 comorbidities (CIRS<sup>a</sup>, <italic>N</italic> = 765)</td><td>2 (2.0)</td><td>4 (4.0)</td><td>6 (1.1)</td><td>12 (1.6)</td></tr><tr><td>ECOG<sup>b</sup> 0–1 (<italic>N</italic> = 751)</td><td>94 (98.9)</td><td>100 (100.0)</td><td>554 (98.9)</td><td>748 (99.1)</td></tr><tr><td>B symptoms (<italic>N</italic> = 762)</td><td>8 (8.1)</td><td>9 (9.1)</td><td>25 (4.4)</td><td>42 (5.5)</td></tr><tr><td>Clinical lymphadenopathy &gt; 1 cm (<italic>N</italic> = 772)</td><td>48 (48.0)</td><td>49 (48.5)</td><td>159 (27.8)</td><td>256 (33.2)</td></tr><tr><td>Radiologic lymphadenopathy &gt; 1 cm (<italic>N</italic> = 772)</td><td>49 (49.0)</td><td>41 (40.6)</td><td>130 (22.8)</td><td>220 (28.5)</td></tr><tr><td>Median WBC, ×10E3/µl (range, <italic>N</italic> = 766)</td><td>28 (7.2–220.0)</td><td>30 (0.1–165.6)</td><td>18 (5.8–239.8)</td><td>20 (0.1–239.8)</td></tr><tr><td>TK &gt; 10 U/L (<italic>N</italic> = 795)</td><td>62 (62.0)</td><td>52 (52.0)</td><td>40 (6.7)</td><td>154 (19.4)</td></tr><tr><td>LDT &lt; 12 months (<italic>N</italic> = 799)</td><td>58 (58.0)</td><td>48 (47.5)</td><td>71 (11.9)</td><td>177 (22.2)</td></tr><tr><td>IGHV unmutated (<italic>N</italic> = 783)</td><td>81 (81.0)</td><td>82 (82.0)</td><td>57 (9.8)</td><td>220 (28.1)</td></tr><tr><td>Cytogenetics (<italic>N</italic> = 795)<sup>c</sup></td><td>100</td><td>100</td><td>595</td><td>795</td></tr><tr><td> Trisomy 12 (<italic>n</italic>, %)</td><td>25 (25.0)</td><td>24 (24.0)</td><td>18 (3.0)</td><td>67 (8.4)</td></tr><tr><td> Del(11q) (<italic>n</italic>, %)</td><td>17 (17.0)</td><td>35 (35.0)</td><td>3 (0.5)</td><td>55 (6.9)</td></tr><tr><td> Del(17p) (<italic>n</italic>, %)</td><td>4 (4.0)</td><td>9 (9.0)</td><td>5 (0.8)</td><td>18 (2.3)</td></tr><tr><td> Not del(17p)/del(11q)/trisomy 12</td><td>54 (54.0)</td><td>32 (32.0)</td><td>569 (95.6)</td><td>655 (82.4)</td></tr><tr><td>Total risk factors (<italic>N</italic> = 799)</td><td>100</td><td>100</td><td>599</td><td>799</td></tr><tr><td> 0</td><td>0 (0.0)</td><td>0 (0.0)</td><td>395 (65.9)</td><td>395 (49.4)</td></tr><tr><td> 1</td><td>0 (0.0)</td><td>1 (1.0)<sup>d</sup></td><td>202 (33.7)</td><td>203 (25.4)</td></tr><tr><td> 2</td><td>58 (58.0)</td><td>55 (55.0)</td><td>2 (0.3)<sup>d</sup></td><td>115 (14.4)</td></tr><tr><td> 3</td><td>34 (34.0)</td><td>36 (36.0)</td><td>0 (0.0)</td><td>70 (8.8)</td></tr><tr><td> 4</td><td>8 (.0)</td><td>8 (8.0)</td><td>0 (0.0)</td><td>16 (2.0)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>CTC grade ≥ 3 adverse events (AE) in patients treated with early FCR (safety population, <italic>n</italic> = 82).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Total (CTC 3–5)<break/><italic>n</italic> (%)</th><th>CTC grade 3<break/><italic>n</italic> (%)</th><th>CTC grade 4<break/><italic>n</italic> (%)</th><th>CTC grade 5<break/><italic>n</italic> (%)</th><th>Unknown grade<break/><italic>n</italic> (%)</th></tr></thead><tbody><tr><td><italic>N</italic> patients with at least one ≥ CTC grade 3 AE</td><td>61 (74.4)</td><td>28 (34.1)</td><td>29 (35.4)</td><td>4 (4.9)</td><td>5 (6.1)</td></tr><tr><td colspan=\"6\">CTC AE category<sup><bold>a</bold></sup></td></tr><tr><td>Blood/bone marrow</td><td>50 (61.0)</td><td>23 (28.0)</td><td>26 (31.7)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td> Neutropenia</td><td>37 (45.1)</td><td>15 (18.3)</td><td>22 (26.8)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Leukopenia</td><td>25 (30.5)</td><td>19 (23.2)</td><td>6 (7.3)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Thrombocytopenia</td><td>5 (6.1)</td><td>1 (1.2)</td><td>4 (4.9)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Anemia</td><td>4 (4.9)</td><td>4 (4.9)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Cytopenia</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Hemolysis</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td>Infection</td><td>18 (22.0)</td><td>16 (19.5)</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td> Respiratory tract infection</td><td>7 (8.5)</td><td>7 (8.5)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Fever/infection of unknown origin</td><td>3 (3.7)</td><td>3 (3.7)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Herpes zoster</td><td>3 (3.7)</td><td>3 (3.7)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Catheter-related infection</td><td>2 (2.4)</td><td>2 (2.4)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> CMV reactivation</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Bursitis</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Candida esophagitis</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Hepatitis B</td><td>1 (1.2)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Sepsis</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Splondylodiscitis</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Viral encephalititis</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td>Metabolic/laboratory</td><td>5 (6.1)</td><td>4 (4.9)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Elevated GOT, GPT, or GGT</td><td>4 (4.9)</td><td>3 (3.7)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Hyperglycemia</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Gastrointestinal</td><td>3 (3.7)</td><td>2 (2.4)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Nausea/vomiting</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Obstipation</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Diarrhea</td><td>1 (1.2)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Vascular</td><td>4 (4.9)</td><td>4 (4.9)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Thrombosis</td><td>2 (2.4)</td><td>2 (2.4)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Thrombosis with consecutive pulmonary embolism</td><td>2 (2.4)</td><td>2 (2.4)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Ruptured aortic aneurysm</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Neurology</td><td>2 (2.4)</td><td>2 (2.4)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Depression</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Orthostasis</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Pain</td><td>2 (2.4)</td><td>2 (2.4)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Herniated disc/radiculopathy</td><td>2 (2.4)</td><td>2 (2.4)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Dermatology/skin</td><td>2 (2.4)</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Exanthema</td><td>2 (2.4)</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Circulatory symptoms/arrhythmia</td><td>2 (2.4)</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Circulatory symptoms/arrhythmia</td><td>2 (2.4)</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Cardiac arrhythmia</td><td>1 (1.2)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Circulatory symptoms/arrythmia</td><td>1 (1.2)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Cardiac general</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Circulatory symptoms/arrythmia</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Constitutional symptoms</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Constitutional symptoms</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Musculoskeletal/soft tissue</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td> Bone fracture (nonpathologic)</td><td>1 (1.2)</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Pulmonary/upper respiratory</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td> Pulmonary fibrosis</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td>Renal/genitourinary</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr><tr><td> Renal insufficiency</td><td>1 (1.2)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (1.2)</td><td>0 (0.0)</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Response to treatment overall and in post hoc analysis of subgroups.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Patients assessed for response (Hi-FCR)</th><th>Overall response<break/><italic>N</italic> (%)</th><th>Complete response<break/><italic>N</italic> (%)</th><th>Partial response<break/><italic>N</italic> (%)</th><th>Stable disease<break/><italic>N</italic> (%)</th><th>Not evaluable/available<break/><italic>N</italic> (%)</th></tr></thead><tbody><tr><td>All patients (ITT, <italic>N</italic> = 100)</td><td>76 (76.0)</td><td>61 (61.0)</td><td>15 (15.0)</td><td>3 (3.0)</td><td>21 (21.0)<sup>a</sup></td></tr><tr><td>Treated patients (SP, <italic>N</italic> = 82)</td><td>76 (92.7)</td><td>61 (74.4)</td><td>15 (18.3)</td><td>3 (3.7)</td><td>3 (3.7)</td></tr><tr><td> Patients with ≥ 3 cycles of FCR (<italic>N</italic> = 75)</td><td>74 (98.7)</td><td>61 (81.3)</td><td>13 (17.3)</td><td>1 (1.3)</td><td>0 (0.0)</td></tr><tr><td> Two risk factors present (<italic>N</italic> = 58)</td><td>44 (75.9)</td><td>36 (62.1)</td><td>8 (13.8)</td><td>1 (1.7)</td><td>13 (22.4)</td></tr><tr><td> Three risk factors present (<italic>N</italic> = 34)</td><td>26 (76.5)</td><td>20 (58.8)</td><td>6 (17.6)</td><td>2 (5.9)</td><td>6 (17.6)</td></tr><tr><td> Four risk factors present (<italic>N</italic> = 8)</td><td>6 (75.0)</td><td>5 (62.5)</td><td>1 (12.5)</td><td>0 (0.0)</td><td>2 (25.0)</td></tr><tr><td> LDT &lt; 12 months (<italic>N</italic> = 58)</td><td>43 (74.1)</td><td>38 (65.5)</td><td>5 (8.6)</td><td>2 (3.4)</td><td>13 (22.4)</td></tr><tr><td> TK &gt; 10 U/L (<italic>N</italic> = 62)</td><td>50 (80.6)</td><td>38 (61.3)</td><td>12 (19.4)</td><td>2 (3.2)</td><td>10 (16.1)</td></tr><tr><td> IGHV unmutated (<italic>N</italic> = 81)</td><td>59 (72.8)</td><td>45 (55.6)</td><td>14 (17.3)</td><td>3 (3.7)</td><td>19 (23.5)</td></tr><tr><td> IGHV mutated (<italic>N</italic> = 19)</td><td>17 (89.5)</td><td>16 (84.2)</td><td>1 (5.3)</td><td>0 (0.0)</td><td>2 (10.5)</td></tr><tr><td> Trisomy 12<sup>b</sup> (<italic>N</italic> = 25)</td><td>18 (72.0)</td><td>16 (64.0)</td><td>2 (8.0)</td><td>0 (0.0)</td><td>7 (28.0)</td></tr><tr><td> Del(11q)<sup>b</sup> (<italic>N</italic> = 17)</td><td>15 (88.2)</td><td>12 (70.6)</td><td>3 (17.6)</td><td>0 (0.0)</td><td>2 (11.8)</td></tr><tr><td> Del(17p)<sup>b</sup> (<italic>N</italic> = 4)</td><td>2 (50.0)</td><td>0 (0.0)</td><td>2 (50.0)</td><td>1 (25.0)</td><td>1 (25.0)</td></tr><tr><td> No trisomy 12/del(11q)/del(17p) (<italic>N</italic> = 54)</td><td>41 (75.9)</td><td>33 (61.1)</td><td>8 (14.8)</td><td>2 (3.7)</td><td>11 (20.4)</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p><sup>a</sup>Cumulative illness rating scale [##REF##7836636##32##].</p><p><sup>b</sup>ECOG = performance status scale according to the Eastern Cooperative Group [##REF##7165009##33##].</p><p><sup>c</sup>According to Döhner et al. [##REF##11136261##24##].</p><p><sup>d</sup>Three patients were allocated to the incorrect risk stratum according to their risk profile presented here. Two of those cases (one Hi-W&amp;W and one Lo-W&amp;W) were caused by entry/capture errors for assigned risk factors in the database; and, these patients were stratified in the correct risk subset. Only one Lo-W&amp;W patient was truly misstratified as a low-risk case, despite the fact that two risk factors had been found present by central diagnostics.</p></table-wrap-foot>", "<table-wrap-foot><p><sup>a</sup>According to CTC v3.0. Adverse events are shown of CTC grade 3, 4, or 5, which occurred at least in patient per event category.</p></table-wrap-foot>", "<table-wrap-foot><p><sup>a</sup>Includes 18 patients who refused initiation of FCR therapy after stratification/randomization.</p><p><sup>b</sup>According to Döhner et al. [##REF##11136261##24##].</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Carmen D. Herling, Florence Cymbalista, Carolin Groß-Ophoff-Müller</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41375_2020_747_Fig1_HTML\" id=\"d32e561\"/>", "<graphic xlink:href=\"41375_2020_747_Fig2_HTML\" id=\"d32e2138\"/>", "<graphic xlink:href=\"41375_2020_747_Fig3_HTML\" id=\"d32e2163\"/>", "<graphic xlink:href=\"41375_2020_747_Fig4_HTML\" id=\"d32e2185\"/>" ]
[ "<media xlink:href=\"41375_2020_747_MOESM1_ESM.docx\"><caption><p>Supplemental files</p></caption></media>" ]
[{"label": ["3."], "mixed-citation": ["Chemotherapeutic options in chronic lymphocytic leukemia: a meta-analysis of the randomized trials. CLL Trialists' Collaborative Group. J Natl Cancer Inst. 1999;91:861\u20138."]}, {"label": ["4."], "mixed-citation": ["Effects of chlorambucil and therapeutic decision in initial forms of chronic lymphocytic leukemia (stage A): results of a randomized clinical trial on 612 patients. The French Cooperative Group on Chronic Lymphocytic Leukemia. Blood. 1990;75:1414-21."]}, {"label": ["12."], "surname": ["Zelenetz", "Gordon", "Wierda", "Abramson", "Advani", "Andreadis"], "given-names": ["AD", "LI", "WG", "JS", "RH", "CB"], "article-title": ["Chronic lymphocytic leukemia/small lymphocytic lymphoma, version 1.2015"], "source": ["J Natl Compr Cancer Netw"], "year": ["2015"], "volume": ["13"], "fpage": ["326"], "lpage": ["62"], "pub-id": ["10.6004/jnccn.2015.0045"]}]
{ "acronym": [], "definition": [] }
33
CC BY
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2024-01-13 23:35:07
Leukemia. 2020 Feb 18; 34(8):2038-2050
oa_package/af/08/PMC7387319.tar.gz
PMC7387320
32457355
[ "<title>Introduction</title>", "<p id=\"Par2\">Clonal hematopoiesis (CH) is defined as the clonal expansion of blood cells derived from a single hematopoietic stem cell. Recent studies suggest that CH is an age-related event, triggered by somatic mutations in leukemia-associated genes. While acquisition of these mutations is considered one of the earliest events in the pathogenesis of different hematologic malignancies, clonal hematopoiesis driver mutations (CHDMs) may be present and cause CH without any clinically distinct phenotype. Three recent whole-exome-sequencing analyses of large populations have revealed age-related hematopoietic clones in apparently healthy individuals, driven by mutations of genes recurrently mutated in myeloid neoplasms and associated with increased risk of hematologic cancer and cardiovascular diseases [##REF##25326804##1##–##REF##25426837##3##]. CH with somatic mutations was observed in 10% of individuals older than 65 years of age, but only in 1% of those younger than 50 years of age [##REF##25426838##2##]. Furthermore, CHDMs were detected in 10% of individuals older than 70 years and in about 20% of those older than 90 years [##REF##25426837##3##]. The most frequently mutated genes include <italic>DNMT3A</italic>, <italic>TET2</italic>, <italic>JAK2</italic>, and <italic>ASXL1</italic>. Based on these three large studies, Steensma et al. proposed the term CHIP (clonal hematopoiesis of indeterminate potential) for individuals harboring somatic mutations of genes frequently mutated in hematologic malignancies with a variant allele frequency (VAF) ≥ 2%, in the absence of definitive morphologic and clinical evidence of a hematologic malignancy [##REF##25931582##4##].</p>", "<p id=\"Par3\">The fact that patients with hematologic malignancies and healthy individuals share a common set of genetic mutations highlights the need to further elucidate the prevalence and kinetics of these mutations within the general population and to understand the underlying mechanisms of clonal selection and stability. This study investigated the prevalence and long-term dynamics of genetic alterations in leukemia-associated genes among elderly individuals without hematologic disorders or cancer. As <italic>DNMT3A</italic> mutations were most frequently detected, blood samples of 160 individuals of all ages of the general population were analyzed for <italic>DNMT3A</italic> mutations. Additionally, relative <italic>DNMT3A</italic> expression levels of younger and older individuals were compared to investigate age-related differences beyond the mutation status. A model of <italic>DNMT3A</italic> activity in hematopoietic stem cells (HSCs) proposed by Challen et al. suggests the crucial role of <italic>DNMT3A</italic> in balancing differentiation and self-renewal of HSCs by upregulation of HSC multipotency genes and downregulation of differentiation genes. Lack of <italic>DNMT3A</italic> function was shown to lead to altered methylation patterns reminiscent of those observed in human malignancies and an increased expression of genes normally restricted to stem cells [##REF##22138693##5##]. Considering the key epigenetic regulatory role and the frequent involvement of DNMT3A in human malignancies, the study presented here finally investigated clonal expansion using bone marrow of healthy individuals with mutant <italic>DNMT3A</italic> in a humanized mouse model.</p>" ]
[ "<title>Materials and methods</title>", "<title>Cohorts of healthy individuals</title>", "<p id=\"Par4\">The first cohort included 50 elderly individuals (male, <italic>n</italic> = 21; median age 84 years, range 80–90 years). Peripheral blood (PB) samples were taken and sequenced for a panel of commonly mutated leukemia-associated genes. The second cohort of 160 healthy people &lt;80 years of age was specifically sequenced for <italic>DNMT3A</italic> mutations (male, <italic>n</italic> = 77; median age 40 years, range 0–79 years). Total leukocytes were isolated from PB after red cell lysis. As a source of constitutional DNA, oral mucosa cells were obtained from all patients using buccal swabs. All procedures were in accordance with the ethical standards of the institutional research committee and with the Declaration of Helsinki. Informed consent was obtained from all individuals included in the study.</p>", "<title>DNA and RNA extractions</title>", "<p id=\"Par5\">Genomic DNA (gDNA) was isolated from PB leukocytes and oral mucosa cells using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. For RNA isolation, 1 ml Trizol<sup>®</sup> reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to 2 × 10<sup>7</sup> cells and mixed thoroughly. Subsequent RNA extraction was performed as described in the literature [##REF##17406285##6##].</p>", "<title>Next-generation sequencing (NGS)</title>", "<p id=\"Par6\">Samples of 50 healthy elderly individuals were genotyped for a panel of 30 commonly mutated leukemia-associated genes by targeted deep NGS [##REF##27169594##7##, ##REF##23592507##8##], using the 454 GS Junior platform (Roche Diagnostics, Mannheim, Germany) at a sensitivity level of 5%. Prior to sequencing, whole genome amplification (WGA) was performed using 20 ng template gDNA and the Repli-g Ultra Fast Mini Kit (Qiagen) in accordance with manufacturer's instructions. Analyses covered genes involved in signaling, transcription, epigenetics, RNA splicing, and the cohesin complex. Another cohort of 160 healthy people &lt;80 years of age was specifically sequenced for <italic>DNMT3A</italic> mutations, using a custom-designed highly sensitive <italic>DNMT3A</italic>-specific deep NGS assay (sensitivity 1%) covering the entire coding region of the <italic>DNMT3A</italic> gene (exons 2–23). For each NGS run 8 <italic>JAK2</italic> p.V617F samples were generated and used as an external sensitivity control. PCR products were resolved on 3% agarose gels and visualized by staining with ethidium bromide. Mutations found in WGA samples were verified using gDNA isolated from leukocytes and buccal swabs to confirm the somatic origin of mutations, in a separate NGS run. All mutational percentages listed refer to the results found in gDNA. NGS data was analyzed with the GS Amplicon Variant Analyzer (AVA) software (version 2.7; Roche). The open access tools Protein Variation Effect Analyzer (PROVEAN) and Polymorphism Phenotyping v2 (PolyPhen-2) were used for evaluation of potential consequences of the identified mutations [##REF##20354512##9##, ##REF##23056405##10##].</p>", "<title>Expression analysis by quantitative real-time PCR (qRT-PCR)</title>", "<p id=\"Par7\">For <italic>DNMT3A</italic> gene expression analyses, cDNA synthesis was performed using M-MLV Reverse transcriptase (Invitrogen GmbH, Karlsruhe, Germany) and a minimum of 1 µg RNA. Analysis by qRT-PCR was performed according to standard protocols using SYBR Green I (Roche), <italic>DNMT3A</italic> as a target, and <italic>β-glucuronidase</italic> (<italic>GUSB</italic>) as a reference gene. The relative expression rate was calculated using the ΔCt-method [##REF##11846609##11##]. Primers were designed using primer3web (version 4.0.0). Primer sequences with the corresponding product size are shown in Table ##SUPPL##0##SI##. Statistical analysis was performed using the Mann–Whitney nonparametric test, provided by GraphPad Prism v6.01 (GraphPad Software, Inc., San Diego, CA, USA).</p>", "<title>Patient-derived xenograft (PDX) model</title>", "<p id=\"Par8\">Bone marrow cells of two healthy elderly individuals (age 69 and 79 years) with <italic>DNMT3A</italic> mutations were transplanted into a humanized mouse model. Primary bone marrow cells of elderly healthy donors were obtained from fractured hip bones after hemiarthroplasty following informed consent according to the Helsinki declaration (approved by the local ethics committee #4753/04-16). Bone marrow cells were extracted from the bone, erythrolysed and cryopreserved in 1x freezing medium (80% FBS + 10% DMSO + 10% IMDM medium). NOD.Cg-Prkdc<sup>scid</sup> Il2rg<sup>tm1Wjl</sup> Tg(CMV-IL3, CSF2, KITLG) 1Eav/MloySzJ (NSGS) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Before transplantation, 8–12-week-old adult mice were irradiated with 2 Gy (single dose). Via the tail vein, 2 × 10<sup>6</sup> cells were injected intravenously. Engraftment of human cells (hCD45+) was analyzed by flow cytometry. Three mice were engrafted for each mutation. The <italic>DNMT3A</italic> mutations were previously detected using the MiniSeq System (TruSight Myeloid Sequencing Panel, VariantStudio Software 3.0, Illumina, San Diego, USA). For analysis, murine bone marrow cells were isolated from the femurs as previously described [##REF##29467485##12##]. Bone marrow samples were then assessed for clonal selection of the known <italic>DNMT3A</italic> mutations by pyrosequencing, according to standard protocols. Sequencing primers are listed in the Table ##SUPPL##0##SII##.</p>" ]
[ "<title>Results</title>", "<title>Somatic mutations in healthy elderly individuals</title>", "<p id=\"Par9\">A total of 16 somatic mutations in leukemia-associated genes were identified in 13 of 50 (26%) hematologically normal elderly individuals (Table ##TAB##0##1##). One subject presented with two and another subject with three mutations of different genes. Ten of 16 mutations (62.5%) affected epigenetic modifier genes (<italic>DNMT3A</italic>, <italic>n</italic> = 8; <italic>TET2</italic>, <italic>n</italic> = 1; <italic>IDH2</italic>, <italic>n</italic> = 1), four somatic mutations affected genes involved in the RNA splicing machinery (<italic>SRSF2</italic>, <italic>n</italic> = 2; <italic>SF3B1</italic>, <italic>n</italic> = 1; <italic>U2AF1</italic>, <italic>n</italic> = 1). Mutations in <italic>TP53</italic> and <italic>NRAS</italic> were identified in two individuals. Detected mutations were verified on non-amplified gDNA by performing a separate NGS run, whilst the somatic origin was confirmed using corresponding buccal swab gDNA. All but one mutation were missense mutations with cytosine to thymine transitions being the most common base pair change (<italic>n</italic> = 7). Mutations occurred at low VAF with a median of 11.7% (range, 1.0–30.7%), indicating that mutations were present in only a subset of blood cells.</p>", "<title>Mutation dynamics and clinical outcome during a 3-year period</title>", "<p id=\"Par10\">Mutation kinetics remained virtually stable over 3 years, with a median VAF of 13.1% (range, 1.0–35.3%) after a 2-year follow-up and a median VAF of 18.8% after a 3-year follow-up (range, 0.9–56.6%) (Table ##TAB##0##1##). The mutations’ dynamics during the 3-year period are depicted in Fig. ##FIG##0##1##. During the 3-year follow-up observation, two subjects with mutations in splicing factor genes died. One subject with an <italic>SF3B1</italic> mutation developed pancreatic cancer, the other subject harboring a <italic>U2AF1</italic> mutation died due to a stroke. All other individuals with mutations were alive without any evidence for a hematologic or oncologic disorder.</p>", "<title>Age-associated increase of somatic <italic>DNMT3A</italic> mutations in healthy individuals</title>", "<p id=\"Par11\">Since <italic>DNMT3A</italic> mutations were the most common mutations identified in elderly individuals (8/16 detected mutations), we sought to study the age-dependent prevalence of <italic>DNMT3A</italic> mutations in more detail. Therefore, an additional cohort of 160 individuals was investigated, including healthy people &lt;80 years of age (male, <italic>n</italic> = 77; median age 40 years, range 0–79 years) using a custom-designed highly sensitive <italic>DNMT3A</italic>-specific deep NGS assay (sensitivity 1%), covering the complete coding region of the <italic>DNMT3A</italic> gene (exons 2–23). The youngest individual with a <italic>DNMT3A</italic> mutation was a 28 years old female. Mutations occurred at low VAFs with a median of 8.2% (range, 1.5–37.3%). No <italic>DNMT3A</italic> mutation was detected in 40 analyzed PB samples of children and adolescents (0–19 years). Somatic <italic>DNMT3A</italic> mutations were found in 2/40 (5.0%) individuals with an age of 20–39 years, 1/40 subjects (2.5%) between 40 and 59 years, and in 3/40 subjects (7.5%) between 60 and 79 years of age (Fig. ##FIG##1##2##). Overall, six somatic <italic>DNMT3A</italic> mutations were identified in 6 of 160 (3.8%) individuals (Table ##TAB##1##2##) &lt;80 years of age. In comparison, the mutation frequency of <italic>DNMT3A</italic> in healthy elderly persons (≥80 years) was with 14% (7/50) much higher. The <italic>DNMT3A</italic> mutation rate in the whole cohort of healthy individuals, both younger and elderly, was 6.2% (13/210) in total.</p>", "<title><italic>DNMT3A</italic> mRNA expression in healthy individuals</title>", "<p id=\"Par12\"><italic>DNMT3A</italic> mRNA expression was analyzed by qRT-PCR in the previously sequenced cohort of younger healthy individuals (20–39 years, <italic>n</italic> = 40) and healthy elderly (≥80 years, <italic>n</italic> = 48). Significantly lower expression (*<italic>p</italic> = 0.017) was found in healthy elderly people (Fig. ##FIG##2##3a##). Taking sequencing results of these two groups into consideration, a marginal albeit not significant difference (<italic>p</italic> = 0.14) was found in expression levels comparing individuals carrying wild type <italic>DNMT3A</italic> (<italic>DNMT3A</italic><sup><italic>wt</italic></sup>, <italic>n</italic> = 79) with those carrying <italic>DNMT3A</italic> somatic mutations (<italic>DNMT3A</italic><sup><italic>mut</italic></sup>, <italic>n</italic> = 9) (Fig. ##FIG##2##3b##).</p>", "<title>Stable kinetics of <italic>DNMT3A</italic> mutations in a PDX model</title>", "<p id=\"Par13\">To further investigate the clonal selection of <italic>DNMT3A</italic> mutations, transplantation of bone marrow cells of two healthy elderly individuals with different somatic <italic>DNMT3A</italic> mutations in a PDX model were performed. Mutations were detected using the MiniSeq System (Illumina), as a part of a subproject analyzing CHDM in bone marrow samples of healthy individuals. Detected <italic>DNMT3A</italic> mutations are shown in Table SIII. Mutations in human bone marrow cells were present at low VAFs of 23.1% and 6.3%, respectively (Table ##TAB##2##3##). Each human sample was transplanted into three mice. After 8 months, bone marrow samples were isolated from recipient mice, representing an aging mouse model of the hematopoietic compartment. The first <italic>DNMT3A</italic> mutation with VAF of 23.1% remained stable during this time in all three recipient mice, with a median VAF of 21.8% (range, 10.8–30.8%). The second <italic>DNMT3A</italic> mutation, initially present with a VAF of 6.3%, displayed stable mutation kinetic in one mouse, whereas in the other two mice no mutation was detectable (Table ##TAB##2##3##).</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par14\">In this study, leukemia-associated mutations were frequently found in healthy elderly individuals. This confirms findings of recent exome-analysis studies who have shown age-related CH in healthy individuals, driven by mutations of genes recurrently mutated in myeloid neoplasms and associated with an increased risk of hematologic cancer and cardiovascular disease. All those studies identified similar genes, with the majority of mutations affecting epigenetic modifiers, such as <italic>DNMT3A</italic>, <italic>TET2</italic>, and <italic>ASXL1</italic> [##REF##25326804##1##–##REF##25426837##3##, ##REF##25627638##13##]. A recent whole-genome sequencing approach on a large population cohort from Iceland also supports the finding that CH is very common in elderly, both with and without candidate driver mutations, suggesting that age-related CH is inevitable [##REF##28483762##14##]. Here, mutations were detected predominantly in epigenetic modifier genes <italic>DNMT3A</italic>, <italic>TET2</italic>, and <italic>IDH2</italic>, which are known to promote self-renewal and block differentiation of HSCs. These CHDMs are considered to be founder events in the evolution of myeloid diseases, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and myeloproliferative neoplasms (MPN) [##REF##27848178##15##, ##REF##21415852##16##]. Defects in RNA splicing genes, such as <italic>SF3B1</italic>, <italic>SRSF2</italic>, and <italic>U2AF1</italic> have been described in pathogenesis of MDS [##REF##24220272##17##], and were also detected within the cohort investigated.</p>", "<p id=\"Par15\">In the study presented here, it was shown that mutation kinetics remain virtually stable over a 3 years follow-up. This suggests that detected CHDMs in healthy people enter a stage of clonal size stability for most people. A recent longitudinal study also showed temporal stability of mutated clones in healthy individuals [##REF##27546487##18##], indicating an occurrence of these mutations in long-lived HSCs or committed progenitors. However, presence of these CHDMs, albeit in the form of the observed low-level cell clones, may increase the risk of acquiring additional mutations and represents the first step in potentially evolving leukemia. Individuals with CHIP have an 11–13 times increased risk of developing hematologic neoplasia, compared with the general population. This is because the acquisition of subsequent, disease driving mutations occurs at a much higher rate in people already affected by clonal expansion, initiated by early mutations. All-cause mortality was also observed with higher incidence among individuals with detectable CH, possibly due to higher risk of death from cardiovascular disease [##REF##25426838##2##, ##REF##25426837##3##]. A recent study demonstrated almost the doubling of the risk of coronary heart disease in humans in case of CHIP in peripheral-blood cells [##REF##28636844##19##]. However, the absolute risk of developing a malignant hematologic disease is low, with the rate of progression being 0.5–1% per year [##REF##25931582##4##]. Further research should focus on the underlying mechanisms which transform clonal haematopoiesis from a seemingly common and benign to a relatively rare malignant state.</p>", "<p id=\"Par16\">In the cohort of healthy elderly individuals <italic>DNMT3A</italic> mutations were the most frequent (14%; 7/50), with one person harboring two different <italic>DNMT3A</italic> mutations in independent clones. This is in accordance with previous studies, which also identified <italic>DNMT3A</italic> as one of the most frequently mutated epigenetic regulator genes [##REF##25326804##1##–##REF##25426837##3##]. Using a custom-designed highly sensitive <italic>DNMT3A</italic> NGS assay age-dependence of <italic>DNMT3A</italic> mutations was assessed. In the whole cohort of young and elderly healthy persons (<italic>n</italic> = 210), 10 out of 13 <italic>DNMT3A</italic> mutations were detected in individuals older than 60 years, displaying an age-dependent increase of <italic>DNMT3A</italic> mutation prevalence. <italic>DNMT3A</italic> mutations are suggested to arise early in AML, leading to a clonally expanded pool of pre-leukemic HSCs [##REF##24522528##20##]. Persistence of low-level <italic>DNMT3A</italic> mutations during remission in AML patients further supports the existence of pre-leukemic stem cells with CHDMs, which then represent a reservoir of pre-leukemic clones that can provoke a relapse [##REF##25371149##21##].</p>", "<p id=\"Par17\">Previous studies have shown that <italic>DNMT3A</italic> is among the most frequently mutated genes in AML, MPN, MDS, and adult-early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) [##REF##21415852##16##, ##REF##21067377##22##–##REF##23603912##26##]. Approximately 22% of cytogenetically normal acute myeloid leukemia (CN-AML) patients carry a <italic>DNMT3A</italic> mutation, from which about 60% are localized in the catalytic domain, at the R882 hotspot [##REF##21067377##22##, ##REF##27841873##27##]. These mutations exhibit dominantly negative functional effects, decreasing enzymatic activity, thus causing hypomethylation of target genes [##REF##24167195##28##–##REF##25693834##30##]. Their presence is correlated with an adverse outcome and a higher therapy-resistance rate in AML [##REF##21067377##22##, ##REF##27841873##27##]. Such hotspot mutations were also detected within the cohort of healthy individuals in this study, suggesting that they are an early event occurring in the pathogenesis of different hematological diseases.</p>", "<p id=\"Par18\">Recent data further suggest a temporal hierarchy in <italic>DNMT3A</italic> and <italic>TET2</italic> mutations’ occurrence. Whereas <italic>DNMT3A</italic> mutations origin in multipotent stem cells, <italic>TET2</italic> mutations occur mainly in myeloid cells [##REF##29764839##31##]. Somatic mutations of <italic>DNMT3A</italic> and <italic>TET2</italic> were also associated with accelerated atherosclerosis, thus increasing the risk of cardiovascular disease mediated by inflammatory mechanisms [##UREF##0##32##, ##REF##29728415##33##]. This finding indicates that CHDMs may predispose to both hematologic and non-hematologic diseases and might represent a valuable predictive and prognostic factor.</p>", "<p id=\"Par19\">Furthermore, gene expression analysis showed significant age-dependent decrease of <italic>DNMT3A</italic> expression. This decrease is independent of the mutation status, implying that other factors apart from genomic alterations, such as changes in the bone marrow niche and an aging cellular background may play an important role in the <italic>DNMT3A</italic> expression rate and thus function. This is in correspondence with the higher prevalence of CH and hematologic malignancies in elderly [##REF##25326804##1##–##REF##25426837##3##]. Decreased <italic>DNMT3A</italic> expression rate with age may contribute to the bias of HSCs toward self-renewal, by affecting expression of downstream targets as shown by Challen et al. who found an upregulation of “HSC fingerprint” genes in <italic>DNMT3A</italic>-null HSCs [##REF##22138693##5##]. These genes, such as <italic>NR4A2</italic>, <italic>PDK4</italic>, <italic>VASN</italic>, and <italic>PRDM16</italic> are normally expressed in HSCs but not in differentiated blood cells [##REF##18371395##34##], suggesting that <italic>DNMT3A</italic> is required to suppress the stem cell program in HSCs to permit differentiation. Furthermore, multipotency genes including <italic>RUNX1</italic>, <italic>GATA3</italic>, <italic>PBX1</italic>, and <italic>CDKN1A</italic> were also highly expressed in <italic>DNMT3A</italic>-null HSCs, opposite to decreased expression of essential differentiation factors, such as those encoded by <italic>FLK2</italic>, <italic>IKAROS</italic>, <italic>SFPI1</italic>, and <italic>MEF2C</italic> [##REF##22138693##5##]. Taken together, altered expression of downstream target genes due to lower <italic>DNMT3A</italic> expression, resulting from mutations or other age-related factors, may promote CH.</p>", "<p id=\"Par20\">To further investigate the potential clonal selection of cells harboring <italic>DNMT3A</italic> mutations a mouse model was established. For that purpose, xenotransplantations of bone marrow cells from two healthy elderly patients with <italic>DNMT3A</italic> mutations were performed and the mutations’ dynamics assessed over a period of 8 months. <italic>DNMT3A</italic> mutations displayed relatively stable kinetics. Mice xenografts from individual A, with an initial VAF of 23.1%, displayed a median VAF of 21.8% (range: 10.8–30.8%), showing stable kinetics. The initial VAF of 6.3% of individual B also remained stable in one out of three transplanted mice (median VAF 6.2%). For the remaining two mice the mutational burden may have been below the detectability level of the pyrosequencing assay (5%). Comparing this data with the longitudinal study on healthy individuals we can conclude that CHDMs, both in humans and a mouse model of aging hematopoiesis, are displaying relatively stable mutations kinetics, without clonal selection of the cells carrying mutations or progressing to overt hematological disease. This again suggests, that the presence of CHDMs itself is insufficient to initiate clonal selection and expansion without an additional influence of other intrinsic and/or extrinsic factors.</p>", "<p id=\"Par21\">In conclusion, these findings indicate that the appearance of low-level clones with mutations in leukemia-associated genes is a common age-associated phenomenon. Occurrence of these mutations within epigenetic regulator genes and the RNA-splicing machinery may represent a premalignant condition in the development of hematologic cancers and predispose to other aging-associated disorders. <italic>DNMT3A</italic> mutations were the most common mutations identified in elderly individuals but were also apparent in younger healthy people, albeit less frequently. Further studies should investigate which compensatory mechanisms inhibit selection of cells carrying mutations, promoting a stage of clonal size stability in healthy elderly individuals in contrast to patients with overt myeloid disorders. Interestingly, this study shows an age-related decrease in <italic>DNMT3A</italic> expression and highlights that in addition to CHDMs, aberrant expression may be a feature of the aging hematopoietic system. Further studies should investigate whether other leukemia-associated genes show similar patterns.</p>" ]
[]
[ "<p id=\"Par1\">Clonal hematopoiesis is frequently observed in elderly people. To investigate the prevalence and dynamics of genetic alterations among healthy elderly individuals, a cohort of 50 people &gt;80 years was genotyped for commonly mutated leukemia-associated genes by targeted deep next-generation sequencing. A total of 16 somatic mutations were identified in 13/50 (26%) individuals. Mutations occurred at low variant allele frequencies (median 11.7%) and remained virtually stable over 3 years without development of hematologic malignancies in affected individuals. With <italic>DNMT3A</italic> mutations most frequently detected, another cohort of 160 healthy people spanning all age groups was sequenced specifically for <italic>DNMT3A</italic> revealing an overall mutation rate of 6.2% (13/210) and an age-dependent increase of mutation prevalence. A significant difference (<italic>p</italic> = 0.017) in the <italic>DNMT3A</italic> expression pattern was detected between younger and healthy elderly people as determined by qRT-PCR. To evaluate the selection of clonal hematopoietic stem cells (HSCs), bone marrow of two healthy individuals with mutant <italic>DNMT3A</italic> was transplanted in a humanized mouse model. Xenografts displayed stable kinetics of <italic>DNMT3A</italic> mutations over 8 months. These findings indicate that the appearance of low-level clones with leukemia-associated mutations is a common age-associated phenomenon, but insufficient to initiate clonal selection and expansion without the additional influence of other factors.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0869-y) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>The excellent technical assistance of Mrs. Anja Waldau and Mrs. Susan Wittig is gratefully acknowledged. Open access funding provided by Projekt DEAL.</p>", "<title>Funding</title>", "<p id=\"Par22\">DM and TE were supported by the Interdisciplinary Center for Clinical Research (IZKF, Universitätsklinikum Jena, Jena, Germany). FHH received funding by the Thuringian state program ProExzellenz (RegenerAging—FSU-I-03/14) of the Thuringian Ministry for Economics, Science and Digital Society (TMWWDG). Open access funding was provided by Projekt DEAL.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par23\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Dynamics of somatic mutations detected in 50 healthy elderly individuals during a 3-year follow up period.</title><p>Two subjects with mutations in splicing factor genes <italic>SF3B1</italic> and <italic>U2AF1</italic> died. For two patients (#15 and #35) the 3-year follow-up samples were not available.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Age-associated increase of <italic>DNMT3A</italic> mutations in healthy individuals.</title><p>Frequency of <italic>DNMT3A</italic> mutations was analyzed within five age cohorts (0–19; 20–39; 40–59; 60–79; &gt;80 years) by a highly sensitive <italic>DNMT3A</italic>-specific deep next-generation sequencing assay.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title><italic>DNMT3A</italic> mRNA expression in healthy individuals.</title><p><bold>a</bold> Age-associated decrease of <italic>DNMT3A</italic> expression (median value depicted; *<italic>p</italic> = 0.017). <bold>b</bold> A marginal albeit not significant difference between <italic>DNMT3A</italic><sup>wt</sup> and <italic>DNMT3A</italic><sup>mut</sup> expression (median value depicted; <italic>p</italic> = 0.14). Statistical analysis was performed with the Mann–Whitney nonparametric test. wt wild type; mut mutant.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Somatic mutations in leukemia-associated genes within a cohort of 50 healthy elderly individuals.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Sample number</th><th>Sex, age</th><th>Gene</th><th>Variant Ensemble</th><th>Protein</th><th>% Reads displaying variant gDNA</th><th>% Reads displaying variant buccal cells</th><th>% Reads displaying variant gDNA (2-year follow-up)</th><th>% Reads displaying variant gDNA (3-year follow-up)</th></tr></thead><tbody><tr><td>#5</td><td>Female, 86</td><td><italic>DNMT3A</italic></td><td>c.1403A&gt;G</td><td>p.K468R</td><td>5.53</td><td>0.00</td><td>5.42</td><td>4.85</td></tr><tr><td>#9</td><td>Male, 81</td><td><italic>SRSF2</italic></td><td>c.144_169del(26)</td><td>p.Y50LfsX11</td><td>10.63</td><td>0.46</td><td>7.12</td><td>8.33</td></tr><tr><td>#10</td><td>Male, 86</td><td><italic>SF3B1</italic></td><td>c.2098A&gt;G</td><td>p.K700E</td><td>6.56</td><td>0.00</td><td>†</td><td>†</td></tr><tr><td rowspan=\"2\">#15</td><td rowspan=\"2\">Female, 83</td><td><italic>DNMT3A</italic></td><td>c.2644C&gt;T</td><td>p.R882C</td><td>4.89</td><td>0.00</td><td>6.76</td><td>─</td></tr><tr><td><italic>DNMT3A</italic></td><td>c.2711C&gt;T</td><td>p.P904L</td><td>3.60</td><td>0.00</td><td>3.54</td><td>─</td></tr><tr><td rowspan=\"3\">#21</td><td rowspan=\"3\">Male, 85</td><td><italic>IDH2</italic></td><td>c.418C&gt;T</td><td>p.R140W</td><td>22.75</td><td>12.47</td><td>28.98</td><td>33.69</td></tr><tr><td><italic>SRSF2</italic></td><td>c.284C&gt;T</td><td>p.P95L</td><td>28.78</td><td>15.6</td><td>35.29</td><td>56.59</td></tr><tr><td><italic>TET2</italic></td><td>c.5618T&gt;C</td><td>p.I1873T</td><td>20.41</td><td>12.74</td><td>21.80</td><td>21.51</td></tr><tr><td>#22</td><td>Female, 85</td><td><italic>DNMT3A</italic></td><td>c.1988C&gt;T</td><td>p.S663W</td><td>1.04</td><td>0.00</td><td>1.02</td><td>0.86</td></tr><tr><td>#24</td><td>Male, 81</td><td><italic>TP53</italic></td><td>c.638G&gt;A</td><td>p.R213Q</td><td>15.43</td><td>0.90</td><td>15.70</td><td>18.50</td></tr><tr><td>#25</td><td>Male, 81</td><td><italic>DNMT3A</italic></td><td>c.2401A&gt;G</td><td>p.M801V</td><td>30.69</td><td>10.82</td><td>30.04</td><td>32.15</td></tr><tr><td>#35</td><td>Female, 86</td><td><italic>NRAS</italic></td><td>c.35G&gt;A</td><td>p.G12D</td><td>6.19</td><td>0.39</td><td>11.72</td><td>─</td></tr><tr><td>#37</td><td>Female, 81</td><td><italic>DNMT3A</italic></td><td>c.1031T&gt;C</td><td>p.L344P</td><td>5.03</td><td>1.58</td><td>5.23</td><td>5.17</td></tr><tr><td>#41</td><td>Female, 82</td><td><italic>DNMT3A</italic></td><td>c.929T&gt;C</td><td>p.I310T</td><td>17.82</td><td>0.00</td><td>17.26</td><td>21.60</td></tr><tr><td>#42</td><td>Male, 85</td><td><italic>U2AF1</italic></td><td>c.101C&gt;T</td><td>p.S34F</td><td>23.16</td><td>1.38</td><td>†</td><td>†</td></tr><tr><td>#43</td><td>Female, 81</td><td><italic>DNMT3A</italic></td><td>c.1988C&gt;T</td><td>p.S663W</td><td>12.67</td><td>0.00</td><td>14.38</td><td>18.80</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Somatic <italic>DNMT3A</italic> mutations within a cohort of 160 healthy younger individuals.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Sample number</th><th>Sex, age</th><th><italic>DNMT3A</italic> exon</th><th>Variant Ensemble</th><th>Protein</th><th>Ensemble entry</th><th>PolyPhen-2 prediction</th><th>PROVEAN prediction</th><th>% Reads displaying variant gDNA</th><th>% Reads displaying variant buccal cells</th></tr></thead><tbody><tr><td>#72</td><td>Female, 28</td><td>23</td><td>c.2645G&gt;A</td><td>p.R882H</td><td>Somatic mutation found in human cancers (COSM52944) and pathogenic SNP (rs147001633)</td><td>Probably damaging</td><td>Deleterious</td><td>18.88</td><td>6.10</td></tr><tr><td>#76</td><td>Male, 31</td><td>14</td><td>c.1651A&gt;G</td><td>p.N551D</td><td>Not displayed in Ensemble</td><td>Probably damaging</td><td>Deleterious</td><td>8.49</td><td>0.00</td></tr><tr><td>#96</td><td>Male, 49</td><td>19</td><td>c.2309C&gt;T</td><td>p.S770L</td><td>Somatic mutation found in human cancers (COSM231549) and SNP (rs758845779)</td><td>Probably damaging</td><td>Deleterious</td><td>2.43</td><td>0.00</td></tr><tr><td>#149</td><td>Male, 63</td><td>23</td><td>c.2644C&gt;T</td><td>p.R882C</td><td>Somatic mutation found in human cancers (COSM53042) and pathogenic SNP (rs377577594)</td><td>Probably damaging</td><td>Deleterious</td><td>8.00</td><td>0.00</td></tr><tr><td>#151</td><td>Female, 69</td><td>8</td><td>c.862delC</td><td>p.R288Gfs*28</td><td>Not displayed in Ensemble</td><td>No predictions available</td><td>No predictions available</td><td>37.30</td><td>0.00</td></tr><tr><td>#158</td><td>Female, 74</td><td>20</td><td>c.2381T&gt;C</td><td>p.F794S</td><td>Not displayed in Ensemble</td><td>Probably damaging</td><td>Deleterious</td><td>1.54</td><td>0.00</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p><italic>DNMT3A</italic> mutation rate in bone marrow of healthy individuals vs. mice xenografts.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Individual A</th><th colspan=\"3\">Mice xenografts (8 months after transplantation)</th></tr><tr><th/><th/><th>Mouse A1</th><th>Mouse A2</th><th>Mouse A3</th></tr></thead><tbody><tr><td colspan=\"5\"><italic>DNMT3A</italic><sup><italic>mut</italic></sup> (S837X)</td></tr><tr><td>VAF (%)</td><td>23.1</td><td>23.8</td><td>10.8</td><td>30.8</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Individual B</th><th colspan=\"3\">Mice xenografts (8 months after transplantation)</th></tr><tr><th/><th/><th>Mouse B1</th><th>Mouse B2</th><th>Mouse B3</th></tr></thead><tbody><tr><td colspan=\"5\"><italic>DNMT3A</italic><sup><italic>mut</italic></sup> (D768H)</td></tr><tr><td>VAF (%)</td><td>6.3</td><td>6.2</td><td>0</td><td>0</td></tr></tbody></table></table-wrap>" ]
[]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<table-wrap-foot><p>† Patient deceased.</p></table-wrap-foot>", "<table-wrap-foot><p><italic>VAF</italic> variant allele frequency.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"41375_2020_869_MOESM1_ESM.docx\"><caption><p>Supplemental Material</p></caption></media>" ]
[{"label": ["32."], "surname": ["Fuster", "MacLauchlan", "Zuriaga", "Polackal", "Ostriker", "Chakraborty"], "given-names": ["JJ", "S", "MA", "MN", "AC", "R"], "article-title": ["Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice"], "source": ["Science (New York, NY)"], "year": ["2017"], "volume": ["355"], "fpage": ["842"], "lpage": ["7"], "pub-id": ["10.1126/science.aag1381"]}]
{ "acronym": [], "definition": [] }
34
CC BY
no
2024-01-13 23:35:08
Leukemia. 2020 May 26; 34(8):2198-2205
oa_package/41/93/PMC7387320.tar.gz
PMC7615482
35770803
[ "<title>Introduction</title>", "<p id=\"P3\">The world has witnessed decades of research and advances in nucleic acid delivery, culminating with the first generation of non-viral nucleic acid therapeutics for Coronavirus Disease (COVID-19), approved by many health agencies across the globe. Before this important landmark, a limited number of viral-based nucleic acid therapeutics have previously been approved as last treatment options for retinal dystrophy, acute lymphoblastic leukemia, and B-cell lymphoma.<sup>[##REF##31887345##1##]</sup> Future nucleic acid therapeutics are in development for other cancers, tissue regeneration, and treatment of different types of inherited and acquired diseases. Most current delivery approaches in the clinic rely on the use of viral vectors due to their high efficiency in delivering nucleic acids intracellularly and escaping immune surveillance mechanisms.<sup>[##REF##30915230##2##–##UREF##0##5##]</sup> However, the high cost of production, limited scalability, and safety concerns due to potentially adverse immunological reactions pose significant barriers toward safe clinical use and approval of nucleic acid therapeutics.<sup>[##REF##30915230##2##–##UREF##0##5##]</sup> For those reasons, non-viral vectors have gained more attention as inexpensive and safer alternatives for nucleic acid delivery. These typically involve negatively charged nucleic acid cargo encapsulation by cationic lipids or polycations to form liposomes and polyplexes, respectively.<sup>[##REF##32554325##4##–##UREF##2##7##]</sup> The primary mechanism of intracellular nucleic acid delivery of non-viral vectors occurs via endocytosis mechanisms mediated by clathrin-coated pits, phagocytosis, micropinocytosis, and membrane fusion; followed by endolysosomal escape to reach the cell nucleus.<sup>[##UREF##3##8##–##REF##15963763##10##]</sup> Synthetic vectors can be engineered to increase cargo loading, biodegrade, target specific cells and exhibit extended shelf-life.<sup>[##UREF##0##5##–##UREF##2##7##,##UREF##4##11##,##UREF##5##12##]</sup> The main limitation of these vectors is the balance between transfection efficiency and cytotoxicity. For example, polyplexes made of poly(ethylene imine) (PEI), a widely used polycation, are highly effective but simultaneously highly cytotoxic due to the quaternary amine in the structure and lack of degradation.<sup>[##UREF##0##5##,##UREF##5##12##–##UREF##6##14##]</sup> Conversely, synthetic strategies aiming to reduce the cytotoxicity of polycations have resulted in decreased transfection levels.<sup>[##UREF##0##5##,##UREF##5##12##–##UREF##6##14##]</sup> Therefore, attempts to develop a non-viral delivery system with high transfection efficiency and minimal cytotoxicity are currently under investigation. An attractive polycationic candidate is poly(CBA<italic toggle=\"yes\">-co-</italic>4-amino-1-butanol) (pABOL), which possesses a disulfide bond in the structure that renders it biodegradable by enzymatic reduction of intracellular esterases such as reduced glutathione (GSH) and thioredoxin reductases.<sup>[##UREF##7##15##,##UREF##8##16##]</sup> Polyplexes comprised of pABOL with various nucleic acid payloads have sizes around a few hundred nanometers have shown no cytotoxicity at optimal concentrations to obtain the highest levels of transfection, comparable to 25 kDa branched PEI.<sup>[##UREF##7##15##,##REF##32267667##17##–##REF##24193511##19##]</sup></p>", "<p id=\"P4\">Nanoneedles and other high-aspect ratio nanostructures are unconventional but interesting systems for intracellular nucleic acid delivery.<sup>[##UREF##9##20##,##REF##34426708##21##]</sup> There has been much debate whether these nanostructures facilitate cargo delivery into the cytosol by directly penetrating the cell membrane, or through other mechanisms.<sup>[##UREF##9##20##–##REF##30091365##23##]</sup> Studies have suggested that for cells seeded onto high-aspect ratio nanostructures, under the influence of gravity alone, the rate of spontaneous penetration of the membrane is relatively low,<sup>[##REF##22166016##24##]</sup> and is strongly influenced by the specific biochemical environment of the nanostructures and cells.<sup>[##UREF##9##20##,##REF##31465200##25##]</sup> Recently, it has been observed a higher proportion of clathrincoated pits and caveolae present at the cell-nanoneedle interface,<sup>[##UREF##9##20##,##UREF##11##26##]</sup>suggesting that endocytosis mechanisms are involved in cargo uptake. Elsewhere, the delivery effect of high-aspect ratio nanostructured surfaces can also be enhanced through the application of external stimuli, those including mechanical interfacing forces and electroporation techniques.<sup>[##UREF##9##20##,##REF##34426708##21##,##UREF##12##27##–##UREF##13##30##]</sup> Regardless, nanoneedles and other high-aspect ratio nanostructures have shown successful delivery of nucleic acids with minimal toxicity in vitro, including plasmid DNA (pDNA) and small interfering RNA (siRNA).<sup>[##UREF##12##27##,##REF##26471006##29##,##UREF##14##31##–##REF##25822693##33##]</sup> In vivo, a study showed that porous nanoneedles increased angiogenesis after delivery of a vascular endothelial growth factor (VEGF) plasmid, with no evidence of toxicity or adverse reactions in the tissue.<sup>[##REF##25822693##33##]</sup> Another aspect to consider is the relative increase in surface area created by nano-structuring a surface. This may impact cargo uptake, in a similar fashion to high-aspect ratio nanoparticles, which have been linked to increased uptake via endocytosis.<sup>[##REF##33666625##34##,##REF##19800115##35##]</sup> Regardless, there is limited control over the amount of nucleic acids loaded and length of release as they are merely adsorbed on the surface of the substrate.</p>", "<p id=\"P5\">Ultrathin coatings, created using layer-by-layer (LbL) self-assembled nanofilms, are highly versatile systems for nucleic acid delivery. The layer assembly of these coatings is typically mediated by the electrostatic interactions of two or more polyelectrolytes, which are deposited in alternating cycles onto the surface of a charged substrate.<sup>[##REF##25908826##36##–##REF##24198464##38##]</sup> The multilayered nature of these coatings allows exquisite control over loading, release kinetics, and porosity (from smooth to highly porous surfaces); while their nanometer-thickness scale preserves the architecture of substrates with complex geometries.<sup>[##REF##27760399##39##,##UREF##15##40##]</sup> This has been used to deliver a variety of cargoes that include proteins,<sup>[##REF##27760399##39##–##UREF##16##42##]</sup>drugs,<sup>[##UREF##17##43##,##REF##25093948##44##]</sup>nucleic acids,<sup>[##UREF##18##45##–##UREF##19##48##]</sup> and particle systems.<sup>[##REF##27114520##49##,##UREF##20##50##]</sup> Therefore, we have combined self-assembled polyplex-polysaccharide nanofilms with high-aspect ratio nanostructures to synergistically enhance nucleic acid delivery and transfection efficiency. We studied the influence of distinct polyplex-coated nanoneedle architectures and other high-aspect ratio structures on transfection efficiency. We also assessed various polyplex-polyelectrolyte combinations, and we found that polysaccharide-based polyanions enhanced the transfection efficiency of pABOL polyplexes, while other polyanions proved to be detrimental to delivery. Interestingly, a few studies have previously claimed that hyaluronate/hyaluronic acid (HA), an anionic polysaccharide and non-sulfated glycosaminoglycan (GAG), resulted in enhanced nucleic acid delivery via liposomes, nanoparticles, and PEI-polyplexes, showing good levels of transfection.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> These studies suggested that the presence of HA in these vectors could enhance nucleic acid delivery by CD44-mediated uptake, where HA binds its receptor CD44, followed by internalization and intracellular release of the vector.<sup>[##REF##34329781##52##–##UREF##23##54##]</sup> A second proposed mechanism is via structural properties of the polymer, where HA would modulate the electrostatic interactions within the polyplex and/or establish hydrogen bonding with nucleic acids, which would facilitate the release from the complex.<sup>[##UREF##21##51##,##UREF##22##53##]</sup> Interestingly, our studied nanofilm combinations showed that enhanced plasmid delivery was not specifically linked to HA, but rather to polysaccharide-based polyanions. Moreover, results showed that these effects were not mediated by interactions with the CD44 receptor. Systematic studies were performed to clarify the role of polysaccharide-based polyanions with enhanced transfection efficiency, expanding the spectrum of possible polyelectrolyte combinations that can be used to develop non-viral vectors with high delivery efficiency and low cytotoxicity.</p>" ]
[]
[ "<title>Results and Discussion</title>", "<title>Plasmid DNA-Polyplex Characterization</title>", "<p id=\"P6\">Polyplexes loaded into the coating were made of a bio-reducible polycationic polymer, poly(cystamine bisacrylamide<italic toggle=\"yes\">-co-</italic>4-amino-1-butanol), hereafter referred to as pABOL, in which its backbone structure contains a disulfide bond that is broken down intracellularly after endocytosis via esterases, releasing the nucleic acid cargoes contained within the polyplex complex.<sup>[##UREF##7##15##,##UREF##8##16##]</sup> Polyplexes were loaded with a pCAG-GFP, a plasmid coding for green fluorescent protein (GFP), by mixing them in a mass ratio of 45:1 (##FIG##0##Figure 1a##), which corresponds to an N/P ratio of 38, which is the ratio of positively charged amino groups in pABOL to the number of negatively charged phosphate groups in the pDNA. This optimized mass ratio was chosen from previous studies using pABOL polyplexes for delivery of nucleic acids.<sup>[##REF##32267667##17##–##REF##24193511##19##]</sup> This method results in polyplexes of 104 ± 3.6 nm in average size, with good polydispersity (PDI: 0.135); and a surface zeta potential of +35.1 ± 6.7 mV (##FIG##0##Figure 1b,c##), making them positively charged, which facilitates electrostatic deposition and loading onto the coating.</p>", "<title>Polyplex-Polysaccharide Coated Nanoneedle Characterization</title>", "<p id=\"P7\">An optimal surface charge of the silicon nanoneedles is a key requirement to allow LbL deposition of the first polycationic chitosan layer. The surface of silicon nanoneedles (50 nm tip, 4.5 μm height) was cleaned from any remaining impurities using isopropanol:acetone (1:1) followed by oxygen plasma treatment. X-ray photoelectron spectroscopy (XPS) revealed that the plasma treatment increased the proportion of silicon oxide (103 eV) over pure silicon (98–100 eV doublet) (##FIG##0##Figure 1d,e##). Silicon oxide is known to provide negatively charged oxide ions.<sup>[##UREF##26##59##,##REF##30900715##60##]</sup> The increase in surface oxidation is also further supported by an increase in oxygen (533 eV) (##FIG##0##Figure 1d,e##). Contact angle measurements showed that the hydrophilicity of the nanoneedle surface significantly increased after plasma treatment (##FIG##0##Figure 1f,g##), which corresponds with the increased SiO<sub>2</sub> seen under XPS.</p>", "<p id=\"P8\">After plasma treatment, nanoneedles were coated using a LbL procedure via cycles of self-assembled deposition of chitosan (polycation) and HA (polyanion), mediated by opposite electrostatic interactions. We used a core coating of 10 bilayers (10B) as a supporting surface for further polyplex loading, which has been shown to ensure a uniform coating with no gaps present.<sup>[##REF##27760399##39##,##UREF##15##40##]</sup> The first polyplex layer, made of net positively charged complexes, was deposited onto the last negatively charged HA layer of the core coating, and additional polyplex layers were loaded within an intermediate coating of 5 bilayers (5B) (##FIG##1##Figure 2a##). The presence and uniformity of the nanofilm were evaluated via confocal microscopy, using tetramethylrhodamine (TAMRA) to stain the coating (reacting to amino groups in chitosan) and DAPI to stain pDNA-polyplexes. Confocal images showed that the entire nanoneedle surface is coated and polyplexes are distributed in the coating (##FIG##1##Figure 2b##; ##SUPPL##0##Figure S1, Supporting Information##). Differences in fluorescence intensity and missing nanoneedles are most likely a consequence of needle fracture during handling or artifacts during nanoneedle fabrication. Another important note is that the architecture of nanoneedles is preserved after the coating procedure, corroborated via SEM imaging (##FIG##1##Figure 2c##), which is essential to study the specific effects of high-aspect ratio nanostructures on transfection efficiency. XPS has been used to provide a more exhaustive confirmation of the components present in the coating (##FIG##1##Figure 2d##). The predominant presence of C-O (286 eV) and O-C=O (288 eV) structures show the polysaccharide structures from hyaluronic acid and chitosan, while the specific presence of nitrogen peaks (400 and 402 eV) confirms the presence of chitosan. The presence of pABOL is confirmed explicitly by a peak in sulfur at 164 eV, but also an increased ratio in protonated nitrogen (<sup>+</sup> N-H) species at 402 eV due to the tertiary amine from the polymer. Due to the comparatively low amount of DNA in the polyplexes (1:45 pDNA:pABOL), further diluted into the coating, the phosphorus signal from pDNA is low but still detected. We have further evaluated the surface composition with increasing numbers of polyplex and seen that the presence of silicon species diminished until complete disappearance by 4 polyplex layers (##SUPPL##0##Figure S2, Supporting Information##). This indicates how the coating increases thickness with more layers until the x-ray beam is not able to penetrate deep enough to detect the underlying silicon substrate. At this particular point, the surface composition and atomic percentages seen under XPS resemble more accurately the real elemental proportion of species in the coating. Since atomic force microscopy (AFM) cannot readily be used on high-aspect ratio structures like nanoneedles, the surface roughness of the coating was assessed on flat silicon substrates. After coating, the surface roughness increased compared to the relatively smooth surface of pristine silicon (##FIG##1##Figure 2e##). It is possible to observe spheric-like shapes of ≈100 nm in diameter on the surface of polyplex-coated flat silicon surfaces, which may correspond to the polyplexes, which are distributed uniformly throughout the surface, and embedded within the coating. This increase in surface roughness translates into an increased surface area, expected to enhance the interactions at the interface between cells and nanoneedles and hence cell uptake of polyplexes.</p>", "<title>Release and Degradation of Polyplex-Polysaccharide Nanofilms</title>", "<p id=\"P9\">To study, quantify and optimize the loading and intracellular delivery of pDNA of the nanofilms as a function of polyplex layers, flat silicon substrates of known surface area (8 × 8 mm<sup>2</sup>) coated with polyplex nanofilms were assayed in a release buffer containing a physiological intracellular concentration of reduced glutathione (10 mM),<sup>[##REF##18158646##61##]</sup> and the pDNA was quantified from aliquots taken at different time points using a PicoGreen assay. Release kinetics analyses showed similar trends for most samples regardless of the number of polyplex layers. Nanofilms with 3P and 4P released ≈40% of their pDNA loaded within the first hour, reaching complete release by 6 h, as evidenced by a plateau in the release curves (##FIG##2##Figure 3a##). Nanofilms with 1P and 2P released ≈20%of their total content by 1 h, also reaching completion by 6 h. As expected, the amount of pDNA loaded in the coating increased with the number of polyplex layers, all of them on the nanogram scale (##FIG##2##Figure 3b##). While the growth of material deposition in LbL films has been described to occur exponentially via dipping methods,<sup>[##REF##15969601##62##,##UREF##27##63##]</sup> there was an unexpected and sudden increase in the amount of pDNA in the 5-polyplex layers sample, accompanied by a high variability. AFM assessments on these samples showed abnormal deposition and the presence of material aggregation in the nanofilms (##SUPPL##0##Figure S3, Supporting Information##), which are not present in 4-polyplex layer nanofilms. Increased washing times or changes in polymer concentration did not change these outcomes, suggesting they occur unavoidably. These anomalies are likely a major contributor to the aberrant loading and release kinetics, hence not considered for statistical analysis.</p>", "<p id=\"P10\">The detection of polyplexes and the clear differences in surface roughness between coated and non-coated silicon substrates, allow feasible tracking of degradation via AFM over time using the same experimental conditions used for release assays. The results are consistent with release assays. The degradation of polyplexes occurs mainly within the first hour, while the presence of coating material seems to remain in the substrate for at least 4 h, then entirely degraded at 6 h, with surface roughness quite comparable to pristine controls (##FIG##2##Figure 3c##).</p>", "<title>Transfection Efficiency of Polyplex-Polysaccharide Nanofilms</title>", "<p id=\"P11\">Transfection efficiency on COS-7 cells was studied as a function of the number of polyplex layers in the nanofilms to observe whether there is a correlation with the amount of released plasmid. The transfection efficiency, which is the percentage of GFP expression adjusted to cell viability, was evaluated 24-h post-transfection via fluorescence microscopy and flow cytometry. Results showed an increasing trend in transfection efficiency with the first four polyplex layers (##FIG##3##Figure 4a,b##), which correlated to an increasing amount of released pDNA. However, the 5-polyplex layers coated nanoneedles were significantly less effective in transfection than the 4-polyplex layers, even though they released more than twice the amount of pDNA. Also, 5-polyplex layers resulted in lower viability (##FIG##3##Figure 4c##). This is likely a consequence of the abnormal nanofilm deposition observed under AFM and aberrant release previously observed. Regardless, 4-polyplex layer coatings resulted in the highest transfection efficiency and were therefore used for all further studies. An optimal number of intermediate chitosanhyaluronic acid bilayers between polyplexes was also assessed to maximize transfection efficiency. We found that each polyplex layer, contained within five intermediate bilayers, resulted in the highest and most reproducible transfection levels (##SUPPL##0##Figure S4, Supporting Information##). No additional bilayers were assessed as this would make the procedure impractical, hence all studies have been done with five intermediate bilayers.</p>", "<p id=\"P12\">We investigated whether the HA-chitosan-polyplex nanofilm enhances plasmid delivery and transfection efficiency compared to polyplexes alone. The transfection efficiency of silicon substrates coated with 4-polyplex layer nanofilms was compared to polyplexes in suspension containing 49 ng of plasmid, equivalent amount of pDNA released on 4-polyplex nanofilms (##FIG##2##Figure 3b##), but also compared to polyplexes containing 200 ng of pDNA, which is equivalent to standard concentrations used with polyplexes (2 μg/500 μL). COS-7 cells were seeded on flat silicon to maintain consistent experimental conditions. Results with polyplexes in suspension containing 49 ng of plasmid resulted in minimal transfection efficiency, compared to 4-polyplex layer nanofilm substrates with a transfection efficiency of 59.3% (##FIG##3##Figure 4d##). Polyplexes containing 200 ng of plasmid increased transfection efficiency to 11.5%, but still significantly lower than 4-polyplex nanofilms. Transfection was also performed in standard culture conditions, with COS-7 cells already adhered to tissue culture plastic (TCP) wells, using polyplexes in suspension containing 2 μg of plasmid in 500 μL, which resulted in transfection efficiency of 41.3%. This shows that adhesion of cells before transfection is important to obtain higher transfection efficiency using polyplexes in suspension, while nanofilms do not present this limitation. Therefore, these results demonstrate consistently that HA-chitosan nanofilms enhance the transfection efficiency of polyplexes, using an amount of plasmid an order of magnitude lower than standard transfection with polyplexes in culture, but also most non-viral commercial vectors, including liposomes and nanoparticles. In fact, an order of magnitude higher in the amount of plasmid in the polyplexes (2 μg) was not enough to equal the transfection efficiency obtained with nanofilms. This matches previous findings, in which HA has been incorporated during polyplex formation and shown to enhance nucleic acid delivery.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> Studies to evaluate whether the specific presence of HA is required for an enhanced transfection have been performed and are discussed later.</p>", "<p id=\"P13\">Intracellular delivery of cargoes by high-aspect ratio structures occurs at the cell-material interface. This highly localized delivery is a consequence of cargoes being physisorbed to the surface and not readily released to the surrounding environment.<sup>[##UREF##9##20##–##REF##30091365##23##]</sup> On the other hand, delivery and release of molecules in LbL coatings rely on the degradation properties of the polymers forming the coating and its architecture. For instance, the release of protein cargoes triggered by enzymatic action in the host tissue has been shown to affect the biological response of cells ≈50 μm from the tissue material interface.<sup>[##REF##27760399##39##]</sup> Alternatively, LbL films containing intracellular nucleic acid cargoes have been shown to depend more on cell uptake and delivery only occurs in cells directly interfacing the surface of the coated substrates.<sup>[##UREF##18##45##–##UREF##19##48##]</sup> To assess whether polyplexes remain within the nanofilms for highly localized surface-mediated transfection or diffuse away from the surface during transfection, we have used two contiguous coated silicon substrates: one loaded with pCAG-GFP (coding for GFP); and the second one containing pCAG-RFP, coding for red fluorescent protein (RFP), placed on top of a PDMS sheet to ensure tight adhesion within a tissue culture well-plate, as shown in ##FIG##3##Figure 4e##. Transfection was done by adding a single-cell suspension to cover both substrates simultaneously, allowing for possible polyplex diffusion, and incubated together for 4 h. After 24 h post-transfection, results showed COS-7 cells expressing either GFP or RFP on their corresponding sides of the coated substrates, with a clear separation of transfected cells in the contiguous edge of the substrates (##FIG##3##Figure 4f##). Therefore, polyplexes remained confined to each side of the substrate. No apparent diffusion or release of polyplexes was observed, confirming a surface-mediated mechanism of transfection, which means that cells or tissues must interface the coating for transfection to occur. The immobilization and possible concentration of polyplexes at the surface of the material may contribute to the observed enhanced transfection efficiency, with an order of magnitude lower in the amount of plasmid compared to polyplexes in suspension. This also demonstrates the potential of nanofilms for spatial patterning of transfection and generation of gradients in multicellular systems and tissue regeneration, respectively.</p>", "<title>Influence of High-Aspect Ratio Nanostructures on Transfection Efficiency</title>", "<p id=\"P14\">Considering that an optimal tissue or cell interface with the coated substrate is essential for an efficient transfection of genes, the surface area would be an important parameter for both the coating and high-aspect ratio nanostructures. Hence we have studied transfection efficiency with nanostructures of increasing surface area and several architectures. In increasing order of relative surface area, we compared transfection efficiency of COS-7 cells on flat silicon, short solid nanoneedles (SnN, height 3 μm), medium solid nanoneedles (MnN, height 4.5 μm), tall solid nanoneedles (TnN, height 7 μm), porous nanoneedles (PnN, height 5.5 μm) and nanowires (nW, height 400 nm). SEM was used to demonstrate that the architecture of these high-aspect ratio nanostructures was preserved after the coating procedure, ensuring that the differences in transfection efficiency are attributed to the nanostructures and not to other artifacts caused by the coating. As shown in ##FIG##4##Figure 5a##, the architecture of all solid nanoneedles was preserved. The overall shape of porous nanoneedles was mainly preserved, however, some features in the nanoneedles seemed to be too small to be preserved and were coated during the procedure. The dimensions of the nanowires were small, but the coating was able to maintain the overall architecture of their features, with the presence of some aggregates.</p>", "<p id=\"P15\">In this study, transfection efficiency was complemented with phalloidin staining to assess differences in cell morphology in response to each nanostructure. Results revealed similar transfection efficiencies for flat silicon, small nN, medium nN, and porous nN, while tall nanoneedles and nanowires show a lower average in transfection efficiency and inconsistent results with higher variance between repeats (##FIG##4##Figure 5b,c##). Considering these findings and their corresponding cell morphological staining, these inconsistencies in transfection for both TnN and nW substrates seem to be associated with poor interfacing area between cells and nanostructures. As previously mentioned, an optimal cell-surface interface is essential for transfection, but it seems that an increased surface area in the substrate does not necessarily translate to a larger area of cell contact. Instead, in this case, the architecture of high-aspect ratio structures seems to be more important, including height, spacing, or porosity. For subsequent experiments, we used medium nanoneedles, which have the lowest variability in transfection efficiency of all solid nanoneedles and better preserved high-aspect ratio structure after coating than porous needles. Medium nanoneedles have also been chosen over flat substrates as we have seen evidence that nanoneedles aid tissue delivery in a number of different contexts,<sup>[##REF##34426708##21##,##REF##25822693##33##,##REF##30430139##64##,##REF##25858596##65##]</sup> but also in our ex vivo studies, as it will be shown in a later section.</p>", "<title>Role of Polyanion Structure on Enhanced Transfection Efficiency</title>", "<p id=\"P16\">As previously mentioned, it has been recently hypothesized that HA enhances nucleic acid delivery.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> The proposed mechanisms have linked this enhancement to (1) the chemical structure of HA – modulating the electrostatic interactions in the polyplex via hydrogen bonding,<sup>[##UREF##21##51##,##UREF##22##53##]</sup> and (2) CD44-mediated cell uptake of the complex via interactions with HA.<sup>[##REF##34329781##52##–##UREF##23##54##]</sup> We first studied the role of the chemical structure of HA on transfection efficiency, comparing coatings made of different polyanions in combination with chitosan. The presence of all polyelectrolytes and pDNA-polyplexes in these coatings was confirmed successfully via XPS (##SUPPL##0##Figure S5, Supporting Information##).</p>", "<p id=\"P17\">HA is composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units, and it is the only non-sulfated member of the GAG family that plays important roles in the extracellular matrix and regulation of cellular responses.<sup>[##REF##30960626##66##–##UREF##28##68##]</sup> Therefore, transfection efficiency was tested in other members of the same family such as chondroitin-4-sulfate (CS), a GAG with low sulfation degree, in which constituent disaccharide units are D-glucuronic acid and N-acetyl-D-galactosamine.<sup>[##UREF##28##68##,##REF##31887904##69##]</sup> Similarly to HA, CS has also been shown to bind CD44.<sup>[##REF##11222505##70##,##REF##9891022##71##]</sup> Heparin (Hep), a GAG with a high sulfation degree, has also been tested. This is composed of sulfated uronic acid and sulfated D-glucosamine.<sup>[##UREF##28##68##,##REF##32255159##72##]</sup> Hep has been shown to bind an exon v3 variant of CD44 to regulate the activity of specific growth factors.<sup>[##REF##9891022##71##,##UREF##29##73##]</sup> The chemical structures of all three GAGs and all studied polyanions are shown in ##FIG##5##Figure 6a##. Transfection with both sulfated GAGs resulted in a drastically reduced efficiency (##FIG##5##Figure 6b##), which indicates that sulfation in the polysaccharide structure is detrimental for transfection, as discussed below. Alginate (Alg) was then chosen due to similar polysaccharide chemical structure and charge density to HA, but formed by D-mannuronate and L-guluronate sugars.<sup>[##REF##22125349##74##]</sup> Results revealed higher transfection efficiency than HA (75.3% versus 64.8%), demonstrating that enhanced transfection is not specific to HA (##FIG##5##Figure 6b##). The role of the polysaccharide backbone in transfection efficiency from both HA and Alg has been assessed by testing coatings with polyglutamic acid (pGlu), a polyaminoacid with the same carboxylate anion but no polysaccharide backbone. Interestingly, results showed poor transfection (8%), suggesting that a polysaccharide backbone in the polyanion would be essential to facilitate transfection.</p>", "<p id=\"P18\">We looked further into the detrimental effect of sulfation on transfection efficiency and evaluated whether the coating is affecting the release of pDNA, as a consequence of the high anionic charge of sulfonates in the polyanion. Release assays with both chondroitin sulfate (CS) and Hep showed lower released amounts of pDNA released, while similar release kinetics were observed (##FIG##5##Figure 6d,e##). No linear dependence between the degree of sulfation and transfection efficiency was observed. These results show that sulfation in the polyanion structure affects the release of pDNA and hence transfection efficiency. Supporting evidence indicates that sulfated polysaccharides disrupt the electrostatic interactions within polyplexes and liposomes. For example, buffers containing dextran sulfate or Hep have been used to unpack nucleic acid cargoes to quantify release.<sup>[##UREF##24##55##,##UREF##25##58##,##REF##21993237##75##,##UREF##30##76##]</sup> It is then possible that part of the pDNA is released from the polyplexes during the coating procedure. However, in other systems, polyplexes coated with CS have been shown to enhance transfection efficiency,<sup>[##UREF##31##77##,##UREF##32##78##]</sup> which suggests that these effects may also be dependent on the architecture of the delivery system, polymer composition, and concentration. However, these differences in release amount do not account for the dramatic decrease in transfection efficiency observed, with 4.4% for CS and 1% for Hep (##FIG##5##Figure 6B##).</p>", "<p id=\"P19\">Polyglutamic acid promotes poor transfection compared to both HA and Alg, in which the most evident structural difference is the presence of a polysaccharide backbone. The abundant hydroxyl groups in the polysaccharide backbone serve as both hydrogen bond acceptors and donors, while pGlu predominantly has hydrogen bond acceptors and a hydrophobic segment. Previous studies using polysaccharides and other synthetic polymers for particle decoration have claimed that hydrogen bonding modifies the strength and density of electrostatic interactions between the nucleic acids and polycations, facilitating the release of cargoes from the polyplex.<sup>[##UREF##5##12##,##UREF##21##51##,##UREF##22##53##]</sup> Alternatively, the higher charge density of polyglutamic acid could disrupt polyplexes as a consequence of the shorter spacing among amino acid units in polyglutamic acid compared to the sugar units in the polysaccharide backbone of both HA and Alg. To test this hypothesis, the transfection efficiency was assessed using gamma polyglutamic acid (γ-pGlu), in which glutamic acid is polymerized in the <italic toggle=\"yes\">γ</italic> position, increasing the distance between carboxylate anions from three to five atoms which decreases the charge density to similar levels to polysaccharides: Alg with five atoms and HA with 10 atoms (##FIG##5##Figure 6a##). The results showed that the transfection efficiency using <italic toggle=\"yes\">γ</italic>-pGlu is low, with similar efficiency to pGlu (##FIG##5##Figure 6b##), suggesting that the enhancement in transfection efficiency observed in polysaccharides is not dictated by charge density in the polyanion. The poor performance of <italic toggle=\"yes\">γ</italic>-pGlu on delivery and transfection efficiency seems to be specific to the nanofilm-polyplex system, considering that <italic toggle=\"yes\">γ</italic>-pGlu has shown to improve cell-uptake and transfection efficiency when incorporated in the fabrication of polyplexes.<sup>[##REF##19232715##79##–##REF##20864162##83##]</sup></p>", "<title>Role of CD44-Mediated Cell Uptake on Enhanced Transfection Efficiency</title>", "<p id=\"P20\">To test the hypothesis of CD44-mediated cell uptake, we have evaluated the transfection efficiency in cell types with and without distinct expression of CD44 (##FIG##6##Figure 7a##). We have quantified the levels of CD44 expression using antibody labeling and flow cytometry analyses (##FIG##6##Figure 7b##). COS-7 cells, a fibroblast cell line from monkey kidney,<sup>[##REF##6260373##84##]</sup> present high expression levels of CD44 (96%). HEK-293, an epithelial cell line from human kidney embryo<sup>[##REF##11967234##85##]</sup> exhibits almost no expression of CD44 (1%). C2C12, a mouse myoblast cell line,<sup>[##REF##8023908##86##]</sup> showed a relatively fair expression of CD44 (37.5%). Human mesenchymal stem cells (MSCs), primary cells derived from bone marrow, as expected by definition,<sup>[##UREF##35##87##]</sup> exhibited a high (95.8%) expression of CD44. Transfection results have shown that the high transfection efficiency of COS-7 is correlated to the high expression of CD44 (##FIG##6##Figure 7c##). However, HEK-293T showed a similar high transfection efficiency to COS-7 cells, regardless of the minimal expression of CD44. C2C12 cells exhibited lower transfection efficiency than both COS-7 and HEK-293T cells. Surprisingly, MSCs showed marginal transfection efficiency (3%), which should have been significantly higher due to the highest expression of CD44 if CD44-mediated uptake were indeed the predominant mechanism at play.</p>", "<p id=\"P21\">Despite the fact that the transfection efficiency does not seem to comply with the CD44-mediated transfection hypothesis given the uncorrelated levels of CD44 expression in most of these cell types, it could be argued that HEK-293T cells could still efficiently uptake polyplexes via conventional endocytosis, or that MSCs are widely known to be inherently difficult to transfect.<sup>[##REF##15277699##88##,##REF##33034446##89##]</sup> For further confirmation, transfection efficiency was assessed in COS-7 cells after blocking the CD44 receptor using an antibody, and compared to normal transfection. This has been done in nanoneedles coated with either HA or Alg, both with high transfection efficiency and polysaccharide-based chemical structures (##FIG##5##Figure 6b##). Blocking the CD44 receptor in COS-7 cells did not disrupt transfection efficiency. In both cases, results did not show decreased transfection efficiency after blocking CD44 receptors in COS-7 cells and unexpectedly, transfection efficiency in nanofilms containing Alg was higher after blocking (##FIG##6##Figure 7e,f##). Therefore, the results in this study consistently confirm that facilitated transfection of HA and Alg does not predominantly occur via CD44-mediated cell uptake. Instead, it relies on the polysaccharide physicochemical properties such as hydrogen bonding to facilitate intracellular delivery of the nucleic cargo.</p>", "<p id=\"P22\">Despite producing some results at odds with the previously-proposed CD44-mediated cell uptake mechanism, our results do confirm that HA facilitates transfection.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> We also demonstrate that transfection is not only facilitated by HA but also by additional polysaccharide-based polyanions such as Alg. We also have to consider experimental differences with prior studies where HA has likely been integrated into the bulk and surface during the formation of polyplexes, liposomes, and particles; in contrast, our procedure would likely integrate HA onto the surface of the polyplex. Discrepancies may also arise from the molecular weight of hyaluronic acid used, cell types studied and CD44 antibodies used for blocking.</p>", "<title>Surface-Mediated Transfection on an Ex Vivo Cardiac Slice Model</title>", "<p id=\"P23\">We evaluated the efficacy of the polyplex coated nanoneedles to transfect tissues on an ex vivo cardiac slice platform. Due to the architecture of nanoneedles and the surface-mediated delivery of the coating, transfection would occur in the outermost layer of cells in the tissue, in which in vivo imaging techniques and histological methods are limited. Cardiac slices, on the other hand, are an ideal ex vivo platform to study surface-mediated transfection on tissues, with many imaging techniques and quantification methods available.<sup>[##REF##30671746##90##,##REF##29189769##91##]</sup> These slices were prepared from left ventricles of rats in 300 μm sections, an optimal thickness to allow nutrient and oxygen supply for prolonged survival.<sup>[##REF##29189769##91##–##REF##31868875##93##]</sup> Polyplex coated nanoneedles or controls were interfaced for 1 h on top of the cardiac slices, fixed to a holder inside of a custom chamber filled with sufficient culture media to be in contact with the bottom side of the slice, allowing the nanoneedle substrate to adhere to the tissue before filling the chamber completely with media (##FIG##7##Figure 8a##). Similar to our in vitro studies, slices were evaluated after 24 h of incubation, during which time the coated nanoneedles stayed in contact with the tissue. The tissue viability was assessed using a Live/ Dead staining. The functionality of the slices after transfection was evaluated via contractility trace analysis from force transducer measurements, then fixed for staining.</p>", "<p id=\"P24\">To assess tissue transfection in the slice, a co-immunolabeling was performed using antibodies against GFP (green, transfected cells), cardiac troponin (cTNT, red, cardiomyocyte marker), vimentin (white, a stromal cell marker to label fibroblast and endothelial cells) and DAPI (blue) as counterstaining. Image analysis was performed to quantify the expression of GFP, as percentage of the total area in the imaged field. After 24 h of transfection, fibroblasts and endothelial cells showed to be transfected, as evidenced by high colocalization of GFP expression and vimentin, mainly localized between cardiomyocyte bundles and in the lining of blood vessels (##FIG##7##Figure 8b-iv,v##). Only a few discrete transfected cardiomyocytes were observed. No transfection was observed in controls groups and coated (no polyplex) nanoneedle groups (##FIG##7##Figure 8b-i,ii##). This selective transfection might be explained by the intrinsic physiological roles of these cells within the cardiac tissue, resulting in distinct cell uptake activity. For example, previous studies have shown that cardiomyocytes exhibit lower cell uptake of extracellular vesicles (EVs) compared to endothelial cells and fibroblasts,<sup>[##REF##27806113##94##,##REF##32969445##95##]</sup> which was hypothesized to be related to the increased ability of endothelial cells and fibroblasts to receive signals from cardiomyocytes via EVs, especially under injury, process that remains to be elucidated.<sup>[##REF##32969445##95##–##REF##30410918##97##]</sup> The observed differences in transfection among groups were correlated with quantification of GFP expression—nanoneedles with 4P-polyplex nanofilms showed 2.62% area of GFP per field, while nanofilm controls and no chip controls showed an area of 0.31% and 0.22%, respectively (##FIG##7##Figure 8c##). These results are in agreement with levels of transfection observed in COS-7 (fibroblasts), HEK-293 (epithelial cells), and C2C12 (myoblasts or muscle cells) in vitro (##FIG##6##Figure 7##), while noting that a direct comparison among these cell lines with cells in the tissue is not intended due to their metabolic and physiological differences. These results also support the highly localized surface-mediated mechanism of delivery, as transfection was only observed on the top side of the cardiac slice, where the tissue was interfacing the polyplex coated nanoneedles. In contrast, limited transfection and a lower area of GFP expression were observed on the contralateral surface (##FIG##7##Figure 8b-vi,c##). In our in vitro experiments, medium nanoneedles were selected over flat nanofilm substrates as hypothesized the penetration of nanoneedles into the tissue would increase surface interaction and transfection efficiency compared to flat. Cardiac slices were transfected with 4-polyplex layer flat nanofilms and results showed that the expression of GFP within the tissue was considerably lower than nanoneedles (##FIG##7##Figure 8b-iii,d##). Therefore, polyplex coated nanoneedles are capable of transfecting tissues with an exquisite level of localization, also ensuring a proper interface with tissues for facilitated transfection.</p>", "<p id=\"P25\">Cell viability assessments showed preserved viability of the cardiac tissue, with no differences in the expression of calcein and number of dead cells among slices treated with polyplex coated needles, coated nanoneedles (no polyplex), and nonanoneedle controls (##SUPPL##0##Figure S6, Supporting Information##). Similarly, force transducer measurements of contractility in the cardiac slices were performed to demonstrate that transfection did not alter the functionality of the tissue. Trace analysis of cardiac contractility showed preserved function of cardiac slices, with no differences in contracting force (##FIG##7##Figure 8e##), time to peak (##FIG##7##Figure 8f##), and time to decay (##FIG##7##Figure 8g,h##) observed among groups. Therefore, transfection via nanoneedles coated with polyplex-nanofilms preserves both the viability and functionality of the tissue.</p>", "<p id=\"P26\">In clinical settings, our surface-mediated transfection system is then advantageous in accessible tissues, where highly localized transfection and confined penetration depth is sought. For example, the epicardium is the outermost layer covering the heart, harboring a population of progenitor cells capable of regeneration in the heart, mainly dormant after birth, which makes the epicardium a highly attractive target for transfection to reactivate gene pathways that stimulate epithelial-to-mesenchymal transition.<sup>[##REF##26907357##98##,##REF##35328640##99##]</sup> Given the highly confined layer architecture of the epicardium, a surface-mediated transfection via the polyplex-nanofilm system would hence be highly suitable for localized and efficient transfection. Transdermal delivery via nanoneedle systems may pose limitations, compared to their microscale counterparts. This route has always been an attractive and suitable target for microneedle-mediated delivery, in which many cargoes (e.g., nucleic acids, proteins, and drugs) have been delivered for therapeutic applications that include melanoma, wound healing, as well as sustained systemic delivery of drugs.<sup>[##REF##32575392##100##–##REF##30348022##102##]</sup> In this context, the combination of nanofilms with microneedles would be well suited for skin interfacing. The limited penetration depth of nanoneedles (a few microns) would be insufficient to reach tissue below the stratum corneum, but may still be appropriate for wound healing, where the stratum corneum is compromised. Similarly, transfection or delivery of cargoes using coated nanoneedles could be performed directly on the surface of small tumors or any other tissues, as long as profound penetration is not required. All these applications would benefit from a clinical perspective by transitioning toward flexible and biodegradable high-aspect ratio nanostructures, as these would interface non-planar tissues more appropriately and allows for implantation into tissues without the need for resection surgeries. Regardless, the fabrication method of nanofilms is highly versatile and hence can be easily combined with a wide variety of delivery systems and medical devices for the delivery of therapeutic cargoes.</p>" ]
[ "<title>Results and Discussion</title>", "<title>Plasmid DNA-Polyplex Characterization</title>", "<p id=\"P6\">Polyplexes loaded into the coating were made of a bio-reducible polycationic polymer, poly(cystamine bisacrylamide<italic toggle=\"yes\">-co-</italic>4-amino-1-butanol), hereafter referred to as pABOL, in which its backbone structure contains a disulfide bond that is broken down intracellularly after endocytosis via esterases, releasing the nucleic acid cargoes contained within the polyplex complex.<sup>[##UREF##7##15##,##UREF##8##16##]</sup> Polyplexes were loaded with a pCAG-GFP, a plasmid coding for green fluorescent protein (GFP), by mixing them in a mass ratio of 45:1 (##FIG##0##Figure 1a##), which corresponds to an N/P ratio of 38, which is the ratio of positively charged amino groups in pABOL to the number of negatively charged phosphate groups in the pDNA. This optimized mass ratio was chosen from previous studies using pABOL polyplexes for delivery of nucleic acids.<sup>[##REF##32267667##17##–##REF##24193511##19##]</sup> This method results in polyplexes of 104 ± 3.6 nm in average size, with good polydispersity (PDI: 0.135); and a surface zeta potential of +35.1 ± 6.7 mV (##FIG##0##Figure 1b,c##), making them positively charged, which facilitates electrostatic deposition and loading onto the coating.</p>", "<title>Polyplex-Polysaccharide Coated Nanoneedle Characterization</title>", "<p id=\"P7\">An optimal surface charge of the silicon nanoneedles is a key requirement to allow LbL deposition of the first polycationic chitosan layer. The surface of silicon nanoneedles (50 nm tip, 4.5 μm height) was cleaned from any remaining impurities using isopropanol:acetone (1:1) followed by oxygen plasma treatment. X-ray photoelectron spectroscopy (XPS) revealed that the plasma treatment increased the proportion of silicon oxide (103 eV) over pure silicon (98–100 eV doublet) (##FIG##0##Figure 1d,e##). Silicon oxide is known to provide negatively charged oxide ions.<sup>[##UREF##26##59##,##REF##30900715##60##]</sup> The increase in surface oxidation is also further supported by an increase in oxygen (533 eV) (##FIG##0##Figure 1d,e##). Contact angle measurements showed that the hydrophilicity of the nanoneedle surface significantly increased after plasma treatment (##FIG##0##Figure 1f,g##), which corresponds with the increased SiO<sub>2</sub> seen under XPS.</p>", "<p id=\"P8\">After plasma treatment, nanoneedles were coated using a LbL procedure via cycles of self-assembled deposition of chitosan (polycation) and HA (polyanion), mediated by opposite electrostatic interactions. We used a core coating of 10 bilayers (10B) as a supporting surface for further polyplex loading, which has been shown to ensure a uniform coating with no gaps present.<sup>[##REF##27760399##39##,##UREF##15##40##]</sup> The first polyplex layer, made of net positively charged complexes, was deposited onto the last negatively charged HA layer of the core coating, and additional polyplex layers were loaded within an intermediate coating of 5 bilayers (5B) (##FIG##1##Figure 2a##). The presence and uniformity of the nanofilm were evaluated via confocal microscopy, using tetramethylrhodamine (TAMRA) to stain the coating (reacting to amino groups in chitosan) and DAPI to stain pDNA-polyplexes. Confocal images showed that the entire nanoneedle surface is coated and polyplexes are distributed in the coating (##FIG##1##Figure 2b##; ##SUPPL##0##Figure S1, Supporting Information##). Differences in fluorescence intensity and missing nanoneedles are most likely a consequence of needle fracture during handling or artifacts during nanoneedle fabrication. Another important note is that the architecture of nanoneedles is preserved after the coating procedure, corroborated via SEM imaging (##FIG##1##Figure 2c##), which is essential to study the specific effects of high-aspect ratio nanostructures on transfection efficiency. XPS has been used to provide a more exhaustive confirmation of the components present in the coating (##FIG##1##Figure 2d##). The predominant presence of C-O (286 eV) and O-C=O (288 eV) structures show the polysaccharide structures from hyaluronic acid and chitosan, while the specific presence of nitrogen peaks (400 and 402 eV) confirms the presence of chitosan. The presence of pABOL is confirmed explicitly by a peak in sulfur at 164 eV, but also an increased ratio in protonated nitrogen (<sup>+</sup> N-H) species at 402 eV due to the tertiary amine from the polymer. Due to the comparatively low amount of DNA in the polyplexes (1:45 pDNA:pABOL), further diluted into the coating, the phosphorus signal from pDNA is low but still detected. We have further evaluated the surface composition with increasing numbers of polyplex and seen that the presence of silicon species diminished until complete disappearance by 4 polyplex layers (##SUPPL##0##Figure S2, Supporting Information##). This indicates how the coating increases thickness with more layers until the x-ray beam is not able to penetrate deep enough to detect the underlying silicon substrate. At this particular point, the surface composition and atomic percentages seen under XPS resemble more accurately the real elemental proportion of species in the coating. Since atomic force microscopy (AFM) cannot readily be used on high-aspect ratio structures like nanoneedles, the surface roughness of the coating was assessed on flat silicon substrates. After coating, the surface roughness increased compared to the relatively smooth surface of pristine silicon (##FIG##1##Figure 2e##). It is possible to observe spheric-like shapes of ≈100 nm in diameter on the surface of polyplex-coated flat silicon surfaces, which may correspond to the polyplexes, which are distributed uniformly throughout the surface, and embedded within the coating. This increase in surface roughness translates into an increased surface area, expected to enhance the interactions at the interface between cells and nanoneedles and hence cell uptake of polyplexes.</p>", "<title>Release and Degradation of Polyplex-Polysaccharide Nanofilms</title>", "<p id=\"P9\">To study, quantify and optimize the loading and intracellular delivery of pDNA of the nanofilms as a function of polyplex layers, flat silicon substrates of known surface area (8 × 8 mm<sup>2</sup>) coated with polyplex nanofilms were assayed in a release buffer containing a physiological intracellular concentration of reduced glutathione (10 mM),<sup>[##REF##18158646##61##]</sup> and the pDNA was quantified from aliquots taken at different time points using a PicoGreen assay. Release kinetics analyses showed similar trends for most samples regardless of the number of polyplex layers. Nanofilms with 3P and 4P released ≈40% of their pDNA loaded within the first hour, reaching complete release by 6 h, as evidenced by a plateau in the release curves (##FIG##2##Figure 3a##). Nanofilms with 1P and 2P released ≈20%of their total content by 1 h, also reaching completion by 6 h. As expected, the amount of pDNA loaded in the coating increased with the number of polyplex layers, all of them on the nanogram scale (##FIG##2##Figure 3b##). While the growth of material deposition in LbL films has been described to occur exponentially via dipping methods,<sup>[##REF##15969601##62##,##UREF##27##63##]</sup> there was an unexpected and sudden increase in the amount of pDNA in the 5-polyplex layers sample, accompanied by a high variability. AFM assessments on these samples showed abnormal deposition and the presence of material aggregation in the nanofilms (##SUPPL##0##Figure S3, Supporting Information##), which are not present in 4-polyplex layer nanofilms. Increased washing times or changes in polymer concentration did not change these outcomes, suggesting they occur unavoidably. These anomalies are likely a major contributor to the aberrant loading and release kinetics, hence not considered for statistical analysis.</p>", "<p id=\"P10\">The detection of polyplexes and the clear differences in surface roughness between coated and non-coated silicon substrates, allow feasible tracking of degradation via AFM over time using the same experimental conditions used for release assays. The results are consistent with release assays. The degradation of polyplexes occurs mainly within the first hour, while the presence of coating material seems to remain in the substrate for at least 4 h, then entirely degraded at 6 h, with surface roughness quite comparable to pristine controls (##FIG##2##Figure 3c##).</p>", "<title>Transfection Efficiency of Polyplex-Polysaccharide Nanofilms</title>", "<p id=\"P11\">Transfection efficiency on COS-7 cells was studied as a function of the number of polyplex layers in the nanofilms to observe whether there is a correlation with the amount of released plasmid. The transfection efficiency, which is the percentage of GFP expression adjusted to cell viability, was evaluated 24-h post-transfection via fluorescence microscopy and flow cytometry. Results showed an increasing trend in transfection efficiency with the first four polyplex layers (##FIG##3##Figure 4a,b##), which correlated to an increasing amount of released pDNA. However, the 5-polyplex layers coated nanoneedles were significantly less effective in transfection than the 4-polyplex layers, even though they released more than twice the amount of pDNA. Also, 5-polyplex layers resulted in lower viability (##FIG##3##Figure 4c##). This is likely a consequence of the abnormal nanofilm deposition observed under AFM and aberrant release previously observed. Regardless, 4-polyplex layer coatings resulted in the highest transfection efficiency and were therefore used for all further studies. An optimal number of intermediate chitosanhyaluronic acid bilayers between polyplexes was also assessed to maximize transfection efficiency. We found that each polyplex layer, contained within five intermediate bilayers, resulted in the highest and most reproducible transfection levels (##SUPPL##0##Figure S4, Supporting Information##). No additional bilayers were assessed as this would make the procedure impractical, hence all studies have been done with five intermediate bilayers.</p>", "<p id=\"P12\">We investigated whether the HA-chitosan-polyplex nanofilm enhances plasmid delivery and transfection efficiency compared to polyplexes alone. The transfection efficiency of silicon substrates coated with 4-polyplex layer nanofilms was compared to polyplexes in suspension containing 49 ng of plasmid, equivalent amount of pDNA released on 4-polyplex nanofilms (##FIG##2##Figure 3b##), but also compared to polyplexes containing 200 ng of pDNA, which is equivalent to standard concentrations used with polyplexes (2 μg/500 μL). COS-7 cells were seeded on flat silicon to maintain consistent experimental conditions. Results with polyplexes in suspension containing 49 ng of plasmid resulted in minimal transfection efficiency, compared to 4-polyplex layer nanofilm substrates with a transfection efficiency of 59.3% (##FIG##3##Figure 4d##). Polyplexes containing 200 ng of plasmid increased transfection efficiency to 11.5%, but still significantly lower than 4-polyplex nanofilms. Transfection was also performed in standard culture conditions, with COS-7 cells already adhered to tissue culture plastic (TCP) wells, using polyplexes in suspension containing 2 μg of plasmid in 500 μL, which resulted in transfection efficiency of 41.3%. This shows that adhesion of cells before transfection is important to obtain higher transfection efficiency using polyplexes in suspension, while nanofilms do not present this limitation. Therefore, these results demonstrate consistently that HA-chitosan nanofilms enhance the transfection efficiency of polyplexes, using an amount of plasmid an order of magnitude lower than standard transfection with polyplexes in culture, but also most non-viral commercial vectors, including liposomes and nanoparticles. In fact, an order of magnitude higher in the amount of plasmid in the polyplexes (2 μg) was not enough to equal the transfection efficiency obtained with nanofilms. This matches previous findings, in which HA has been incorporated during polyplex formation and shown to enhance nucleic acid delivery.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> Studies to evaluate whether the specific presence of HA is required for an enhanced transfection have been performed and are discussed later.</p>", "<p id=\"P13\">Intracellular delivery of cargoes by high-aspect ratio structures occurs at the cell-material interface. This highly localized delivery is a consequence of cargoes being physisorbed to the surface and not readily released to the surrounding environment.<sup>[##UREF##9##20##–##REF##30091365##23##]</sup> On the other hand, delivery and release of molecules in LbL coatings rely on the degradation properties of the polymers forming the coating and its architecture. For instance, the release of protein cargoes triggered by enzymatic action in the host tissue has been shown to affect the biological response of cells ≈50 μm from the tissue material interface.<sup>[##REF##27760399##39##]</sup> Alternatively, LbL films containing intracellular nucleic acid cargoes have been shown to depend more on cell uptake and delivery only occurs in cells directly interfacing the surface of the coated substrates.<sup>[##UREF##18##45##–##UREF##19##48##]</sup> To assess whether polyplexes remain within the nanofilms for highly localized surface-mediated transfection or diffuse away from the surface during transfection, we have used two contiguous coated silicon substrates: one loaded with pCAG-GFP (coding for GFP); and the second one containing pCAG-RFP, coding for red fluorescent protein (RFP), placed on top of a PDMS sheet to ensure tight adhesion within a tissue culture well-plate, as shown in ##FIG##3##Figure 4e##. Transfection was done by adding a single-cell suspension to cover both substrates simultaneously, allowing for possible polyplex diffusion, and incubated together for 4 h. After 24 h post-transfection, results showed COS-7 cells expressing either GFP or RFP on their corresponding sides of the coated substrates, with a clear separation of transfected cells in the contiguous edge of the substrates (##FIG##3##Figure 4f##). Therefore, polyplexes remained confined to each side of the substrate. No apparent diffusion or release of polyplexes was observed, confirming a surface-mediated mechanism of transfection, which means that cells or tissues must interface the coating for transfection to occur. The immobilization and possible concentration of polyplexes at the surface of the material may contribute to the observed enhanced transfection efficiency, with an order of magnitude lower in the amount of plasmid compared to polyplexes in suspension. This also demonstrates the potential of nanofilms for spatial patterning of transfection and generation of gradients in multicellular systems and tissue regeneration, respectively.</p>", "<title>Influence of High-Aspect Ratio Nanostructures on Transfection Efficiency</title>", "<p id=\"P14\">Considering that an optimal tissue or cell interface with the coated substrate is essential for an efficient transfection of genes, the surface area would be an important parameter for both the coating and high-aspect ratio nanostructures. Hence we have studied transfection efficiency with nanostructures of increasing surface area and several architectures. In increasing order of relative surface area, we compared transfection efficiency of COS-7 cells on flat silicon, short solid nanoneedles (SnN, height 3 μm), medium solid nanoneedles (MnN, height 4.5 μm), tall solid nanoneedles (TnN, height 7 μm), porous nanoneedles (PnN, height 5.5 μm) and nanowires (nW, height 400 nm). SEM was used to demonstrate that the architecture of these high-aspect ratio nanostructures was preserved after the coating procedure, ensuring that the differences in transfection efficiency are attributed to the nanostructures and not to other artifacts caused by the coating. As shown in ##FIG##4##Figure 5a##, the architecture of all solid nanoneedles was preserved. The overall shape of porous nanoneedles was mainly preserved, however, some features in the nanoneedles seemed to be too small to be preserved and were coated during the procedure. The dimensions of the nanowires were small, but the coating was able to maintain the overall architecture of their features, with the presence of some aggregates.</p>", "<p id=\"P15\">In this study, transfection efficiency was complemented with phalloidin staining to assess differences in cell morphology in response to each nanostructure. Results revealed similar transfection efficiencies for flat silicon, small nN, medium nN, and porous nN, while tall nanoneedles and nanowires show a lower average in transfection efficiency and inconsistent results with higher variance between repeats (##FIG##4##Figure 5b,c##). Considering these findings and their corresponding cell morphological staining, these inconsistencies in transfection for both TnN and nW substrates seem to be associated with poor interfacing area between cells and nanostructures. As previously mentioned, an optimal cell-surface interface is essential for transfection, but it seems that an increased surface area in the substrate does not necessarily translate to a larger area of cell contact. Instead, in this case, the architecture of high-aspect ratio structures seems to be more important, including height, spacing, or porosity. For subsequent experiments, we used medium nanoneedles, which have the lowest variability in transfection efficiency of all solid nanoneedles and better preserved high-aspect ratio structure after coating than porous needles. Medium nanoneedles have also been chosen over flat substrates as we have seen evidence that nanoneedles aid tissue delivery in a number of different contexts,<sup>[##REF##34426708##21##,##REF##25822693##33##,##REF##30430139##64##,##REF##25858596##65##]</sup> but also in our ex vivo studies, as it will be shown in a later section.</p>", "<title>Role of Polyanion Structure on Enhanced Transfection Efficiency</title>", "<p id=\"P16\">As previously mentioned, it has been recently hypothesized that HA enhances nucleic acid delivery.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> The proposed mechanisms have linked this enhancement to (1) the chemical structure of HA – modulating the electrostatic interactions in the polyplex via hydrogen bonding,<sup>[##UREF##21##51##,##UREF##22##53##]</sup> and (2) CD44-mediated cell uptake of the complex via interactions with HA.<sup>[##REF##34329781##52##–##UREF##23##54##]</sup> We first studied the role of the chemical structure of HA on transfection efficiency, comparing coatings made of different polyanions in combination with chitosan. The presence of all polyelectrolytes and pDNA-polyplexes in these coatings was confirmed successfully via XPS (##SUPPL##0##Figure S5, Supporting Information##).</p>", "<p id=\"P17\">HA is composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units, and it is the only non-sulfated member of the GAG family that plays important roles in the extracellular matrix and regulation of cellular responses.<sup>[##REF##30960626##66##–##UREF##28##68##]</sup> Therefore, transfection efficiency was tested in other members of the same family such as chondroitin-4-sulfate (CS), a GAG with low sulfation degree, in which constituent disaccharide units are D-glucuronic acid and N-acetyl-D-galactosamine.<sup>[##UREF##28##68##,##REF##31887904##69##]</sup> Similarly to HA, CS has also been shown to bind CD44.<sup>[##REF##11222505##70##,##REF##9891022##71##]</sup> Heparin (Hep), a GAG with a high sulfation degree, has also been tested. This is composed of sulfated uronic acid and sulfated D-glucosamine.<sup>[##UREF##28##68##,##REF##32255159##72##]</sup> Hep has been shown to bind an exon v3 variant of CD44 to regulate the activity of specific growth factors.<sup>[##REF##9891022##71##,##UREF##29##73##]</sup> The chemical structures of all three GAGs and all studied polyanions are shown in ##FIG##5##Figure 6a##. Transfection with both sulfated GAGs resulted in a drastically reduced efficiency (##FIG##5##Figure 6b##), which indicates that sulfation in the polysaccharide structure is detrimental for transfection, as discussed below. Alginate (Alg) was then chosen due to similar polysaccharide chemical structure and charge density to HA, but formed by D-mannuronate and L-guluronate sugars.<sup>[##REF##22125349##74##]</sup> Results revealed higher transfection efficiency than HA (75.3% versus 64.8%), demonstrating that enhanced transfection is not specific to HA (##FIG##5##Figure 6b##). The role of the polysaccharide backbone in transfection efficiency from both HA and Alg has been assessed by testing coatings with polyglutamic acid (pGlu), a polyaminoacid with the same carboxylate anion but no polysaccharide backbone. Interestingly, results showed poor transfection (8%), suggesting that a polysaccharide backbone in the polyanion would be essential to facilitate transfection.</p>", "<p id=\"P18\">We looked further into the detrimental effect of sulfation on transfection efficiency and evaluated whether the coating is affecting the release of pDNA, as a consequence of the high anionic charge of sulfonates in the polyanion. Release assays with both chondroitin sulfate (CS) and Hep showed lower released amounts of pDNA released, while similar release kinetics were observed (##FIG##5##Figure 6d,e##). No linear dependence between the degree of sulfation and transfection efficiency was observed. These results show that sulfation in the polyanion structure affects the release of pDNA and hence transfection efficiency. Supporting evidence indicates that sulfated polysaccharides disrupt the electrostatic interactions within polyplexes and liposomes. For example, buffers containing dextran sulfate or Hep have been used to unpack nucleic acid cargoes to quantify release.<sup>[##UREF##24##55##,##UREF##25##58##,##REF##21993237##75##,##UREF##30##76##]</sup> It is then possible that part of the pDNA is released from the polyplexes during the coating procedure. However, in other systems, polyplexes coated with CS have been shown to enhance transfection efficiency,<sup>[##UREF##31##77##,##UREF##32##78##]</sup> which suggests that these effects may also be dependent on the architecture of the delivery system, polymer composition, and concentration. However, these differences in release amount do not account for the dramatic decrease in transfection efficiency observed, with 4.4% for CS and 1% for Hep (##FIG##5##Figure 6B##).</p>", "<p id=\"P19\">Polyglutamic acid promotes poor transfection compared to both HA and Alg, in which the most evident structural difference is the presence of a polysaccharide backbone. The abundant hydroxyl groups in the polysaccharide backbone serve as both hydrogen bond acceptors and donors, while pGlu predominantly has hydrogen bond acceptors and a hydrophobic segment. Previous studies using polysaccharides and other synthetic polymers for particle decoration have claimed that hydrogen bonding modifies the strength and density of electrostatic interactions between the nucleic acids and polycations, facilitating the release of cargoes from the polyplex.<sup>[##UREF##5##12##,##UREF##21##51##,##UREF##22##53##]</sup> Alternatively, the higher charge density of polyglutamic acid could disrupt polyplexes as a consequence of the shorter spacing among amino acid units in polyglutamic acid compared to the sugar units in the polysaccharide backbone of both HA and Alg. To test this hypothesis, the transfection efficiency was assessed using gamma polyglutamic acid (γ-pGlu), in which glutamic acid is polymerized in the <italic toggle=\"yes\">γ</italic> position, increasing the distance between carboxylate anions from three to five atoms which decreases the charge density to similar levels to polysaccharides: Alg with five atoms and HA with 10 atoms (##FIG##5##Figure 6a##). The results showed that the transfection efficiency using <italic toggle=\"yes\">γ</italic>-pGlu is low, with similar efficiency to pGlu (##FIG##5##Figure 6b##), suggesting that the enhancement in transfection efficiency observed in polysaccharides is not dictated by charge density in the polyanion. The poor performance of <italic toggle=\"yes\">γ</italic>-pGlu on delivery and transfection efficiency seems to be specific to the nanofilm-polyplex system, considering that <italic toggle=\"yes\">γ</italic>-pGlu has shown to improve cell-uptake and transfection efficiency when incorporated in the fabrication of polyplexes.<sup>[##REF##19232715##79##–##REF##20864162##83##]</sup></p>", "<title>Role of CD44-Mediated Cell Uptake on Enhanced Transfection Efficiency</title>", "<p id=\"P20\">To test the hypothesis of CD44-mediated cell uptake, we have evaluated the transfection efficiency in cell types with and without distinct expression of CD44 (##FIG##6##Figure 7a##). We have quantified the levels of CD44 expression using antibody labeling and flow cytometry analyses (##FIG##6##Figure 7b##). COS-7 cells, a fibroblast cell line from monkey kidney,<sup>[##REF##6260373##84##]</sup> present high expression levels of CD44 (96%). HEK-293, an epithelial cell line from human kidney embryo<sup>[##REF##11967234##85##]</sup> exhibits almost no expression of CD44 (1%). C2C12, a mouse myoblast cell line,<sup>[##REF##8023908##86##]</sup> showed a relatively fair expression of CD44 (37.5%). Human mesenchymal stem cells (MSCs), primary cells derived from bone marrow, as expected by definition,<sup>[##UREF##35##87##]</sup> exhibited a high (95.8%) expression of CD44. Transfection results have shown that the high transfection efficiency of COS-7 is correlated to the high expression of CD44 (##FIG##6##Figure 7c##). However, HEK-293T showed a similar high transfection efficiency to COS-7 cells, regardless of the minimal expression of CD44. C2C12 cells exhibited lower transfection efficiency than both COS-7 and HEK-293T cells. Surprisingly, MSCs showed marginal transfection efficiency (3%), which should have been significantly higher due to the highest expression of CD44 if CD44-mediated uptake were indeed the predominant mechanism at play.</p>", "<p id=\"P21\">Despite the fact that the transfection efficiency does not seem to comply with the CD44-mediated transfection hypothesis given the uncorrelated levels of CD44 expression in most of these cell types, it could be argued that HEK-293T cells could still efficiently uptake polyplexes via conventional endocytosis, or that MSCs are widely known to be inherently difficult to transfect.<sup>[##REF##15277699##88##,##REF##33034446##89##]</sup> For further confirmation, transfection efficiency was assessed in COS-7 cells after blocking the CD44 receptor using an antibody, and compared to normal transfection. This has been done in nanoneedles coated with either HA or Alg, both with high transfection efficiency and polysaccharide-based chemical structures (##FIG##5##Figure 6b##). Blocking the CD44 receptor in COS-7 cells did not disrupt transfection efficiency. In both cases, results did not show decreased transfection efficiency after blocking CD44 receptors in COS-7 cells and unexpectedly, transfection efficiency in nanofilms containing Alg was higher after blocking (##FIG##6##Figure 7e,f##). Therefore, the results in this study consistently confirm that facilitated transfection of HA and Alg does not predominantly occur via CD44-mediated cell uptake. Instead, it relies on the polysaccharide physicochemical properties such as hydrogen bonding to facilitate intracellular delivery of the nucleic cargo.</p>", "<p id=\"P22\">Despite producing some results at odds with the previously-proposed CD44-mediated cell uptake mechanism, our results do confirm that HA facilitates transfection.<sup>[##REF##26996815##47##,##UREF##21##51##–##UREF##25##58##]</sup> We also demonstrate that transfection is not only facilitated by HA but also by additional polysaccharide-based polyanions such as Alg. We also have to consider experimental differences with prior studies where HA has likely been integrated into the bulk and surface during the formation of polyplexes, liposomes, and particles; in contrast, our procedure would likely integrate HA onto the surface of the polyplex. Discrepancies may also arise from the molecular weight of hyaluronic acid used, cell types studied and CD44 antibodies used for blocking.</p>", "<title>Surface-Mediated Transfection on an Ex Vivo Cardiac Slice Model</title>", "<p id=\"P23\">We evaluated the efficacy of the polyplex coated nanoneedles to transfect tissues on an ex vivo cardiac slice platform. Due to the architecture of nanoneedles and the surface-mediated delivery of the coating, transfection would occur in the outermost layer of cells in the tissue, in which in vivo imaging techniques and histological methods are limited. Cardiac slices, on the other hand, are an ideal ex vivo platform to study surface-mediated transfection on tissues, with many imaging techniques and quantification methods available.<sup>[##REF##30671746##90##,##REF##29189769##91##]</sup> These slices were prepared from left ventricles of rats in 300 μm sections, an optimal thickness to allow nutrient and oxygen supply for prolonged survival.<sup>[##REF##29189769##91##–##REF##31868875##93##]</sup> Polyplex coated nanoneedles or controls were interfaced for 1 h on top of the cardiac slices, fixed to a holder inside of a custom chamber filled with sufficient culture media to be in contact with the bottom side of the slice, allowing the nanoneedle substrate to adhere to the tissue before filling the chamber completely with media (##FIG##7##Figure 8a##). Similar to our in vitro studies, slices were evaluated after 24 h of incubation, during which time the coated nanoneedles stayed in contact with the tissue. The tissue viability was assessed using a Live/ Dead staining. The functionality of the slices after transfection was evaluated via contractility trace analysis from force transducer measurements, then fixed for staining.</p>", "<p id=\"P24\">To assess tissue transfection in the slice, a co-immunolabeling was performed using antibodies against GFP (green, transfected cells), cardiac troponin (cTNT, red, cardiomyocyte marker), vimentin (white, a stromal cell marker to label fibroblast and endothelial cells) and DAPI (blue) as counterstaining. Image analysis was performed to quantify the expression of GFP, as percentage of the total area in the imaged field. After 24 h of transfection, fibroblasts and endothelial cells showed to be transfected, as evidenced by high colocalization of GFP expression and vimentin, mainly localized between cardiomyocyte bundles and in the lining of blood vessels (##FIG##7##Figure 8b-iv,v##). Only a few discrete transfected cardiomyocytes were observed. No transfection was observed in controls groups and coated (no polyplex) nanoneedle groups (##FIG##7##Figure 8b-i,ii##). This selective transfection might be explained by the intrinsic physiological roles of these cells within the cardiac tissue, resulting in distinct cell uptake activity. For example, previous studies have shown that cardiomyocytes exhibit lower cell uptake of extracellular vesicles (EVs) compared to endothelial cells and fibroblasts,<sup>[##REF##27806113##94##,##REF##32969445##95##]</sup> which was hypothesized to be related to the increased ability of endothelial cells and fibroblasts to receive signals from cardiomyocytes via EVs, especially under injury, process that remains to be elucidated.<sup>[##REF##32969445##95##–##REF##30410918##97##]</sup> The observed differences in transfection among groups were correlated with quantification of GFP expression—nanoneedles with 4P-polyplex nanofilms showed 2.62% area of GFP per field, while nanofilm controls and no chip controls showed an area of 0.31% and 0.22%, respectively (##FIG##7##Figure 8c##). These results are in agreement with levels of transfection observed in COS-7 (fibroblasts), HEK-293 (epithelial cells), and C2C12 (myoblasts or muscle cells) in vitro (##FIG##6##Figure 7##), while noting that a direct comparison among these cell lines with cells in the tissue is not intended due to their metabolic and physiological differences. These results also support the highly localized surface-mediated mechanism of delivery, as transfection was only observed on the top side of the cardiac slice, where the tissue was interfacing the polyplex coated nanoneedles. In contrast, limited transfection and a lower area of GFP expression were observed on the contralateral surface (##FIG##7##Figure 8b-vi,c##). In our in vitro experiments, medium nanoneedles were selected over flat nanofilm substrates as hypothesized the penetration of nanoneedles into the tissue would increase surface interaction and transfection efficiency compared to flat. Cardiac slices were transfected with 4-polyplex layer flat nanofilms and results showed that the expression of GFP within the tissue was considerably lower than nanoneedles (##FIG##7##Figure 8b-iii,d##). Therefore, polyplex coated nanoneedles are capable of transfecting tissues with an exquisite level of localization, also ensuring a proper interface with tissues for facilitated transfection.</p>", "<p id=\"P25\">Cell viability assessments showed preserved viability of the cardiac tissue, with no differences in the expression of calcein and number of dead cells among slices treated with polyplex coated needles, coated nanoneedles (no polyplex), and nonanoneedle controls (##SUPPL##0##Figure S6, Supporting Information##). Similarly, force transducer measurements of contractility in the cardiac slices were performed to demonstrate that transfection did not alter the functionality of the tissue. Trace analysis of cardiac contractility showed preserved function of cardiac slices, with no differences in contracting force (##FIG##7##Figure 8e##), time to peak (##FIG##7##Figure 8f##), and time to decay (##FIG##7##Figure 8g,h##) observed among groups. Therefore, transfection via nanoneedles coated with polyplex-nanofilms preserves both the viability and functionality of the tissue.</p>", "<p id=\"P26\">In clinical settings, our surface-mediated transfection system is then advantageous in accessible tissues, where highly localized transfection and confined penetration depth is sought. For example, the epicardium is the outermost layer covering the heart, harboring a population of progenitor cells capable of regeneration in the heart, mainly dormant after birth, which makes the epicardium a highly attractive target for transfection to reactivate gene pathways that stimulate epithelial-to-mesenchymal transition.<sup>[##REF##26907357##98##,##REF##35328640##99##]</sup> Given the highly confined layer architecture of the epicardium, a surface-mediated transfection via the polyplex-nanofilm system would hence be highly suitable for localized and efficient transfection. Transdermal delivery via nanoneedle systems may pose limitations, compared to their microscale counterparts. This route has always been an attractive and suitable target for microneedle-mediated delivery, in which many cargoes (e.g., nucleic acids, proteins, and drugs) have been delivered for therapeutic applications that include melanoma, wound healing, as well as sustained systemic delivery of drugs.<sup>[##REF##32575392##100##–##REF##30348022##102##]</sup> In this context, the combination of nanofilms with microneedles would be well suited for skin interfacing. The limited penetration depth of nanoneedles (a few microns) would be insufficient to reach tissue below the stratum corneum, but may still be appropriate for wound healing, where the stratum corneum is compromised. Similarly, transfection or delivery of cargoes using coated nanoneedles could be performed directly on the surface of small tumors or any other tissues, as long as profound penetration is not required. All these applications would benefit from a clinical perspective by transitioning toward flexible and biodegradable high-aspect ratio nanostructures, as these would interface non-planar tissues more appropriately and allows for implantation into tissues without the need for resection surgeries. Regardless, the fabrication method of nanofilms is highly versatile and hence can be easily combined with a wide variety of delivery systems and medical devices for the delivery of therapeutic cargoes.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"P27\">The polyplex-polysaccharide coating based on LbL assembly of nanofilms is a suitable technology to provide uniform nanometerscale coatings that preserve the architecture of nanoneedles and other high-aspect ratio nanostructures. The multilayered nature of the coating provides fine control over release kinetics and amount of pDNA loaded, which was directly correlated to transfection efficiency, with maximum efficiency at 4-polyplex layers in this particular case. A proper interface between cells and nanostructures is essential for the surface-mediated delivery of the coating, as evidenced by the highly localized delivery of polyplexes with different cargoes. High-aspect ratio nanostructures of increasing surface area were not necessarily correlated to increased cell contact, but nanostructures with poor contact with cells resulted in inconsistent transfection efficiency. While in vitro, high-aspect ratio nanostructures and flat substrates with similar cell contact exhibited comparable transfection efficiency, nanoneedles coated with polyplex nanofilms showed significantly higher transfection efficiency in tissues, compared to nanofilms in flat substrates, which is likely due to increased penetration into the tissue.</p>", "<p id=\"P28\">The presence of polysaccharides in the coating enhanced transfection efficiency compared to polyplexes alone, with an amount of plasmid an order of magnitude lower than standard culture transfection. Our results show that enhanced delivery is not restricted to HA as claimed in previous studies but is also seen in polysaccharide-based polyanions such as Alg. However, the use of sulfated anionic polysaccharides is detrimental, as they disrupt polyplexes and nucleic acid release. Moreover, the predominant mechanism leading to this enhancement does not occur via CD44-mediated cell uptake. Instead, the essential role of polysaccharides facilitating delivery and transfection seems to involve hydrogen bonding regulation of the electrostatic interactions with nucleic acids within the polyplexes. In ex vivo cardiac slices, transfection is observed mainly in fibroblast and endothelial cells, while cardiomyocytes do not seem to be consistently transfected, which is in agreement with in vitro studies. Transfection was predominantly observed on the side of the slice interfacing polyplex-coated nanoneedles. Viability and functional assessments show that tissue viability and function after nanoneedle transfection remain unchanged. Therefore, gene transfection in tissues via nanoneedles coated with polyplex nanofilms is biocompatible, efficient, and highly localized. This is potentially an attractive approach for clinical settings where surgical procedures enable direct tissue-nanoneedle interfacing. While further testing and trials are required, the system has potential as an off-the-shelf type device that can be maintained dry-frozen before use. Beyond nanoneedles and high-aspect ratio nanostructures, the use of this coating technology in combination with microneedles and other devices opens an entire spectrum of different clinical approaches for nucleic acid delivery with precise spatial control.</p>" ]
[ "<p id=\"P1\">Present address: Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China</p>", "<p id=\"P2\">Non-viral vectors represent versatile and immunologically safer alternatives for nucleic acid delivery. Nanoneedles and high-aspect ratio nanostructures are unconventional but interesting delivery systems, in which delivery is mediated by surface interactions. Herein, nanoneedles are synergistically combined with polysaccharide-polyplex nanofilms and enhanced transfection efficiency is observed, compared to polyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations are assessed and it is found that transfection efficiency is enhanced when using polysaccharide-based polyanions, rather than being only specific for hyaluronic acid, as suggested in earlier studies. Moreover, results show that enhanced transfection is not mediated by interactions with the CD44 receptor, previously hypothesized as a major mechanism mediating enhancement via hyaluronate. In cardiac tissue, nanoneedles are shown to increase the transfection efficiency of nanofilms compared to flat substrates; while in vitro, high transfection efficiencies are observed in nanostructures where cells present large interfacing areas with the substrate. The results of this study demonstrate that surface-mediated transfection using this system is efficient and safe, requiring amounts of nucleic acid with an order of magnitude lower than standard culture transfection. These findings expand the spectrum of possible polyelectrolyte combinations that can be used for the development of suitable non-viral vectors for exploration in further clinical trials.</p>" ]
[ "<title>Experimental Section</title>", "<title>Materials</title>", "<p id=\"P29\">HA, chondroitin sulfate A, Hep, chitosan, Alg, and pGlu were purchased from Sigma Aldrich, UK. HPLC-grade water, isopropanol, and acetone were purchased from VWR, Germany. γ-pGlu was purchased from Carbosynth Limited, UK. Cell culture materials, anti-human/monkey CD44, anti-vimentin antibody, phalloidin, secondary antibodies, and DAPI were purchased from Thermo Fisher Scientific, UK. Anti-mouse CD44 was purchased from BD Biosciences. pCAG-GFP and pCAG-dsRFP were obtained from Addgene (MA, USA) and developed by Cepko C, et al.<sup>[##REF##14603031##103##]</sup> Fluc-pcDNA3 was obtained from Addgene (MA, USA) and developed by Safran M, et al.<sup>[##REF##16373502##104##]</sup> pABOL was obtained in-house and synthesized as previously described.<sup>[##UREF##7##15##,##UREF##8##16##]</sup> P-type doped silicon wafers with 0.01 Ω cm resistivity were obtained from University Wafers, USA. Anti-GFP and anti-cardiac troponin T were obtained from Abcam, UK.</p>", "<title>Fabrication of High-Aspect Ratio Nanostructures</title>", "<p id=\"P30\">Silicon nanoneedle and nanowire patterns were generated using reactive ion etching (RIE) and photolithography as previously reported.<sup>[##REF##25822693##33##,##REF##32330008##105##,##REF##34100486##106##]</sup> Briefly, a 1200 Å layer of low-stress silicon nitride was deposited using low-pressure chemical vapor deposition (Scottish Microelectronic Centre, The University of Edinburgh, UK). Dot arrays with varying diameters and spacings were transferred to a hard mask via photolithography, using a MA6 mask aligner (Suss Microtech, Germany), NR9-250P photoresist, and RD6 developer (Futurrex, USA). RIE was done on silicon wafers using 50 sccm of CF<sub>3</sub> gas, 5 sccm of O<sub>2</sub> gas, 55 mTorr of pressure, and 140 W of power for 150 s. The dot-patterned wafer was mounted on a 6 inch-diameter carrier wafer using a Crystalbond 555 adhesive stick for DRIE using a deep reactive ion etcher (Surface Technology Systems, UK). Each DRIE cycle consisted of i) 130 sccm of SF<sub>6</sub> gas and 6 sccm of O<sub>2</sub> gas with a process pressure of 15 mTorr and power of 800 W for an 8 s etch phase and ii) 85 sccm of C<sub>4</sub>F<sub>8</sub> gas with a process pressure of 14 mTorr and power of 600 W for a deposition phase of 6.5 s. To produce structures of 3–7 μm height, between 30 and 45 cycles were conducted. To fabricate porous nanoneedles, the patterned substrate was cleaned in 10% HF, then coated with Ag using 1 mM AgNO<sub>3</sub> in 10% HF for 2 min and washed in water and isopropanol. Porosity was obtained using metal-assisted chemical etching (MACE) in 10% HF, 122 mM H<sub>2</sub>O<sub>2</sub> for 8 min 30 s. Sharpening of the structures was done via RIE using SF<sub>6</sub> plasma for 3 min 45 s at a pressure of 100 mTorr, with gas flux of 20 sccm and forward plasma bias power of 200 W. To fabricate nanowires, the native silicon oxide layer was removed using 2.83 M HF, then immediately immersed into a 2.83 M HF and 0.02 M AgNO<sub>3</sub> for an electroless deposition of silver during 1 min. The reaction was stopped with sequential washes of water, isopropanol, and N<sub>2</sub> drying. MACE was done with 2.83 M HF and 0.081 M H<sub>2</sub>O<sub>2</sub> for 2 min and then stopped. Residual silver was removed by immersing the substrates into a solution of gold etchant (Sigma Aldrich, USA), for 10 min, then washed sequentially as described above. The processed wafers were then diced into 8 × 8 mm squares for further use.</p>", "<title>Polyplex Fabrication and Characterization</title>", "<p id=\"P31\">pCAG-GFP plasmid and pABOL were mixed in a mass ratio of 1:45 in 20 mM HEPES, 5% w/v D-glucose, pH 7.4, ratio optimized in previous studies.<sup>[##REF##32267667##17##–##REF##24193511##19##]</sup> For each polyplex layer, 2 μg of pCAG-GFP was diluted in 15 μL of buffer and 90 μg of pABOL were diluted in 55 μL of buffer, then mixed and vortexed for 30 s. This procedure was also used to fabricate polyplexes containing either pCAG-dsRFP or Fluc-pcDNA3. Polyplex size and zeta potential were assessed via dynamic light scattering using a Zetasizer Nano ZSZEN3600 (Malvern Instruments, Worcestershire, UK). To do so, polyplexes in triplicate were diluted to 1 mL with HPLC-grade water, measured three times each at 25 °C.</p>", "<title>Fabrication of Self-Assembled Polyplex-Polysaccharide Nanofilms</title>", "<p id=\"P32\">To provide a more hydrophilic and negatively charged surface, patterned silicon substrates were treated with oxygen plasma, using oxygen gas at 1 mBar steady-state pressure, 100 W for 5 min in a PlasmaPrep5 instrument (Gala Instrumente GmBH). An automated layer by layer coating was done using an in-house customized 3D printer (GeeTech M2 Creator 2, China). The LbL coating was done by dipping the patterned substrates in 2 mg mL<sup>−1</sup> of chitosan in 0.5% acetic acid for 10 min and three washes in HPLC-grade water, 1 min each. This was followed by dipping the substrates into a 1 mg mL<sup>−1</sup> polyanion solution in water (HA, CS, Hep, Alg, pGlu, or <italic toggle=\"yes\">γ</italic>-pGlu) for 10 min and three washes in HPLC-grade water, 1 min each. This cycle was repeated ten times to obtain a 10-bilayer core coating. To deposit a monolayer of DNA polyplexes onto the coated nanopatterns, 70 μL of a freshly prepared solution of polyplexes was placed on top of the substrate and incubated for 1 h at room temperature (RT), followed by three washes in HPLC-grade water. Additional chitosan and polyanion monolayers were deposited for a total of 5 bilayers (including polyplex), before adding the following pDNA polyplex layer. This was performed in cycles to obtain nanopatterned substrates coated with 1 to 5 polyplex layers. Core coated substrates were stored at 4 °C, while substrates containing DNA polyplexes were stored at –20 °C before use.</p>", "<title>Characterization of Polyplex-Polysaccharide Nanofilms</title>", "<p id=\"P33\">The presence and uniformity of the coating were observed via confocal microscopy, using TAMRA to stain the amino groups in the coating and DAPI to stain the DNA polyplexes. The presence of each polymer (elemental coating surface composition) in the coating was assessed via XPS on a Thermo-Fisher K Alpha XPS system (Waltham, MA, USA). To acquire the overall elemental composition, a survey of 2 scans was performed with a constant analyzer energy of 200 eV, dwell time of 25 ms, 0.5 eV step size, and an X-ray spot size of 400 μm. Single element spectra were obtained using a constant analyzer energy of 20 eV, dwell time of 50 ms, 0.1 eV step size, an X-ray spot size of 400 μm, 10 scans for silicon/ carbon/oxygen, and 20 scans for nitrogen/sulfur. Carbon peak was used as reference, with a value of 284 eV for adventitious carbon. Spectra data were analyzed using Avantage software V5.9925, (Thermo Scientific, Waltham, MA, USA). The preservation of the patterned nanostructures after coating was corroborated via scanning electron microscopy (SEM). To do so, samples were mounted and sputtered with a 10 nm layer of chromium (Q150, Quorum) and imaged using a LEO Gemini 1525 FEGSEM (Zeiss, Germany) with an accelerating voltage of 5 keV. Surface roughness and presence of polyplexes in the coating were assessed via AFM, using an Agilent 5500 AFM system (Agilent Technologies, USA), equipped with silicon nitride cantilevers (MikroMasch AFM Tips, Germany) in intermittent contact mode. Gwyddion V2.49 software was utilized for data processing.</p>", "<title>Degradation and Release Studies</title>", "<p id=\"P34\">These assays used 1 to 5 polyplex-coated flat silicon substrates of 8 × 8 mm, immersed into 500 μL of a PBS pH 7.4 buffer containing 10 mM of reduced glutathione at 37 °C. Degradation of polyplexes (4P layers) and the coating was assessed through changes in surface topography and porosity via AFM, using an Agilent 5500 (Agilent Technologies, USA). Topographical images were recorded in dry flat silicon substrates every 1 h using silicon nitride cantilevers (MikroMasch AFM Tips, Germany) in intermittent contact mode. Gwyddion V2.49 software was utilized for data processing. For release assays, aliquots were taken every hour and replaced with fresh glutathione buffered solution. Aliquots were stored at −20 °C until quantification. Released pCAG-GFP in these aliquots was quantified using PicoGreen assay, following kit instructions.</p>", "<title>Cell Transfection</title>", "<p id=\"P35\">COS-7 and HEK-293T were cultured in DMEM High Glucose (4.5 g L<sup>−1</sup>) +Glutamax medium supplemented with 10% v/v FBS and 1% v/v Penicillin-Streptomycin (PS), at 37 °C and 5% v/v CO<sub>2</sub>. C2C12 were cultured in DMEM High Glucose (4.5 g L<sup>−1</sup>) +Glutamax medium supplemented with 10% FBS and 1% v/v PS, at 37 °C and 5% CO<sub>2</sub>. Mesenchymal stem cells (MSCs) were cultured in MesenPRO RS Medium with 2% v/v Mesen-Pro Growth Supplement, 1% w/v L-glutamine and 1% v/v PS, at 37 °C and 5% CO<sub>2</sub>.</p>", "<p id=\"P36\">Cells in plates with 80–90% confluency were used for surface-mediated transfection, detached using 0.25% w/v Trypsin-EDTA, followed by two washes in Opti-MEM medium with 1% v/v Penicillin-Streptomycin. Cells were counted and resuspended in a concentration of 6 × 10<sup>5</sup> cells mL<sup>−1</sup> in Opti-MEM medium + 1% v/v PS. pCAG-GFP polyplex coated substrates of 8 × 8 mm were placed inside of 24-well plates and 3 × 10<sup>4</sup> cells (50 μL) were placed on the top of the substrates, covering the whole coated surface of the patterned nanostructures. Cells were incubated for 4 h at 37 °C and 5% CO<sub>2</sub> to allow cell adhesion and transfection, and then 500 μL of the standard medium was added to fill the well. After 24 h of incubation at 37 °C and 5% CO<sub>2</sub>, GFP<sup>+</sup> cells were imaged under fluorescence microscopy or detached for quantification via flow cytometry. Comparisons with polyplexes alone were done by adding polyplexes (containing 49 ng and 200 ng of pCAG-GFP, maintaining same mass ratio) during cell seeding on top of silicon substrates to keep consistency, while a standard polyplex transfection was done on a 48-well TCP plate, using polyplexes (2 μg of pCAG-GFP) suspended in 500 μL of Opti-MEM medium + 1% v/v PS and added to COS-7 cells seeded 24 h in advance.</p>", "<p id=\"P37\">Fluorescent imaging was done in an Olympus IX71 microscope (Olympus Life Sciences, UK), cells were washed three times with PBS, fixed in 4% PFA (w/v) for 15 min on ice, and permeabilized with 0.5% (v/v) Triton-X100 for 10 min at RT, then washed three times with PBS. The cytoskeleton of cells (actin) was stained with rhodamine-labeled phalloidin (1:2000) in PBS for 1 h at RT, followed by three PBS washes. Nuclei counterstaining was done with DAPI (1:1000) in PBS, 15 min at RT before imaging. Images were analyzed with ImageJ V1.51 (NIH, USA). For flow cytometry analyses, cells in the substrates were washed with PBS and detached with 0.25% v/v Trypsin-EDTA. Cells were resuspended in 250 μL of PBS buffer containing 1 mM EDTA, 25 mM HEPES, and 1% FBS (flow cytometry buffer), then 15 μL of To-Pro-3 Iodide (ThermoFisher, UK) were added before runs to evaluate cell viability. Each test was done with 10<sup>4</sup> events, using 488/530 nm laser for GFP and 640/670 nm laser for To-Pro-3 Iodide. Data were acquired on a LSR Fortessa Cell Analyser (BD BioSciences, UK) and analyzed using Flowjo V7.0.</p>", "<title>Surface-Mediated Transfection Assessment</title>", "<p id=\"P38\">Localized surface-mediated transfection was assessed using two 8 × 8 mm flat or nanoneedle (4.5 μm height) substrates: one loaded with 4-polyplex layers of pCAG-GFP (green) and another one coated with 4-polyplex layers of pCAG-RFP (red), using the same coating procedure described previously. Both nanoneedle substrates were put next to each other on top of a PDMS sheet that keeps the substrates fixed inside of a 6-well plate. Transfection was done as described above, adding 6 × 10<sup>4</sup> COS-7 cells (100 μL) on the top of the two substrates, hence an 8 × 16 mm area. After 4 h of incubation, substrates were individually put in 24-well plates filled with DMEM + Glutamax medium, 10% v/v FBS, 1% v/v PS, and incubated at 37 °C and 5% CO<sub>2</sub> for 24 h. Transfected cells with either GFP or RFP were imaged on the whole surface of both chips using tiling and stitching on an Axio Observer.Z1 inverted widefield microscope (Zeiss, Germany).</p>", "<title>CD44 Expression and Blocking</title>", "<p id=\"P39\">A CD44 monoclonal antibody (Thermo Scientific, UK) was used to determine the expression of CD44 in COS-7, HEK-293, C2C12, and MSCs, as well as block the receptor during transfection. Cells were detached and washed in flow cytometry buffer. 1 × 10<sup>5</sup> cells in 300 μL of buffer were incubated with primary CD44 antibody (1:50) at 4 °C for 30 min. Cells were centrifuged at 300 g and washed with buffer twice, followed by incubation with secondary Alexa Fluor 555 antibody (1:250) at 4 °C for 30 min. Cells were washed three times with flow cytometry buffer before measurements. Similarly, cells were blocked with a solution of CD44 antibody (1:50) at 4 °C for 30 min. Control groups were incubated in PBS without antibody under the same conditions. After blocking, cells were washed twice with Opti-MEM and 3 × 10<sup>4</sup> cells were seeded on coated nanoneedle substrates as previously described.</p>", "<title>Coated Nanoneedle-Based Transfection on Cardiac Slices</title>", "<p id=\"P40\">Cardiac slices were prepared from Sprague–Dawley male rats (300–350 g) as previously described.<sup>[##REF##29189769##91##]</sup> All animal procedures were performed under license by the UK Home Office, in agreement with the United Kingdom Animals (Scientific Procedures) Act 1986 and guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU). The heart and the surrounding tissues of the animal were excised and immersed in cold Tyrode’s solution (pH 7.4), containing 1000 IU mL<sup>−1</sup> of Hep. The left ventricle was isolated from the rest of the heart and extra-heart tissues, then opened with an incision down the interventricular septum and flattened via incisions to the papillary muscles. The tissue was mounted on a 2.5 cm<sup>2</sup> specimen-holder coated in 4% agarose, epicardial side down, using surgical glue (Histoacryl Octyl Micro, Braun Surgical S.A, Catalonia). The specimen holder was placed in a vibratome bath filled with cold Tyrode’s solution, bubbled with filtered 100% O<sub>2</sub>. Using a high precision vibratome (7000 amz-2, Campden Instruments) with a ceramic blade, the tissue was sliced (300 μm thickness) longitudinal to the fiber orientation from the endocardium down. About four to six slices were obtained per heart. Once the slice was obtained, fiber alignment was visualized under light microscopy to cut an aligned squared slice. Custom-made plastic 3D printed rectangular holders were attached perpendicular to the fibers along the width of the slice using surgical glue. The slice was placed on custom-made stainless-steel stretchers and stretched at physiological load (17.5% stretch, equivalent to 2.2 μm sarcomere length). All length measurements were taken with calipers. Stretched slices were then placed in groups of four in custom-made culture chambers and super fused with 60 mL of oxygenated media. The culture media (Medium-199) with Earl’s salts was prepared by adding 0.1% v/v ITS (insulin-transferrin-selenium) +3% v/v PS. Hormones (4 nM adrenaline, 4 nM noradrenaline, 100 nM dexamethasone, and 2.15 nM triiodothyronine (T3)) with the addition of 20 μg mL<sup>−1</sup> ascorbic acid were also added to the media to maintain the physiological properties of the slices during culture.</p>", "<p id=\"P41\">Polyplex coated nanoneedle chips substrates and controls (8 × 8 mm) were interfaced with the upper side of the cardiac slice for 1 h, while the media was filled to the level of the slice, to prevent substrates from floating and allow tissue adhesion. After 1 h of incubation at 37 °C and 5% CO<sub>2</sub>, the chamber was filled with media and cultured for 24 h under electrical field stimulation, using carbon electrodes at 1 Hz, 10 ms pulse width, and 15 V. After transfection, slices were mounted on a force transducer system (Harvard Apparatus) to assess viability and function, under field stimulation at 1 Hz, 10 ms pulse and 15 V. Cardiac muscle contraction traces generated (milli newtons versus time) were analyzed using pClamp V11 software (Molecular Devices, CA) to obtain normalized contraction force (mN mm<sup>−2</sup>, normalized to slice area), time to peak (s), time to decay 50% and 90% (s). Slices were then fixed with 4% w/v PFA for 15 min and rinsed in PBS for immunolabeling. Slices were blocked and permeabilized for 3 h RT with a PBS buffer containing 5% w/v BSA (Bovine Serum Albumin), 10% v/v FBS, 5% v/v Horse Serum and 1.5% v/v Triton X-100. Blocking buffer was removed and replaced with 1:10 blocking buffer:PBS containing the following primary antibodies: rabbit anti-GFP (1:1000), mouse anti-cTNT (1:500), and chicken anti-vimentin (1:3000). Slices were incubated overnight with primary antibodies at 4 °C. After incubation, slices were washed three times with PBS (30 min each) and put in a PBS buffer containing 1% w/v BSA + 0.3% v/v Triton X-100 and the following secondary antibodies: donkey anti-rabbit Alexa Fluor 488 (1:2000), donkey anti-mouse Alexa Fluor 594 (1:500), goat anti-chicken Alexa Fluor 647 (1:2000). Secondary antibodies were incubated at RT for 2 h, then washed three times with PBS (30 min each). A DAPI solution in PBS (1:1000) was added and incubated for 15 min at RT and washed three times (2 min each). For viability testing, a Live/dead staining was done following kit instructions, using nanoneedles coated with polyplexes containing Fluc-pcDNA3, to eliminate the presence of green fluorescence due to transfection. Slices were imaged using a Zeiss Axiovert Confocal Microscope (Zeiss, Germany). Image analysis to quantify area percentage and dead cells was performed in ImageJ V1.51 (NIH, USA).</p>", "<title>Statistical Analyses</title>", "<p id=\"P42\">Comparisons of means were performed using a two-tailed <italic toggle=\"yes\">t</italic>-test or one-way analysis of variance (ANOVA), using at least <italic toggle=\"yes\">p</italic> &lt; 0.05 as statistical significance criteria, followed by Post Hoc tests to perform multiple comparisons. These tests were performed on GraphPad Prism V6 (California, USA). Shapiro–Wilk was used to test normality, using SPSS V28 (IBM, USA).</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>D.H. acknowledges the funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska- Curie grant agreement No 839111. D.H. and M.M.S. acknowledge the support from the Imperial College British Heart Foundation Centre for Cardiac Regeneration (RM/17/1/33377) and the BHF Centre for Regenerative Medicine (RM/21/290002). L.B. and M.M.S. acknowledge the support from the BHF Centre for Research Excellence (RE/13/4/30184) and the Kusuma Trust. H.S. was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1A6A3A03007397) and the European Commission (H2020-MSCA-IF-2017, 797311). M.B. and M.M.S. acknowledge the support from the Rosetrees Trust. L.O. and M.M.S. acknowledge the financial support from the Engineering and Physical Sciences Research Council (EPSRC) Programme Grant “Engineering Growth Factor Microenvironments – A New Therapeutic Paradigm for Regenerative Medicine” (EP/P001114/1). C.L.G. was supported by a StratNeuro postdoctoral grant. S.G.H. acknowledges the support from a Cancer Research UK award (C71717/A30035). S.G.H. and M.M.S. acknowledge the support from the ERC Seventh Framework Programme Consolidator Grant “Naturale CG” (616417) and the Wellcome Trust Senior Investigator Award (098411/Z/12/Z). M.M.S. acknowledges the UK Regenerative Medicine Platform grant “Acellular/Smart Materials – 3D Architecture” (MR/R015651/1). The authors acknowledge the Sir Alexander Fleming Building Flow Cytometry Facility, the Facility for Imaging by Light Microscopy (FILM), and the Harvey Flower Facility at Imperial College London. The authors acknowledge the technical support of Steve Etienne, Lorella Rossi, and Vijayalakshmi Krishnan at the London Centre for Nanotechnology (LCN). The authors acknowledge Dr. Akemi Nogiwa Valdez for manuscript and data management support. Raw data is available on reasonable request from <email>[email protected]</email>.</p>", "<title>Data Availability Statement</title>", "<p id=\"P43\">The data that support the findings of this study are available from the corresponding author upon reasonable request.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>a) Schematics of polyplex formation between pDNA and pABOL in a mass ratio of 1:45. b) Particle diameter of polyplexes via dynamic light scattering (DLS) and c) zeta potential of polyplexes. Three samples shown (<italic toggle=\"yes\">N</italic> = 3) correspond to the mean of three measurements. d) Silicon and oxygen spectra using X-ray photoelectron spectroscopy (XPS) on the surface of pristine silicon nanoneedles and O<sub>2</sub> plasma-treated silicon nanoneedles. e) XPS surface composition of pristine silicon and O<sub>2</sub> plasma-treated silicon nanoneedles. Results are the mean of three samples (<italic toggle=\"yes\">N</italic> = 3) ± SEM. f) Representative images of contact angle measurements on i) pristine silicon nanoneedles and ii) O<sub>2</sub> plasma-treated silicon nanoneedles. Scale bars represent 2 mm. g) Contact angle values as the mean of three replicates (<italic toggle=\"yes\">N</italic> = 3) ± SEM. Statistical significance difference as (****) <italic toggle=\"yes\">p</italic> &lt; 0.0001, using a two-tailed <italic toggle=\"yes\">t</italic>-test.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>a) Polysaccharide-polyplex nanofilm coating procedure of silicon nanoneedles and high-aspect ratio nanostructures. b) Confocal microscopy representative image of silicon nanoneedles coated with hyaluronate-chitosan-pDNA polyplex nanofilms. The nanofilm was stained with TAMRA (red) and pDNA-polyplexes were stained with DAPI (blue). Scale bars represent 20 μm. Separate images for each channel can be found in ##SUPPL##0##Figure S1, Supporting Information##. c) Scanning electron microscopy (SEM) images of i) pristine nanoneedles and ii) polyplex-nanofilm coated nanoneedles. Scale bars represent 10 μm. d) Carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon spectra obtained via XPS from pristine, coated (no polyplex) and polyplex-coated (4 layers) nanoneedles. e) Surface topography characterization via atomic force microscopy (AFM) for pristine, coated (no polyplex) and polyplex coated (4 layers) flat silicon substrates. Scale bars represent 2 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>a) Cumulative release of pCAG-GFP plasmid DNA (nanograms) versus time (hours) and b) total amount of pCAG-GFP released at 24 h from 8 × 8 mm silicon substrates coated with hyaluronate-chitosan nanofilms containing 1 to 5 layers of polyplexes (1P to 5P) in PBS buffer pH 7.4 containing 10 mM of reduced glutathione at 37 °C. Points and bars represent the mean of three replicates (<italic toggle=\"yes\">N</italic> = 3) ± SEM. Statistical significance as (*) <italic toggle=\"yes\">p</italic> &lt; 0.05 and (**) <italic toggle=\"yes\">p</italic> &lt; 0.01, using one-way ANOVA with Sidak’s test to compare 1P to 4P. (ns) non-significant difference. 5P was not considered in the analysis. Linear trend post-test with <italic toggle=\"yes\">p</italic> &lt; 0.0001. c) Nanofilm degradation over time (hours) in function of surface topography via AFM assessments. Scale bars represent 2 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>a) Fluorescent microscopy images of COS-7 cells transfected and expressing pCAG-GFP plasmid (green) using silicon substrates coated with nanofilms containing 1 to 5 polyplex layers (1P to 5P). DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm. b) Quantification of transfection efficiency and c) viability via flow cytometry analyses of COS-7 cells transfected via silicon substrates coated with nanofilms containing 1 to 5 polyplex layers (1P to 5P) after 24 h incubation. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3). Statistical significance as (*) <italic toggle=\"yes\">p</italic> &lt; 0.05 and (**) <italic toggle=\"yes\">p</italic> &lt;0.01, using one-way ANOVA and Sidak’s test. (#) statistical significance with <italic toggle=\"yes\">p</italic> &lt; 0.05, using one-way ANOVA with Sidak’s test to compare 5P to all other groups. (ns) non-significant difference. d) Transfection efficiency of COS-7 cells seeded on silicon substrates coated with 4-polyplex layers versus polyplexes in suspension containing 49 and 200 ng of pDNA on top of silicon substrates; and a standard culture transfection of polyplexes using 2 μg of pCAG-GFP in TCP. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3). Statistical significance as (****) <italic toggle=\"yes\">p</italic> &lt; 0.0001, using one-way ANOVA and Sidak’s test. e) Schematics of the procedure to assess surface-mediated transfection using two contiguous silicon substrates on COS-7 cells, one containing pCAG-GFP (green, left) and the other pCAG-RFP (red, right). f) Tiled and stitched fluorescent microscopy images from the whole surface of two contiguous flat and nanoneedle substrates, expressing GFP (green) or RFP (red). Scale bars represent 2 mm.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p>a) SEM images of pristine high-aspect ratio nanostructures (top) and nanostructures coated with 4-polyplex nanofilms (bottom). SnN, MnN, TnN, and PnN correspond to small, medium, tall, and porous nanoneedles, respectively. nW corresponds to nanowires. Scale bars represent 10μm. b) Fluorescent microscopy images of COS-7 cells transfected on distinct high-aspect ratio nanostructures coated with 4-polyplex nanofilms. GFP<sup>+</sup> cells are shown in green, while the cytoskeleton of cells has been stained with phalloidin (red) and cell nuclei counterstained with DAPI (blue). Scale bars represent 100 μm. c) Quantification of transfection efficiency and d) viability via flow cytometry analyses of COS-7 cells transfected with different coated high-aspect ratio nanostructures. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3). Statistical significance was found using one-way ANOVA (<italic toggle=\"yes\">p</italic> &lt; 0.05), but significances between specific means were not found with Sidak’s post-test.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p>a) Chemical structures of the polyanions assessed in the study, with their anionic groups highlighted in a different color. b) Transfection efficiency and c) viability of COS-7 cells transfected using nanoneedles with polyplex nanofilms containing distinct polyanions. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3–4). Statistical significance as (**) <italic toggle=\"yes\">p</italic> &lt; 0.01 and (****) <italic toggle=\"yes\">p</italic> &lt; 0.0001, using one-way ANOVA and Sidak’s test. (#) <italic toggle=\"yes\">p</italic> &lt; 0.05 compared to alginate. d) Cumulative release of pCAG-GFP plasmid DNA (nanograms) versus time (hours) and e) total amount of pCAG-GFP released at 24 h, from silicon substrates coated with 4-polyplex nanofilms containing hyaluronate (HA), chondroitin sulfate (CS) and heparin (Hep); in PBS buffer pH 7.4 containing 10 mM of reduced glutathione at 37 °C. Points and bars represent the mean of three replicates (<italic toggle=\"yes\">N</italic> = 3) ± SEM. Statistical significance as (***) <italic toggle=\"yes\">p</italic> &lt; 0.001, using one-way ANOVA and Sidak’s test.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p>a) Images of cell types studied and b) their level of CD44 expression receptor. Scale bars represent 200 μm. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3). c) Transfection efficiency and d) viability of distinct cell types transfected with nanoneedles coated with 4-polyplex nanofilms. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3). Statistical significance as (**) <italic toggle=\"yes\">p</italic> &lt; 0.01, (***) <italic toggle=\"yes\">p</italic> &lt; 0.001 and (****) <italic toggle=\"yes\">p</italic> &lt; 0.0001, using one-way ANOVA and Sidak’s test. e) Transfection efficiency of COS-7 cells before and after blocking of receptor CD44, using hyaluronate-chitosan-polyplex coated nanoneedles and f) alginate-chitosan-polyplex coated nanoneedles. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 3–8). Statistical significance as (*) <italic toggle=\"yes\">p</italic> &lt; 0.05, using a two-tailed <italic toggle=\"yes\">t</italic>-test. (ns) non-statistical difference.</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p>a) Polyplex-coated nanoneedle substrate interfacing cardiac slice on custom-made holders. Scale bar represents 8 mm. b) Confocal microscopy images of cardiac slices at 24 h of transfection and immunolabeled with anti-GFP (green), anti-cTNT (red), anti-vimentin (white), and DAPI (blue). Representative images correspond to i) control no chip, ii) coated (no polyplex) nanoneedles, iii) polyplex-coated flat substrates, iv,v) polyplex-coated nanoneedles, and vi) the non-interfacing side of a slice with polyplex-coated nanoneedles. Bars represent 100 μm. Separate images showing green and blue channels are shown in ##SUPPL##0##Figure S7, Supporting Information##. The level of GFP expression in these groups was quantified via image analysis, c) as percentage of total area, d) while a comparison between GFP expression on nanoneedles and flat substrates coated with 4P-polyplex nanofilms was performed separately. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 4). Statistical significance as (*) <italic toggle=\"yes\">p</italic> &lt; 0.05, (**) <italic toggle=\"yes\">p</italic> &lt; 0.01 and (***) <italic toggle=\"yes\">p</italic> &lt; 0.001, using two-tailed <italic toggle=\"yes\">t</italic>-tests to compare two groups or one-way ANOVA with Sidak’s test for multiple groups. Traces from force transducer analyses showing e) contraction force in mN mm<sup>−2</sup>, f) time to peak, g) time to 50% decay, and h) time to 90% decay. Bars represent the mean ± SEM (<italic toggle=\"yes\">N</italic> = 5–7). Differences were not statistically significant: e) <italic toggle=\"yes\">p</italic> = 0.052, f) <italic toggle=\"yes\">p</italic> = 0.837, g) <italic toggle=\"yes\">p</italic> = 0.930, and h) <italic toggle=\"yes\">p</italic> = 0.684, using one-way ANOVA (<italic toggle=\"yes\">p</italic> &lt; 0.05).</p></caption></fig>" ]
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[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supplementary Information</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN2\" fn-type=\"COI-statement\"><p id=\"P44\">\n<bold>Conflict of Interest</bold>\n</p><p id=\"P45\">The authors declare no conflict of interest.</p></fn><fn id=\"FN3\" fn-type=\"con\"><p id=\"P46\">\n<bold>Author Contributions</bold>\n</p><p id=\"P47\">The manuscript was written through the contributions of all authors. All authors have approved to the final version of the manuscript.</p></fn></fn-group>" ]
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[{"label": ["[5]"], "person-group": ["\n"], "surname": ["Chen", "Huang", "Law", "Chu", "Chen", "Lo"], "given-names": ["CK", "PK", "WC", "CH", "NTL", "W"], "source": ["Int J Nanomed"], "year": ["2020"], "volume": ["15"], "fpage": ["2131"]}, {"label": ["[6]"], "person-group": ["\n"], "surname": ["Lostal\u00e9-Seijo", "Montenegro"], "given-names": ["I", "J"], "source": ["Nat Rev Chem"], "year": ["2018"], "volume": ["2"], "fpage": ["258"]}, {"label": ["[7]"], "person-group": ["\n"], "surname": ["Mohammadinejad", "Dehshahri", "Madamsetty", "Zahmatkeshan", "Tavakol", "Makvandi", "Khorsandi", "Pardakhty", "Ashrafizadeh", "Afshar"], "given-names": ["R", "A", "VS", "M", "S", "P", "D", "A", "M", "EG"], "source": ["J Controlled Release"], "year": ["2020"], "volume": ["325"], "fpage": ["249"]}, {"label": ["[8]"], "person-group": ["\n"], "surname": ["Xiang", "Tong", "Shi", "Fernandes", "Jin", "Dai", "Zhang"], "given-names": ["S", "H", "Q", "JC", "T", "K", "X"], "source": ["J Controlled Release"], "year": ["2012"], "volume": ["158"], "fpage": ["371"]}, {"label": ["[11]"], "person-group": ["\n"], "surname": ["Khalil", "Sato", "Harashima"], "given-names": ["IA", "Y", "H"], "source": ["Expert Opin Drug Delivery"], "year": ["2019"], "volume": ["16"], "fpage": ["1037"]}, {"label": ["[12]"], "person-group": ["\n"], "surname": ["Albuquerque", "Alavarse", "Carlan da Silva", "Zilse", "Barth", "Bellettini", "Giacomelli"], "given-names": ["LJC", "AC", "MC", "MS", "MT", "IC", "FC"], "source": ["Macromol Biosci"], "year": ["2018"], "volume": ["18"], "elocation-id": ["1700299"]}, {"label": ["[14]"], "person-group": ["\n"], "surname": ["Deng", "Yue", "Jin", "Chen", "Kung", "Lin", "Wu"], "given-names": ["R", "Y", "F", "Y", "H-F", "MC", "C"], "source": ["J Controlled Release"], "year": ["2009"], "volume": ["140"], "fpage": ["40"]}, {"label": ["[15]"], "person-group": ["\n"], "surname": ["Lin", "Zhong", "Lok", "Jiang", "Hennink", "Feijen", "Engbersen"], "given-names": ["C", "Z", "MC", "X", "WE", "JJ", "F"], "source": ["Bioconjugate Chem"], "year": ["2007"], "volume": ["18"], "fpage": ["138"]}, {"label": ["[16]"], "person-group": ["\n"], "surname": ["Gurnani", "Blakney", "Yeow", "Bouton", "Shattock", "Stevens", "Alexander"], "given-names": ["P", "AK", "J", "CR", "RJ", "MM", "C"], "source": ["Polym Chem"], "year": ["2020"], "volume": ["11"], "fpage": ["5861"]}, {"label": ["[20]"], "person-group": ["\n"], "surname": ["Higgins", "Becce", "Belessiotis-Richards", "Seong", "Sero", "Stevens"], "given-names": ["SG", "M", "A", "HJ", "E", "MM"], "source": ["Adv Mater"], "year": ["2020"], "volume": ["32"], "elocation-id": ["1903862"]}, {"label": ["[22]"], "person-group": ["\n"], "surname": ["He", "Hu", "Xu", "Li", "Zhao", "Xie"], "given-names": ["G", "N", "AM", "X", "Y", "X"], "source": ["Adv Funct Mater"], "year": ["2020"], "volume": ["30"], "elocation-id": ["1909890"]}, {"label": ["[26]"], "person-group": ["\n"], "surname": ["Gopal", "Chiappini", "Penders", "Leonardo", "Seong", "Rothery", "Korchev", "Shevchuk", "Stevens"], "given-names": ["S", "C", "J", "V", "H", "S", "Y", "A", "MM"], "source": ["Adv Mater"], "year": ["2019"], "volume": ["31"], "elocation-id": ["1806788"]}, {"label": ["[27]"], "person-group": ["\n"], "surname": ["Elnathan", "Delalat", "Brodoceanu", "Alhmoud", "Harding", "Buehler", "Nelson", "Isa", "Kraus", "Voelcker"], "given-names": ["R", "B", "D", "H", "FJ", "K", "A", "L", "T", "NH"], "source": ["Adv Funct Mater"], "year": ["2015"], "volume": ["25"], "fpage": ["7215"]}, {"label": ["[30]"], "person-group": ["\n"], "surname": ["Choi", "Lee", "Kim", "Gujrati", "Kim", "Lee", "Kim", "Kim", "Saw", "Jon"], "given-names": ["M", "SH", "WB", "V", "D", "J", "JI", "H", "PE", "S"], "source": ["Adv Healthcare Mater"], "year": ["2016"], "volume": ["5"], "fpage": ["101"]}, {"label": ["[31]"], "person-group": ["\n"], "surname": ["Yamagishi", "Matsumoto", "Kato", "Honda", "Morikawa", "Iwata", "Kobayashi", "Nakamura"], "given-names": ["A", "D", "Y", "Y", "M", "F", "T", "C"], "source": ["Appl Sci"], "year": ["2019"], "volume": ["9"], "fpage": ["965"]}, {"label": ["[40]"], "person-group": ["\n"], "surname": ["Hachim", "Iftikhar", "LoPresti", "Nolfi", "Ravichandar", "Skillen", "Brown"], "given-names": ["D", "A", "ST", "AL", "S", "C", "BN"], "source": ["J Controlled Release"], "year": ["2019"], "volume": ["305"], "fpage": ["65"]}, {"label": ["[42]"], "person-group": ["\n"], "surname": ["Mao", "Ma", "Zhou", "Gao", "Shen"], "given-names": ["Z", "L", "J", "C", "J"], "source": ["Bioconjugate Chem"], "year": ["2005"], "volume": ["16"], "fpage": ["1316"]}, {"label": ["[43]"], "person-group": ["\n"], "surname": ["Smith", "Riollano", "Leung", "Hammond"], "given-names": ["RC", "M", "A", "PT"], "source": ["Angew Chem, Int Ed"], "year": ["2009"], "volume": ["48"], "fpage": ["8974"]}, {"label": ["[45]"], "person-group": ["\n"], "surname": ["Jewell", "Zhang", "Fredin", "Lynn"], "given-names": ["CM", "J", "NJ", "DM"], "source": ["Controlled J Release"], "year": ["2005"], "volume": ["106"], "fpage": ["214"]}, {"label": ["[48]"], "person-group": ["\n"], "surname": ["Xie", "Ding", "Budry", "Mao"], "given-names": ["L", "X", "R", "G"], "source": ["Int J Nanomed"], "year": ["2018"], "volume": ["13"], "fpage": ["4943"]}, {"label": ["[50]"], "person-group": ["\n"], "surname": ["Correa", "Choi", "Shi", "Hammond"], "given-names": ["S", "KY", "A", "PT"], "source": ["Front Bioeng Biotechnol"], "year": ["2016"], "pub-id": ["10.3389/conf.FBIOE.2016.01.00555"]}, {"label": ["[51]"], "person-group": ["\n"], "surname": ["Oliveira", "Bitoque", "Silva"], "given-names": ["AV", "DB", "GA"], "source": ["J Nanomater"], "year": ["2014"], "volume": ["2014"], "elocation-id": ["246347"]}, {"label": ["[53]"], "person-group": ["\n"], "surname": ["Paidikondala", "Rangasami", "Nawale", "Casalini", "Perale", "Kadekar", "Mohanty", "Salminen", "Oommen", "Varghese"], "given-names": ["M", "VK", "GN", "T", "G", "S", "G", "T", "OP", "OP"], "source": ["Angew Chem, Int Ed"], "year": ["2019"], "volume": ["58"], "fpage": ["2815"]}, {"label": ["[54]"], "person-group": ["\n"], "surname": ["Sato", "Nakata", "Yang", "Torizuka", "Kishimoto", "Ishihara"], "given-names": ["T", "M", "Z", "Y", "S", "M"], "source": ["Gene J Med"], "year": ["2017"], "volume": ["19"], "fpage": ["e2968"]}, {"label": ["[55]"], "person-group": ["\n"], "surname": ["Aldawsari", "Dhaliwal", "Aljaeid", "Alhakamy", "Banjar", "Amiji"], "given-names": ["HM", "HK", "BM", "NA", "ZM", "MM"], "source": ["Mol Pharmaceutics"], "year": ["2019"], "volume": ["16"], "fpage": ["128"]}, {"label": ["[58]"], "person-group": ["\n"], "surname": ["Teng", "Wang", "Chen", "Huang"], "given-names": ["W", "Q", "Y", "H"], "source": ["Int J Nanomed"], "year": ["2014"], "volume": ["9"], "fpage": ["5013"]}, {"label": ["[59]"], "person-group": ["\n"], "surname": ["Fowkes", "Burgess"], "given-names": ["F", "T"], "source": ["Surf Sci"], "year": ["1969"], "volume": ["13"], "fpage": ["184"]}, {"label": ["[63]"], "person-group": ["\n"], "surname": ["Jiang", "Liu", "Luo", "Zhang", "Shao", "Shi"], "given-names": ["C", "X", "C", "Y", "L", "F"], "source": ["J Mater Chem A"], "year": ["2014"], "volume": ["2"], "elocation-id": ["14048"]}, {"label": ["[68]"], "person-group": ["\n"], "surname": ["Hachim", "Whittaker", "Kim", "Stevens"], "given-names": ["D", "TE", "H", "MM"], "source": ["J Controlled Release"], "year": ["2019"], "volume": ["313"], "fpage": ["131"]}, {"label": ["[73]"], "person-group": ["\n"], "surname": ["Bennett", "Jackson", "Simon", "Tanczos", "Peach", "Modrell", "Stamenkovic", "Plowman", "Aruffo"], "given-names": ["KL", "DG", "JC", "E", "R", "B", "I", "G", "A"], "source": ["Cell J Biol"], "year": ["1995"], "volume": ["128"], "fpage": ["687"]}, {"label": ["[76]"], "person-group": ["\n"], "surname": ["Danielsen", "Strand", "de Lange Davies", "Stokke"], "given-names": ["S", "S", "C", "BT"], "source": ["Biochim Biophy Acta s Gen Subj"], "year": ["2005"], "volume": ["1721"], "fpage": ["44"]}, {"label": ["[77]"], "person-group": ["\n"], "surname": ["Naik", "Sharma", "Nisakar", "Purohit", "Ganguli"], "given-names": ["RJ", "R", "D", "G", "M"], "source": ["Biochim Biophy Acta s Biomembr"], "year": ["2015"], "volume": ["1848"], "fpage": ["1053"]}, {"label": ["[78]"], "person-group": ["\n"], "surname": ["Uchida", "Itaka", "Chen", "Osada", "Miyata", "Ishii", "Harada-Shiba", "Kataoka"], "given-names": ["S", "K", "Q", "K", "K", "T", "M", "K"], "source": ["J Controlled Release"], "year": ["2011"], "volume": ["155"], "fpage": ["296"]}, {"label": ["[80]"], "person-group": ["\n"], "surname": ["Kurosaki", "Kitahara", "Kawakami", "Higuchi", "Yamaguchi", "Nakagawa", "Kodama", "Hamamoto", "Hashida", "Sasaki"], "given-names": ["T", "T", "S", "Y", "A", "H", "Y", "T", "M", "H"], "source": ["J Controlled Release"], "year": ["2010"], "volume": ["142"], "fpage": ["404"]}, {"label": ["[82]"], "person-group": ["\n"], "surname": ["Zhang", "Qiu", "Wu", "Liu", "Zhou", "Tang", "Liu", "Zhou", "Shen"], "given-names": ["Z", "N", "S", "X", "Z", "J", "Y", "R", "Y"], "source": ["Adv Mater"], "year": ["2021"], "volume": ["33"], "elocation-id": ["2102219"]}, {"label": ["[87]"], "person-group": ["\n"], "surname": ["Ramos", "S\u00e1nchez-Abarca", "Munti\u00f3n", "Preciado", "Puig", "L\u00f3pez-Ruano", "Hern\u00e1ndez-Hern\u00e1ndez", "Redondo", "Ortega", "Rodr\u00edguez"], "given-names": ["TL", "LI", "S", "S", "N", "G", "\u00c1", "A", "R", "C"], "source": ["Cell Commun Signaling"], "year": ["2016"], "volume": ["14"], "fpage": ["2"]}]
{ "acronym": [], "definition": [] }
106
CC BY
no
2024-01-13 00:12:46
Small. 2022 Sep 1; 18(36):e2202303
oa_package/6f/c1/PMC7615482.tar.gz
PMC7615494
33951318
[ "<title>Introduction</title>", "<p id=\"P2\">There is currently an unmet clinical need for medical devices that can help heal non-union bone fractures, which are damaged bones that cannot heal on their own or under conventional treatment. Metal implants can be used but are often too stiff, potentially causing bone loss due to stress shielding,<sup>[##UREF##0##1##]</sup> or not transmitting mechanical cues to osteogenic cells, or they become encapsulated by fibrous tissue, leading to micromotion.<sup>[##REF##23258381##2##]</sup> Bioactive ceramics (e.g., synthetic hydroxyapatite) and bioactive glasses (e.g., Bioglass) can bond directly to bone, and stimulate high quality bone growth,<sup>[##REF##10397902##3##]</sup> but they are stiff and brittle.<sup>[##REF##16102812##4##,##REF##21544222##5##]</sup> Inorganic–organic sol-gel hybrids are of great interest for bone repair and have shown potential to surpass bioactive glass scaffolds due to tailorable mechanical properties, such as the ability to withstand cyclic loading.<sup>[##UREF##1##6##,##UREF##2##7##]</sup> These properties stem from the nanoscale interlocking of inorganic and organic co-networks and covalent bonds between the co-networks.<sup>[##UREF##3##8##,##REF##22922331##9##]</sup> Inorganic–organic hybrids have been previously produced via the sol-gel process, using natural polymers functionalized with an organosilane coupling agent, such as glycidoxypropyl trimethoxysilane (GPTMS),<sup>[##REF##32261285##10##–##UREF##5##13##]</sup> to enable covalent bonding between the co-networks. However, controlling the chemical reaction between the coupling agent, for example, the epoxy ring of GPTMS to nucleophiles on the polymer chain, is difficult.<sup>[##UREF##6##14##]</sup> Natural polymers also come with the challenge of whether there is a reproducible source. Synthetic polymers are much more versatile for hybrid synthesis. Copolymers of methyl methacrylate (MMA) and 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) have shown excellent potential as an organic source, as TMSPMA can form bonds between the organic and silica co-networks,<sup>[##REF##26464180##15##,##REF##32261149##16##]</sup> and the copolymer-silica hybrids have shown biocompatibility in vivo<sup>[##REF##24001050##17##]</sup> and in vitro.<sup>[##REF##19304322##18##]</sup> Copolymers of poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA) with different architectures enabled synthesis of hybrids with tailorable mechanical properties, for example, star polymer architectures, which resemble flexible star gels,<sup>[##UREF##7##19##]</sup> within which the arms and crosslinking core can be modified independently.<sup>[##UREF##8##20##,##REF##27299693##21##]</sup> Until now, the poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA) hybrids have not been made into porous scaffolds suitable for bone repair. An ideal bone scaffold must have an open pore network with interconnections between the pores in excess of 100 μm to allow a passageway for vascularized bone ingrowth.<sup>[##REF##16102812##4##,##REF##10880079##22##]</sup> 3D printing is advantageous over foaming,<sup>[##UREF##4##12##]</sup> electro-spinning,<sup>[##UREF##9##23##]</sup> and freeze drying<sup>[##REF##32261285##10##]</sup> techniques, because the pores and their inter-connects can be precisely controlled.<sup>[##REF##33251199##24##]</sup> If 3D grid-like structure printing is possible, the channels can be wide open while the scaffold can have the compressive strength of bone.<sup>[##REF##21745606##25##]</sup> An advantage of using the sol-gel process is that the hybrid sol can be printed directly, in a layer-by-layer extrusion printing process.<sup>[##UREF##2##7##]</sup> Previously, 3D printed silica/poly-tetrahydrofuran/poly-<italic toggle=\"yes\">ε</italic>-caprolactone (SiO<sub>2</sub>- pTHF-PCL) hybrid scaffolds were found to provoke human bone marrow derived stem cells down a chondrogenic route, when pore channel sizes were ≈250 μm.<sup>[##REF##32100748##26##]</sup> They were also able to withstand cyclic loads, and even self-heal. However, control of the “ink” gelation and identifying a “printing window” (time within which the ink can be printed but also hold its own weight) are challenging.</p>", "<p id=\"P3\">Here, printability of poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-<italic toggle=\"yes\">star</italic>-SiO<sub>2</sub> hybrid formulations, developed in a previous study,<sup>[##UREF##8##20##]</sup> was investigated. The stars consisted of linear poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA) arms with a molar ratio of MMA<sub>100</sub>-TMSPMA<sub>10</sub> linked with an ethylene glycol dimethacrylate (EGDMA) core. The effect of different inorganic:organic ratios (in wt%); 50:50, 40:60, and 30:70, of the 3D printed hybrids on bone repair in vivo were examined.</p>" ]
[]
[ "<title>Results and Discussion</title>", "<title>Hybrid Confirmation</title>", "<p id=\"P4\">Poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-<italic toggle=\"yes\">star</italic> copolymer was synthesized through reversible addition-fragmentation chain-transfer (RAFT) polymerization technique with the arm-first approach, and then it was mixed with hydrolyzed silica network precursor (tetraethyl orthosilicate, TEOS) in three different inorganic:organic ratios of 50:50, 40:60, and 30:70. Hybrid scaffolds were 3D printed by direct ink writing and the compositions will be referred to as S50, S60, and S70 (the number corresponds to the organic content wt%, ##FIG##0##Figure 1A,B##). The pink coloration of the scaffolds was from dithiol bonds of the RAFT agent used. In the previous study, hybrid monolith cylinders were made successfully only when the inorganic content was 30 wt% or lower as higher inorganic content was susceptible to cracking. This was due to shrinkage during drying and capillary stresses causing fracture.<sup>[##REF##22922331##9##]</sup> Here, capillary stress was reduced through 3D printing, resulting in a higher surface area to volume ratio of the scaffold structure (thin struts rather than a bulk material).<sup>[##UREF##10##27##]</sup> The actual inorganic:organic ratios were confirmed by thermogravimetry (TGA, ##FIG##0##Figure 1C##). All the hybrid scaffolds showed thermal decomposition at 360°C, due to oxidation of the polymer.<sup>[##REF##32261149##16##,##UREF##8##20##,##UREF##11##28##]</sup> Residual mass remaining from S50, S60, and S70 were close to our targeted inorganic content of 50.5%, 42.1%, and 30.2% respectively. Fourier transform infrared spectroscopy (FTIR) was conducted to confirm the scaffolds’ molecular structure (##FIG##0##Figure 1D##). The FTIR spectra of the scaffolds were similar to those of monoliths produced previously.<sup>[##UREF##8##20##]</sup> The TGA and FTIR results confirmed that both polymer and silica network were present in the scaffolds.</p>", "<title>3D Printing Process and Scaffold Characterization</title>", "<p id=\"P5\">3D printing inorganic-organic hybrids with consistent quality was a challenging process.Height of the 3D printing syringes and their nozzles varied (± ≈1-2 mm), due to variation in supply from the manufacturer, therefore position of the ink syringe had to be adjusted every printing session. An ideal gap between the syringe nozzle to the printing platform was 0.2 mm. Due to the fluid to viscous (sol to gel) transition during the gelation process, there was a printing window, defined as the time period after mixing the polymer and the sol, when hybrid ink was viscous enough to retain its shape after printing but still fluid enough to be extruded from the nozzle.<sup>[##UREF##12##29##]</sup> Additionally, viscosity of the hybrid ink gradually increased during printing, due to covalent bonding formation between polymer to silica network and solvent evaporation (i.e., ethanol, THF, and trace of water). The printing window for all the compositions tested in this study was 1h, but decreased as organic content increased (i.e., S70 gelled faster than other compositions).</p>", "<p id=\"P6\">Scanning electron microscope (SEM) images of the horizontal and vertical cross-sections of the scaffolds are shown in ##FIG##1##Figure 2##. The actual inter-strut distance was narrower than the printed distance due to the shrinkage. The shrinkage resulted from the evaporation of solvents during drying stage as well as polymerization of the hybrid from the condensation reaction.<sup>[##UREF##5##13##]</sup> An ideal inter-strut distance was determined by printing S60 hybrids with different inter-strut dimensions. Printed scaffolds were optimal when printed with a pore size between 100 and 200 μm. A pore size of 500 μm, which is in the range of trabecular bone pore sizes, was produced with a nominal 1 mm inter-strut distance. However, as shown in ##SUPPL##1##Figure S1A,B##, Supporting Information, the struts were sagging between underlying struts and collapsing in the <italic toggle=\"yes\">z</italic> direction; resulting in poor interconnectivity. A nominal inter-strut distance of 0.7 mm also produced horizontally merged and collapsed porous structure, despite having vertical pore size of ≈300-500 μm (##SUPPL##1##Figure S1C,D##, Supporting Information). 3D printing with 0.5 mm inter-strut distance enabled fabrication of scaffolds with improved interconnected porous structure in both horizontal and vertical directions (##FIG##1##Figure 2##). Hence, hybrid scaffolds with different compositions were printed with 0.2 mm printing nozzle (strut diameter) and 0.5 mm inter-strut distance (pore size). After drying, the strut diameter shrank to ≈100 μm and pore size was between 100 and 200 μm (##TAB##0##Table 1##), meeting the hypothesized minimal requirements for vascularization.<sup>[##REF##16102812##4##,##REF##18373428##30##]</sup> The horizontal cross-section SEM images (##FIG##1##Figure 2A,B,C##) displayed vertical channels running through the scaffolds with ≈190 μm widths for S70 and S60 scaffolds. S50, on the other hand, had pore width of 180 μm, as scaffolds with the higher inorganic content were more susceptible to shrinkage. The vertical cross-section images (##FIG##1##Figure 2D,E,F##) also showed inter-connectivity of pores and bonding between the struts of each layer. However, lower pore sizes were observed compared to the horizontal cross-section. S70 had an average pore size of 111 μm, which is due to higher organic content, resulting in less stiff ink that compressed under its own weight while printing. S50 also showed merging of the layers producing average pore size of 121 μm; possibly caused by higher inorganic content inhibiting to gel. From the SEM images, S60 had the most defined porous structure. The pore interconnectivity and porosity of S60 was confirmed geometrically, using helium pycnometry to obtain the skeletal density. The percent porosity was 60.5 ± 1.1%, which is within the range of trabecular bone.<sup>[##UREF##13##31##]</sup> The qualitative pore inter-connectivity was confirmed through micro-CT scans. The reconstructed 3D image of the central region (##FIG##1##Figure 2G##) and volumetric rendering of S60 (##SUPPL##0##Video S1##, Supporting Information) also showed inter-connected porous structure.</p>", "<p id=\"P7\">Uniaxial compression tests were performed. An ideal bone substitute should have mechanical properties that are similar to that of bone.<sup>[##UREF##0##1##]</sup>The stress-strain curves (##FIG##1##Figure 2H##) indicate that all the scaffolds are within the range of trabecular bone, which is reported to have Young’s modulus values in the range of 50-500 MPa and compressive strength of 2-12 MPa (shaded region in ##FIG##1##Figure 2H##).<sup>[##UREF##0##1##,##REF##22311079##32##]</sup> The general trend of Young’s modulus and failure stress of the scaffolds increased as inorganic content of the hybrids increased, which agrees with previous studies on poly(MMA)-silica hybrids.<sup>[##UREF##14##33##]</sup> As shown in ##TAB##0##Table 1##, S50 had the highest failure stress of 26.3 MPa at a strain to failure of 8.9%. S70 had the lowest failure stress which was more than threefold lower than other compositions, while failure strain value was 7.4%. S60 had the most synergistic properties, that is, flexibility from polymer and strength from silica matrix, with the highest strain to failure of 11.5%, failure stress of 22.4 MPa and Young’s modulus of 247 MPa. Cyclic compressive loading tests were also performed on S60 to confirm ductility for 12 MPa compressive stress, which is at the upper end of the compressive strength range of trabecular bone. As ##FIG##1##Figure 2I## shows, the scaffold did not appear to fracture and the only noticeable hysteresis was observed during the first cycle, which is common in such tests, possibly from an uneven top/bottom surface of the scaffold. Cyclic deformations decreased after each cycle, which was less than total of 0.5% strain stretch.</p>", "<p id=\"P8\">The mechanical properties of the scaffolds not only meet those required of trabecular bone, but also surpassed previous 3D printed bone substitutes. In terms of previously printed hybrid scaffolds, hybrids with methacrylic copolymers have been 3D printed previously,<sup>[##UREF##10##27##]</sup> but their pore channel size was 2 mm and mechanical properties were only extrapolated from three point bending tests of bulk samples. Gelatin-SiO<sub>2</sub> hybrid with 25 wt% gelatin was 3D printed with pore channel size of ≈550 μm and low porosity (30%), perhaps due to high shrinkage.<sup>[##UREF##15##34##]</sup> Despite having higher inorganic content and denser porous structure, the scaffold had compressive strength of ≈5 MPa with failure strain of ≈5% (estimated from published stress/strain graphs<sup>[##UREF##15##34##]</sup>). SiO<sub>2</sub>-pTHF-PCL hybrids, with 75 wt% organic component, was printed with a pore channel size of 200 μm (42% porosity) and were highly elastic, with a 36% strain to failure at a compressive strength of 1.2 MPa, which was seen as ideal for cartilage regeneration but too flexible for bone repair.<sup>[##UREF##2##7##]</sup></p>", "<title>Cytotoxicity and Pre-Osteoblast Adherence Evaluations on the Hybrid Scaffolds</title>", "<p id=\"P9\">For an ideal scaffold for bone repair, the scaffold material must also be non-cytotoxic and promote osteogenic cell adherence.<sup>[##REF##15119934##35##]</sup> Cytotoxicity of the scaffolds were evaluated by ISO 10993-12.<sup>[##UREF##16##36##]</sup> MTT metabolic assay (##FIG##2##Figure 3A##) confirmed that all the scaffolds passed ISO standards for cytotoxicity, with viability of the cells exposed to extractables of the hybrid scaffolds at &gt;87% to that of the control media, which proved that most of the solvents were removed by drying. The MC3T3 pre-osteoblasts were able to adhere on the scaffolds within 24 h. As shown in ##FIG##2##Figure 3B,C,D##, major cytoskeletal constituents of intermediate filaments and microfilaments were observed along with DAPI staining of nuclei. Although methacrylic polymers are known to be hydrophobic, MC3T3 cells were able to attach on S70 which had the highest amount of polymer. This was expected since monolithic hybrids with the same composition have shown cell attachment previously.<sup>[##UREF##1##6##,##UREF##8##20##]</sup> Total DNA quantification of MC3T3 cells on each scaffold (Hoechst stain) (##FIG##2##Figure 3E##) revealed that the scaffolds had cell attachment rate of 59 ± 7%, 56 ± 8%, and 36 ± 7% for S50, S60, and S70, respectively (##TAB##0##Table 1##). The improvement in cell adherence as inorganic content increased was possibly due to increase in stiffness and improvement in hydrophilicity, due to surface silanol groups, which corroborates with a previous study on hybrid monoliths with a range of degree of hydrophilicity.<sup>[##UREF##1##6##]</sup></p>", "<title>S60 Hybrid Scaffold as a Bone Substitute</title>", "<p id=\"P10\">The osteogenic properties of S60 were evaluated through a calvarial defect model, since: printability of S60 was superior to other compositions; its material interface was pre-osteoblast friendly; and its mechanical properties were comparable to trabecular bone. A critical sized defect (diameter <italic toggle=\"yes\">(φ):</italic> 8 mm) rat calvarial model was used.<sup>[##REF##23018195##37##]</sup> Representative micro-computed tomography (<italic toggle=\"yes\">μ</italic>CT) images are shown in ##FIG##3##Figure 4##. The images include 3D reconstructions and 2D cross section slices through the center of the defects. Defects containing S60 implants were compared to the defects without scaffolds (control group). Reconstructed 3D <italic toggle=\"yes\">μCT</italic> images after 8 and 16 weeks post-surgery showed new bone formation in both the control group and defect model with implanted S60 (##FIG##3##Figure 4A,B##). At 8 weeks, new bone formation in the control group was small, and it started from edges of the host bone, while 2D slice across the center of the defect (coronal section) showed no bone ingrowth. More new bone tissue intruded into the porous structure of S60, and bone ingrowth along the interface of the scaffold-bone was confirmed. At 16 weeks, bone was present in both the control defect and in defect containing scaffold. The quantitative analysis of <italic toggle=\"yes\">μCT</italic> images indicated that the percentage of new bone volume relative to total defect volume (BV/TV) in the S60 group was 12.4%, which was lower than the control group of 19.6% (##FIG##3##Figure 4C##). This was likely due to new bone formation in the defect interface of bony borders in the control group while the scaffold took up space in the defect site. The defect with S60 implant displayed both peripheral and central distribution of new bone formation. Since the innate healing capacity that originates from the defect margin is known to be constant regardless of the defect size,<sup>[##REF##20827327##38##]</sup> osteogenic evaluation of the central area of a defect is an important factor in criticalsized bone defect models. Following a previous study on evaluating bone regeneration<sup>[##REF##21212786##39##]</sup> bone formation of the central area was evaluated by the BV to central volume, that is, <italic toggle=\"yes\">φ</italic> 5 mm within <italic toggle=\"yes\">φ</italic> 8 mm defect (##FIG##3##Figure 4D##). S60 was able to enhance bone formation compared to that of the control model in both 8 and 16 week post surgeries. The 3D porous structure with osteoblast friendly surface<sup>[##REF##32263492##40##]</sup> possibly allowed more suitable environment for bone formation.</p>", "<p id=\"P11\">Results from the <italic toggle=\"yes\">μCT</italic> analysis were further confirmed by histological assessment. ##FIG##4##Figure 5A,B## shows the histological images of the specimens at 8 and 16 weeks of post implantation. Hematoxylin and eosin (H &amp; E) and Masson’s trichrome (MT) staining were utilized to further evaluate the bone regeneration patterns within S60. The control group showed bone in-growth pattern only from the peripheral region, while S60 groups revealed bone regeneration from the peripheral region plus in situ bone islands formation in the center of defect. At 16 weeks, thin connective tissue formation was detected in control defects, whereas bone-like tissue formation was observed in the areas grafted with S60. Substantial soft connective tissue, stained as blue color in MT staining, was observed in defects of the control group. On the other hand, red colored staining, representing matured bone, was apparent in the central region of the defects after 16 weeks of S60 implantation. Based on the MT staining, mature bone tissue formation was quantified through measuring non-collagenous tissue region; area with red staining, of the central defect area <italic toggle=\"yes\">(φ</italic> 5 mm within <italic toggle=\"yes\">φ</italic> 8 mm defect) (##FIG##4##Figure 5C##). More matured bone was present for defects with S60 compared to the defects of control group at 8 and 16 weeks post implantation. These histology quantification results corroborated with the bone volume to central volume (BV/CV) <italic toggle=\"yes\">μCT</italic> results.</p>", "<p id=\"P12\">Angiogenesis and mature vessel formation within the calvarial defect site, with and without S60 scaffold implantation, were evaluated since angiogenesis and vascular tissue formation must be induced for bone regeneration. In order to confirm angiogenic activity and vessel maturation, histology sections were examined by double immunofluorescence staining of von Willebrand factor (vWF) and alpha smooth muscle actin <italic toggle=\"yes\">(a</italic>-SMA) (##FIG##5##Figure 6A##). As shown in ##FIG##5##Figure 6B##, vWF-positive cell density within a S60 implanted defect was threefold and twofold higher than that of the control group at 8 and 16 weeks post-surgery, respectively. This indicates that S60 scaffold enhanced new blood vessel formation and angiogenic activity. Additionally, <italic toggle=\"yes\">a</italic>-SMA positive vessel density for S60 group was eightfold and threefold higher than that of the control group at 8 and 16 weeks after implantation, respectively (##FIG##5##Figure 6C##). The maturation index; based on double staining results (##FIG##5##Figure 6D##), confirmed that implanted S60 scaffold enhanced vessel maturation. For the control group, vessel density (vWF and <italic toggle=\"yes\">a</italic>-SMA positive staining) and maturation index increased from 8 to 16 weeks post-surgery. On the other hand, S60 did not show significant differences between 8 and 16 weeks post implantation. The vessel density and maturation index values were higher than that of the control group at all the time points. Thus, the results imply that the S60 scaffold encourages angiogenesis and vessel maturation, starting from an early stage. This could be due to silica release from S60 scaffold, or the surface chemistry and 3D architecture. The degradation mechanism of sol-gel bioactive glasses is well established.<sup>[##REF##22922331##9##]</sup> Silica released from mesoporous silica microspheres was shown to up-regulate hypoxia inducing factor <italic toggle=\"yes\">1α</italic> (HIF-1<italic toggle=\"yes\">α</italic>) expression and stabilize the activation through down-regulating HIF-prolyl hydroxylase 2 in human umbilical vein endothelial cells culture. This process successively induced key angiogenic factors, such as basic fibroblast growth factor, vascular endothelial growth factor, and endothelial nitric oxide synthase,<sup>[##REF##27918936##41##]</sup> which could explain angiogenic characteristics of S60. Poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-SiO<sub>2</sub> hybrids have been shown to release silica. For example, a similar inorganic:organic composition to our S60 hybrid lost mass over a period of 100 days degradation in PBS.<sup>[##REF##24001050##17##]</sup> However, the Si concentrations reported were 10 to 25 μg mL<sup>-1</sup>, which is higher than that released by our scaffolds (4.8 ± 0.1 μg mL<sup>-1</sup> released over 16 weeks, ##SUPPL##1##Figure S2##, Supporting Information). As the Si release is small, the surface chemistry and pore architecture are also likely to contribute to the angiogenic effect.</p>", "<p id=\"P13\">To verify osteogenic differentiation and bone production as a result of S60 scaffold implantation, immunostaining of osteogenic markers, that is, collagen Type I and osteocalcin, were evaluated (##FIG##6##Figure 7A##). Collagen Type I is one of the main components of bone that gives strength by forming oriented layers, while osteocalcin acts as an essential linker between the organic and inorganic components in the bone matrix. Both markers were more highly expressed in defects containing S60 scaffolds compared to that of the control group, as shown in ##FIG##6##Figure 7B,C##, regardless of the post-implantation time points. The osteogenic markers confirmed that S60 enhanced bone regeneration, due to the release of silica species which stimulate collagen Type I and osteoblastic differentiation<sup>[##REF##12633784##42##]</sup>,or a combination of 3D architecture and surface chemistry of the scaffold.</p>", "<p id=\"P14\">Overall, S60 was biocompatible and able to promote new vascularized bone tissue formation within the pore channels with the support of its surface, which promoted cell adhesion. Bone growth was starting in the central part of the defect area, that is, inside S60, which was not contiguous to calvarial defect edges. The combination of S60 scaffold induced both blood vessel and bone growth.</p>", "<p id=\"P15\">Macrophage polarization in the defect site and S60 at 8 and 16 weeks post-surgery were assessed, since macrophages are known to be involved in the bone healing process.<sup>[##UREF##17##43##]</sup> Immunolabeling of CD68 (pan macrophage) and CD206 (M2 marker) was performed to identify the number, location, and phenotypic profiles of macrophages (##FIG##7##Figure 8A##). Generally, macrophages are known to polarize into two groups; M1 and M2, depending on the microenvironment. M1 macrophages act as a pro-inflammatory agent, while M2 macrophages repair damaged tissues. More macrophages were found in defects with S60 compared to the control group regardless of the time points (##FIG##7##Figure 8B##), which is not surprising, as a material was present. M2 macrophage ratio was also higher in the S60 samples compared to the control group (##FIG##7##Figure 8C##). Of note, M1 macrophages (CD 206 negative cells) appeared in the control group after 8 weeks post-surgery, although it is considered beyond the early stage of inflammation. It is well-known that M2 macrophages play crucial roles in promoting bone repair and improving biomaterial to tissue integration.<sup>[##REF##30555559##44##]</sup> A previous study was able to show that soluble silica released from bioactive glass stimulated macrophage polarization toward M2 phenotype, which concurs with the results from this study.<sup>[##REF##32264109##45##]</sup></p>" ]
[ "<title>Results and Discussion</title>", "<title>Hybrid Confirmation</title>", "<p id=\"P4\">Poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-<italic toggle=\"yes\">star</italic> copolymer was synthesized through reversible addition-fragmentation chain-transfer (RAFT) polymerization technique with the arm-first approach, and then it was mixed with hydrolyzed silica network precursor (tetraethyl orthosilicate, TEOS) in three different inorganic:organic ratios of 50:50, 40:60, and 30:70. Hybrid scaffolds were 3D printed by direct ink writing and the compositions will be referred to as S50, S60, and S70 (the number corresponds to the organic content wt%, ##FIG##0##Figure 1A,B##). The pink coloration of the scaffolds was from dithiol bonds of the RAFT agent used. In the previous study, hybrid monolith cylinders were made successfully only when the inorganic content was 30 wt% or lower as higher inorganic content was susceptible to cracking. This was due to shrinkage during drying and capillary stresses causing fracture.<sup>[##REF##22922331##9##]</sup> Here, capillary stress was reduced through 3D printing, resulting in a higher surface area to volume ratio of the scaffold structure (thin struts rather than a bulk material).<sup>[##UREF##10##27##]</sup> The actual inorganic:organic ratios were confirmed by thermogravimetry (TGA, ##FIG##0##Figure 1C##). All the hybrid scaffolds showed thermal decomposition at 360°C, due to oxidation of the polymer.<sup>[##REF##32261149##16##,##UREF##8##20##,##UREF##11##28##]</sup> Residual mass remaining from S50, S60, and S70 were close to our targeted inorganic content of 50.5%, 42.1%, and 30.2% respectively. Fourier transform infrared spectroscopy (FTIR) was conducted to confirm the scaffolds’ molecular structure (##FIG##0##Figure 1D##). The FTIR spectra of the scaffolds were similar to those of monoliths produced previously.<sup>[##UREF##8##20##]</sup> The TGA and FTIR results confirmed that both polymer and silica network were present in the scaffolds.</p>", "<title>3D Printing Process and Scaffold Characterization</title>", "<p id=\"P5\">3D printing inorganic-organic hybrids with consistent quality was a challenging process.Height of the 3D printing syringes and their nozzles varied (± ≈1-2 mm), due to variation in supply from the manufacturer, therefore position of the ink syringe had to be adjusted every printing session. An ideal gap between the syringe nozzle to the printing platform was 0.2 mm. Due to the fluid to viscous (sol to gel) transition during the gelation process, there was a printing window, defined as the time period after mixing the polymer and the sol, when hybrid ink was viscous enough to retain its shape after printing but still fluid enough to be extruded from the nozzle.<sup>[##UREF##12##29##]</sup> Additionally, viscosity of the hybrid ink gradually increased during printing, due to covalent bonding formation between polymer to silica network and solvent evaporation (i.e., ethanol, THF, and trace of water). The printing window for all the compositions tested in this study was 1h, but decreased as organic content increased (i.e., S70 gelled faster than other compositions).</p>", "<p id=\"P6\">Scanning electron microscope (SEM) images of the horizontal and vertical cross-sections of the scaffolds are shown in ##FIG##1##Figure 2##. The actual inter-strut distance was narrower than the printed distance due to the shrinkage. The shrinkage resulted from the evaporation of solvents during drying stage as well as polymerization of the hybrid from the condensation reaction.<sup>[##UREF##5##13##]</sup> An ideal inter-strut distance was determined by printing S60 hybrids with different inter-strut dimensions. Printed scaffolds were optimal when printed with a pore size between 100 and 200 μm. A pore size of 500 μm, which is in the range of trabecular bone pore sizes, was produced with a nominal 1 mm inter-strut distance. However, as shown in ##SUPPL##1##Figure S1A,B##, Supporting Information, the struts were sagging between underlying struts and collapsing in the <italic toggle=\"yes\">z</italic> direction; resulting in poor interconnectivity. A nominal inter-strut distance of 0.7 mm also produced horizontally merged and collapsed porous structure, despite having vertical pore size of ≈300-500 μm (##SUPPL##1##Figure S1C,D##, Supporting Information). 3D printing with 0.5 mm inter-strut distance enabled fabrication of scaffolds with improved interconnected porous structure in both horizontal and vertical directions (##FIG##1##Figure 2##). Hence, hybrid scaffolds with different compositions were printed with 0.2 mm printing nozzle (strut diameter) and 0.5 mm inter-strut distance (pore size). After drying, the strut diameter shrank to ≈100 μm and pore size was between 100 and 200 μm (##TAB##0##Table 1##), meeting the hypothesized minimal requirements for vascularization.<sup>[##REF##16102812##4##,##REF##18373428##30##]</sup> The horizontal cross-section SEM images (##FIG##1##Figure 2A,B,C##) displayed vertical channels running through the scaffolds with ≈190 μm widths for S70 and S60 scaffolds. S50, on the other hand, had pore width of 180 μm, as scaffolds with the higher inorganic content were more susceptible to shrinkage. The vertical cross-section images (##FIG##1##Figure 2D,E,F##) also showed inter-connectivity of pores and bonding between the struts of each layer. However, lower pore sizes were observed compared to the horizontal cross-section. S70 had an average pore size of 111 μm, which is due to higher organic content, resulting in less stiff ink that compressed under its own weight while printing. S50 also showed merging of the layers producing average pore size of 121 μm; possibly caused by higher inorganic content inhibiting to gel. From the SEM images, S60 had the most defined porous structure. The pore interconnectivity and porosity of S60 was confirmed geometrically, using helium pycnometry to obtain the skeletal density. The percent porosity was 60.5 ± 1.1%, which is within the range of trabecular bone.<sup>[##UREF##13##31##]</sup> The qualitative pore inter-connectivity was confirmed through micro-CT scans. The reconstructed 3D image of the central region (##FIG##1##Figure 2G##) and volumetric rendering of S60 (##SUPPL##0##Video S1##, Supporting Information) also showed inter-connected porous structure.</p>", "<p id=\"P7\">Uniaxial compression tests were performed. An ideal bone substitute should have mechanical properties that are similar to that of bone.<sup>[##UREF##0##1##]</sup>The stress-strain curves (##FIG##1##Figure 2H##) indicate that all the scaffolds are within the range of trabecular bone, which is reported to have Young’s modulus values in the range of 50-500 MPa and compressive strength of 2-12 MPa (shaded region in ##FIG##1##Figure 2H##).<sup>[##UREF##0##1##,##REF##22311079##32##]</sup> The general trend of Young’s modulus and failure stress of the scaffolds increased as inorganic content of the hybrids increased, which agrees with previous studies on poly(MMA)-silica hybrids.<sup>[##UREF##14##33##]</sup> As shown in ##TAB##0##Table 1##, S50 had the highest failure stress of 26.3 MPa at a strain to failure of 8.9%. S70 had the lowest failure stress which was more than threefold lower than other compositions, while failure strain value was 7.4%. S60 had the most synergistic properties, that is, flexibility from polymer and strength from silica matrix, with the highest strain to failure of 11.5%, failure stress of 22.4 MPa and Young’s modulus of 247 MPa. Cyclic compressive loading tests were also performed on S60 to confirm ductility for 12 MPa compressive stress, which is at the upper end of the compressive strength range of trabecular bone. As ##FIG##1##Figure 2I## shows, the scaffold did not appear to fracture and the only noticeable hysteresis was observed during the first cycle, which is common in such tests, possibly from an uneven top/bottom surface of the scaffold. Cyclic deformations decreased after each cycle, which was less than total of 0.5% strain stretch.</p>", "<p id=\"P8\">The mechanical properties of the scaffolds not only meet those required of trabecular bone, but also surpassed previous 3D printed bone substitutes. In terms of previously printed hybrid scaffolds, hybrids with methacrylic copolymers have been 3D printed previously,<sup>[##UREF##10##27##]</sup> but their pore channel size was 2 mm and mechanical properties were only extrapolated from three point bending tests of bulk samples. Gelatin-SiO<sub>2</sub> hybrid with 25 wt% gelatin was 3D printed with pore channel size of ≈550 μm and low porosity (30%), perhaps due to high shrinkage.<sup>[##UREF##15##34##]</sup> Despite having higher inorganic content and denser porous structure, the scaffold had compressive strength of ≈5 MPa with failure strain of ≈5% (estimated from published stress/strain graphs<sup>[##UREF##15##34##]</sup>). SiO<sub>2</sub>-pTHF-PCL hybrids, with 75 wt% organic component, was printed with a pore channel size of 200 μm (42% porosity) and were highly elastic, with a 36% strain to failure at a compressive strength of 1.2 MPa, which was seen as ideal for cartilage regeneration but too flexible for bone repair.<sup>[##UREF##2##7##]</sup></p>", "<title>Cytotoxicity and Pre-Osteoblast Adherence Evaluations on the Hybrid Scaffolds</title>", "<p id=\"P9\">For an ideal scaffold for bone repair, the scaffold material must also be non-cytotoxic and promote osteogenic cell adherence.<sup>[##REF##15119934##35##]</sup> Cytotoxicity of the scaffolds were evaluated by ISO 10993-12.<sup>[##UREF##16##36##]</sup> MTT metabolic assay (##FIG##2##Figure 3A##) confirmed that all the scaffolds passed ISO standards for cytotoxicity, with viability of the cells exposed to extractables of the hybrid scaffolds at &gt;87% to that of the control media, which proved that most of the solvents were removed by drying. The MC3T3 pre-osteoblasts were able to adhere on the scaffolds within 24 h. As shown in ##FIG##2##Figure 3B,C,D##, major cytoskeletal constituents of intermediate filaments and microfilaments were observed along with DAPI staining of nuclei. Although methacrylic polymers are known to be hydrophobic, MC3T3 cells were able to attach on S70 which had the highest amount of polymer. This was expected since monolithic hybrids with the same composition have shown cell attachment previously.<sup>[##UREF##1##6##,##UREF##8##20##]</sup> Total DNA quantification of MC3T3 cells on each scaffold (Hoechst stain) (##FIG##2##Figure 3E##) revealed that the scaffolds had cell attachment rate of 59 ± 7%, 56 ± 8%, and 36 ± 7% for S50, S60, and S70, respectively (##TAB##0##Table 1##). The improvement in cell adherence as inorganic content increased was possibly due to increase in stiffness and improvement in hydrophilicity, due to surface silanol groups, which corroborates with a previous study on hybrid monoliths with a range of degree of hydrophilicity.<sup>[##UREF##1##6##]</sup></p>", "<title>S60 Hybrid Scaffold as a Bone Substitute</title>", "<p id=\"P10\">The osteogenic properties of S60 were evaluated through a calvarial defect model, since: printability of S60 was superior to other compositions; its material interface was pre-osteoblast friendly; and its mechanical properties were comparable to trabecular bone. A critical sized defect (diameter <italic toggle=\"yes\">(φ):</italic> 8 mm) rat calvarial model was used.<sup>[##REF##23018195##37##]</sup> Representative micro-computed tomography (<italic toggle=\"yes\">μ</italic>CT) images are shown in ##FIG##3##Figure 4##. The images include 3D reconstructions and 2D cross section slices through the center of the defects. Defects containing S60 implants were compared to the defects without scaffolds (control group). Reconstructed 3D <italic toggle=\"yes\">μCT</italic> images after 8 and 16 weeks post-surgery showed new bone formation in both the control group and defect model with implanted S60 (##FIG##3##Figure 4A,B##). At 8 weeks, new bone formation in the control group was small, and it started from edges of the host bone, while 2D slice across the center of the defect (coronal section) showed no bone ingrowth. More new bone tissue intruded into the porous structure of S60, and bone ingrowth along the interface of the scaffold-bone was confirmed. At 16 weeks, bone was present in both the control defect and in defect containing scaffold. The quantitative analysis of <italic toggle=\"yes\">μCT</italic> images indicated that the percentage of new bone volume relative to total defect volume (BV/TV) in the S60 group was 12.4%, which was lower than the control group of 19.6% (##FIG##3##Figure 4C##). This was likely due to new bone formation in the defect interface of bony borders in the control group while the scaffold took up space in the defect site. The defect with S60 implant displayed both peripheral and central distribution of new bone formation. Since the innate healing capacity that originates from the defect margin is known to be constant regardless of the defect size,<sup>[##REF##20827327##38##]</sup> osteogenic evaluation of the central area of a defect is an important factor in criticalsized bone defect models. Following a previous study on evaluating bone regeneration<sup>[##REF##21212786##39##]</sup> bone formation of the central area was evaluated by the BV to central volume, that is, <italic toggle=\"yes\">φ</italic> 5 mm within <italic toggle=\"yes\">φ</italic> 8 mm defect (##FIG##3##Figure 4D##). S60 was able to enhance bone formation compared to that of the control model in both 8 and 16 week post surgeries. The 3D porous structure with osteoblast friendly surface<sup>[##REF##32263492##40##]</sup> possibly allowed more suitable environment for bone formation.</p>", "<p id=\"P11\">Results from the <italic toggle=\"yes\">μCT</italic> analysis were further confirmed by histological assessment. ##FIG##4##Figure 5A,B## shows the histological images of the specimens at 8 and 16 weeks of post implantation. Hematoxylin and eosin (H &amp; E) and Masson’s trichrome (MT) staining were utilized to further evaluate the bone regeneration patterns within S60. The control group showed bone in-growth pattern only from the peripheral region, while S60 groups revealed bone regeneration from the peripheral region plus in situ bone islands formation in the center of defect. At 16 weeks, thin connective tissue formation was detected in control defects, whereas bone-like tissue formation was observed in the areas grafted with S60. Substantial soft connective tissue, stained as blue color in MT staining, was observed in defects of the control group. On the other hand, red colored staining, representing matured bone, was apparent in the central region of the defects after 16 weeks of S60 implantation. Based on the MT staining, mature bone tissue formation was quantified through measuring non-collagenous tissue region; area with red staining, of the central defect area <italic toggle=\"yes\">(φ</italic> 5 mm within <italic toggle=\"yes\">φ</italic> 8 mm defect) (##FIG##4##Figure 5C##). More matured bone was present for defects with S60 compared to the defects of control group at 8 and 16 weeks post implantation. These histology quantification results corroborated with the bone volume to central volume (BV/CV) <italic toggle=\"yes\">μCT</italic> results.</p>", "<p id=\"P12\">Angiogenesis and mature vessel formation within the calvarial defect site, with and without S60 scaffold implantation, were evaluated since angiogenesis and vascular tissue formation must be induced for bone regeneration. In order to confirm angiogenic activity and vessel maturation, histology sections were examined by double immunofluorescence staining of von Willebrand factor (vWF) and alpha smooth muscle actin <italic toggle=\"yes\">(a</italic>-SMA) (##FIG##5##Figure 6A##). As shown in ##FIG##5##Figure 6B##, vWF-positive cell density within a S60 implanted defect was threefold and twofold higher than that of the control group at 8 and 16 weeks post-surgery, respectively. This indicates that S60 scaffold enhanced new blood vessel formation and angiogenic activity. Additionally, <italic toggle=\"yes\">a</italic>-SMA positive vessel density for S60 group was eightfold and threefold higher than that of the control group at 8 and 16 weeks after implantation, respectively (##FIG##5##Figure 6C##). The maturation index; based on double staining results (##FIG##5##Figure 6D##), confirmed that implanted S60 scaffold enhanced vessel maturation. For the control group, vessel density (vWF and <italic toggle=\"yes\">a</italic>-SMA positive staining) and maturation index increased from 8 to 16 weeks post-surgery. On the other hand, S60 did not show significant differences between 8 and 16 weeks post implantation. The vessel density and maturation index values were higher than that of the control group at all the time points. Thus, the results imply that the S60 scaffold encourages angiogenesis and vessel maturation, starting from an early stage. This could be due to silica release from S60 scaffold, or the surface chemistry and 3D architecture. The degradation mechanism of sol-gel bioactive glasses is well established.<sup>[##REF##22922331##9##]</sup> Silica released from mesoporous silica microspheres was shown to up-regulate hypoxia inducing factor <italic toggle=\"yes\">1α</italic> (HIF-1<italic toggle=\"yes\">α</italic>) expression and stabilize the activation through down-regulating HIF-prolyl hydroxylase 2 in human umbilical vein endothelial cells culture. This process successively induced key angiogenic factors, such as basic fibroblast growth factor, vascular endothelial growth factor, and endothelial nitric oxide synthase,<sup>[##REF##27918936##41##]</sup> which could explain angiogenic characteristics of S60. Poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-SiO<sub>2</sub> hybrids have been shown to release silica. For example, a similar inorganic:organic composition to our S60 hybrid lost mass over a period of 100 days degradation in PBS.<sup>[##REF##24001050##17##]</sup> However, the Si concentrations reported were 10 to 25 μg mL<sup>-1</sup>, which is higher than that released by our scaffolds (4.8 ± 0.1 μg mL<sup>-1</sup> released over 16 weeks, ##SUPPL##1##Figure S2##, Supporting Information). As the Si release is small, the surface chemistry and pore architecture are also likely to contribute to the angiogenic effect.</p>", "<p id=\"P13\">To verify osteogenic differentiation and bone production as a result of S60 scaffold implantation, immunostaining of osteogenic markers, that is, collagen Type I and osteocalcin, were evaluated (##FIG##6##Figure 7A##). Collagen Type I is one of the main components of bone that gives strength by forming oriented layers, while osteocalcin acts as an essential linker between the organic and inorganic components in the bone matrix. Both markers were more highly expressed in defects containing S60 scaffolds compared to that of the control group, as shown in ##FIG##6##Figure 7B,C##, regardless of the post-implantation time points. The osteogenic markers confirmed that S60 enhanced bone regeneration, due to the release of silica species which stimulate collagen Type I and osteoblastic differentiation<sup>[##REF##12633784##42##]</sup>,or a combination of 3D architecture and surface chemistry of the scaffold.</p>", "<p id=\"P14\">Overall, S60 was biocompatible and able to promote new vascularized bone tissue formation within the pore channels with the support of its surface, which promoted cell adhesion. Bone growth was starting in the central part of the defect area, that is, inside S60, which was not contiguous to calvarial defect edges. The combination of S60 scaffold induced both blood vessel and bone growth.</p>", "<p id=\"P15\">Macrophage polarization in the defect site and S60 at 8 and 16 weeks post-surgery were assessed, since macrophages are known to be involved in the bone healing process.<sup>[##UREF##17##43##]</sup> Immunolabeling of CD68 (pan macrophage) and CD206 (M2 marker) was performed to identify the number, location, and phenotypic profiles of macrophages (##FIG##7##Figure 8A##). Generally, macrophages are known to polarize into two groups; M1 and M2, depending on the microenvironment. M1 macrophages act as a pro-inflammatory agent, while M2 macrophages repair damaged tissues. More macrophages were found in defects with S60 compared to the control group regardless of the time points (##FIG##7##Figure 8B##), which is not surprising, as a material was present. M2 macrophage ratio was also higher in the S60 samples compared to the control group (##FIG##7##Figure 8C##). Of note, M1 macrophages (CD 206 negative cells) appeared in the control group after 8 weeks post-surgery, although it is considered beyond the early stage of inflammation. It is well-known that M2 macrophages play crucial roles in promoting bone repair and improving biomaterial to tissue integration.<sup>[##REF##30555559##44##]</sup> A previous study was able to show that soluble silica released from bioactive glass stimulated macrophage polarization toward M2 phenotype, which concurs with the results from this study.<sup>[##REF##32264109##45##]</sup></p>" ]
[ "<title>Conclusion</title>", "<p id=\"P16\">Poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-<italic toggle=\"yes\">star</italic>-SiO<sub>2</sub> hybrid “ink” with three different inorganic:organic compositions were successfully 3D printed via direct ink writing. 3D printing allowed production of hybrid scaffolds with controlled interconnected porous structure with pore size in excess of 100 μm. S60 had the most ideal printability, while its porosity and mechanical properties fell within the range of trabecular bone. In vitro and in vivo studies confirmed vascularized bone regeneration ability. Additionally, S60 showed a preferable M1/M2 macrophage profile, which led to pro-healing microenvironment coupled with augmented osteogenesis. The authors believe that this study opens up more possibilities for hybrid materials to be applied toward bone repairing biomaterials.</p>" ]
[ "<p id=\"P1\">Inorganic–organic hybrid biomaterials made with star polymer poly(methyl methacrylate-<italic toggle=\"yes\">co</italic>-3-(trimethoxysilyl)propyl methacrylate) and silica, which show promising mechanical properties, are 3D printed as bone substitutes for the first time, by direct ink writing of the sol. Three different inorganic:organic ratios of poly(methyl methacrylate-<italic toggle=\"yes\">co</italic>-3-(trimethoxysilyl)propyl methacrylate)-<italic toggle=\"yes\">star</italic>-SiO<sub>2</sub> hybrid inks are printed with pore channels in the range of 100-200 μm. Mechanical properties of the 3D printed scaffolds fall within the range of trabecular bone, and MC3T3 pre-osteoblast cells are able to adhere to the scaffolds in vitro, regardless of their compositions. Osteogenic and angiogenic properties of the hybrid scaffolds are shown using a rat calvarial defect model. Hybrid scaffolds with 40:60 inorganic:organic composition are able to instigate new vascularized bone formation within its pore channels and polarize macrophages toward M2 phenotype. 3D printing inorganic-organic hybrids with sophisticated polymer structure opens up possibilities to produce novel bone graft materials.</p>" ]
[ "<title>Experimental Section</title>", "<title>Star Polymer Synthesis</title>", "<p id=\"P17\">MMA (99%), TMSPMA (98%), ethylene glycol dimethacrylate (EGDMA, 98%), cumyl dithiobenzoate (CDB, 98%), toluene (99%), and tetrahydrofuran (THF, 99.9%) were obtained from Sigma–Aldrich. Azobisisobutyronitrile (AIBN, 98%) was obtained from Molekula, and recrystallized in ethanol prior to the polymerization. <italic toggle=\"yes\">n</italic>-hexane was obtained from Fisher Chemical.</p>", "<p id=\"P18\">Poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA)-<italic toggle=\"yes\">star</italic> was synthesized using RAFT polymerization technique, following a same process as the previous study.<sup>[##UREF##8##20##]</sup> Linear, or arm of the star polymer, poly(MMA-<italic toggle=\"yes\">co</italic>-TMSPMA) was first synthesized with the molar ratio of MMA<sub>100</sub>-TMSPMA<sub>10</sub> using toluene as the solvent.</p>", "<p id=\"P19\">AIBN was used as the initiator and CDB was used as the RAFT agent with a molar ratio of 1:2. The reagents were introduced in a round bottle flask in the overall molar ratio of AIBN<sub>1</sub>:RAFT<sub>2</sub>:MMA<sub>480</sub>:TMSPMA<sub>48</sub>. RAFT polymerization was performed at 70 °C in an argon atmosphere under continuous stirring and stopped at 50% conversion. The polymer was then precipitated in excess of <italic toggle=\"yes\">n</italic>-hexane, and re-dissolved in toluene. The obtained polymer was dried using rotary evaporator to measure the mass of the polymer synthesized.</p>", "<p id=\"P20\">The arm of the star polymer was introduced in a round bottle flask with a bi-functional branching agent (EGDMA), AIBN, and toluene as a solvent in the molar ratio of ARM<sub>1</sub>:EGDMA<sub>8</sub>:AIBN<sub>0.3</sub>. The polymerization was performed at 70 °C in an argon atmosphere for 18 h under continuous stirring. The star polymer was then precipitated in excess of <italic toggle=\"yes\">n</italic>-hexane and ethanol, and then dissolved in THF. The obtained polymer was dried using rotary evaporator to measure the mass ofthe polymer synthesized and re-dissolved in THF.</p>", "<title>Hybrid Ink Synthesis</title>", "<p id=\"P21\">Tetraethyl orthosilicate (TEOS, 98%) was obtained from ABCR GmbH &amp; Co. KG, and 1<sub>M</sub> hydrochloric acid (HCl) from Fisher Chemical.</p>", "<p id=\"P22\">Inorganic source of the hybrid was prepared by hydrolyzing TEOS with the molar ratio of TEOS<sub>1</sub>:water<sub>3.7</sub>:HCl<sub>0.01</sub>. The reagents were stirred vigorously for 40-60 min, full hydrolysis of TEOS was visually confirmed; from cloudy to clear. Then, the star polymer (immersed in THF, 50% v/v) was mixed with hydrolyzed TEOS according to three different inorganic:oganic wt% ratios of 50:50, 40:60, and 30:70. The mixture was left to continuously stir (40-60 min) until it was viscous enough to be printed.</p>", "<p id=\"P23\">The appropriate viscosity was determined visually and indicated by being able to draw thread of the hybrid gel from the bulk mixture using a syringe needle. The partially gelled hybrid ink was then transferred to luer lock syringe and aged at room temperature for another 40 min before printing.</p>", "<title>3D Printing</title>", "<p id=\"P24\">A polyethylene tubing (Harvard Apparatus PolyE, internal diameter of 1.78 mm) was attached to the Luer lock syringe that contained the hybrid ink. At the opposite end of the tubing, polypropylene printing nozzle (Nordson EFD, SmoothFlow tapered tip, 0.2 mm) was connected. The syringe-tubing-nozzle was connected to the syringe pump (Harvard Apparatus PHD ULTRA) with constant applied force (flow rate of 0.04 ml s<sup>-1</sup>), and the nozzle was guided by a 3D printer (Ultimaker Original) with the control by computer aided direct-write program (Cura 13.10) at a speed of 10 mm s<sup>-1</sup>.The program was set upto print 9 mm × 9 mm × 8 mm with a 90° mesh architecture where each layer contained an array of parallel struts that were aligned perpendicular to the previous layer. The inter-strut distance was set to 0.5 mm. Each layer consisted of a total of 19 struts and each scaffold consisted of 40 layers. After printing was complete, the hybrid scaffolds were placed in a sealed Nalgene container and aged slowly at room temperature for 2 days and in a 40 °C oven for another 2 days. Then, the scaffolds were dried in 60 °C oven for one day.</p>", "<p id=\"P25\">Rokit Invivo 3D printer was used for fabricating 3D scaffolds for the in vivo (calvarial defect model) study. Printing ink, or sol stage hybrid mixture, was transferred to 10 mL (12mL) NORM-JECT Luer Lock syringe with Nordson EFD SmoothFlow tapered tip (0.2 mm). The syringe was placed in the Bio Dispenser and cylinder with 10 mm (diameter) × 2mm (height) was printed with NewCreatorK 3D printing software. The slicer setting was 0.1 mm layer height, 60% fill density, Grid infill pattern, 10 mm s<sup>-1</sup> printing speed, 10 mm s<sup>-1</sup> traveling speed, and 300% input flow.</p>", "<title>Hybrid Scaffold Characterization</title>", "<p id=\"P26\">The functional groups of the hybrid compositions were confirmed by Fourier transform infrared spectroscopy (Nicolet iS10, Thermo Scientific) with an attenuated total reflectance module set up. 32 scans were averaged to a 4 cm<sup>-1</sup> resolution.</p>", "<p id=\"P27\">Inorganic–organic wt% ratios were analyzed by thermogravimetry analysis (Netzsch Sta 449c). The ground down samples were placed in a platinum crucible and heated to 800 °C at 10 °C min<sup>-1</sup> with constant flow of air.</p>", "<p id=\"P28\">The printed hybrid scaffold’s microstructure was confirmed by scanning electron microscope (JEOL JSM-6010LA Analytical SEM). The electron beam voltage was accelerating at 20 keV with a spot size of 60. The working distance was set to 15 mm, and secondary electron imaging mode was used. Pore sizes were measured using ImageJ image processing software. 40 pores of each compositions’ horizontal and vertical cross-sections were measured using particle analysis function.</p>", "<p id=\"P29\">The skeletal volume and density of the scaffold was measured by gas pycnometer measurement (Ultrapycnometer 1000, Quantachrome Corporation). Four S60 samples were weighted and analyzed together for 10 repetitions.</p>", "<p id=\"P30\">The mechanical properties of the samples were confirmed by a uniaxial compression test (Zwick Roell Z2.5). The samples were loaded perpendicular to the force applied direction regarding their printed layers. 2 kN load cell was used, pre-load was set to 0.05 N, and the compression speed was 0.2 mm min<sup>-1</sup>. Three 3D printed hybrid scaffolds of each composition were analyzed. The test scaffolds were ground down to make surfaces flat and cube structure as possible. Their average dimensions were length: 4.9 ± 0.3 mm, width: 5.0 ± 0.3 mm, and height: 4.7 ± 0.3 mm. Cyclic compressive loading test was set up similar to the compression test, but the maximum force applied was limited to 12 MPa, and point of load removal cycle was set at standard travel of 0 mm with 60 s hold time. 10 cycles were performed on a ground down S60 hybrid scaffold with a dimension of 4.4 × 4.5 × 4.4 mm.</p>", "<p id=\"P31\">Dissolution study of S60 hybrid scaffold was confirmed by measuring silica release profile in phosphate-buffered saline (PBS, pH 7.4, 1X) solution. S60 scaffolds (<italic toggle=\"yes\">n</italic> = 3) were immersed in PBS solution with a ratio of 30 mg inorganic content (Si content from the scaffold) to 20 mLof PBS in a Nalgene PMP jar. Dissolution containers were kept in an incubating orbital shaker (37 °C) and agitated at 110 rpm. The samples were incubated for 1, 2, and 4 weeks. At the end of each time point, the dissolution containers were removed from the incubator. 1 mL of the media was collected using a pipette and replaced by 1 mL of fresh PBS solution for compensation. The collected 1 mL sample solution was mixed with 9 mL of 2m nitric acid to prepare for inductively coupled plasma optical emission spectroscopy (ICP-OES, Thermo Scientific iCAP 6500 Duo) analysis. Si 1000 ppm ICP standard was prepared to 0.5, 1, 5, 10 ppm for the calibration curve.</p>", "<title>MC3T3-E1 Cell Culture</title>", "<p id=\"P32\">Reagents used for cell culture were purchased from Thermo Fisher Scientific and Sigma–Aldrich UK unless otherwise specified. MC3T3-E1 osteoblast precursor cell line (ATCC, UK) were monolayer expanded in basal media (<italic toggle=\"yes\">α</italic>-MEM supplemented with 10% (v/v) FCS and 1% (v/v) penicillin-streptomycin) and maintained at 37 °C in 5% CO<sub>2</sub> and humidified atmosphere until confluent. Cells were passaged using 500 μg mL<sup>-1</sup> trypsin-EDTA for cytotoxicity and cell attachment assays.</p>", "<title>Preparation of Cytotoxicity Test Dissolution Products</title>", "<p id=\"P33\">The hybrid scaffolds in powder form were fabricated and used for the preparation of dissolution products following the guidelines within ISO10993-12<sup>[##UREF##18##46##]</sup> (Biological evaluation of medical devices Part 12: Sample preparation and reference materials). Dissolution products released by the hybrid scaffolds at 0.2g mL<sup>-1</sup> in <italic toggle=\"yes\">α</italic>-MEM over 72-h at 37 °C were prepared. Dissolution products of medical grade polyethylene (PE) and polyurethane (PU) containing 0.1% (w/w) zinc diethyldithiocarbamate (ZDEC) were also prepared and served as non-cytotoxic negative control and cytotoxic positive control respectively. All dissolution products were sterilized through a membrane with 0.2 μm pores and, dilution series (25%, 50%, 75% and 100%) were prepared and supplemented with 10% (v/v) FCS prior to use in cytotoxicity assays.</p>", "<title>Cell Viability Analysis</title>", "<p id=\"P34\">Cellular viability in response to the dissolution products was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay in accordance to ISO10993- 5<sup>[##UREF##16##36##]</sup> (Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity). Briefly, 1 × 104 MC3T3-E1 cells were seeded onto each well in 96-well plates. Following 24 h of culture in basal <italic toggle=\"yes\">α</italic>-MEM, cells were exposed to the dissolution products of hybrid scaffolds or controls for further 24 h. MTT powder was dissolved in plain <italic toggle=\"yes\">α</italic>-MEM at the concentration level of 1 mg mL<sup>-1</sup>. After the incubation period, culture media was replaced by MTT solution. Cells were incubated for 2 h until purple formazan precipitates became visible. MTT solution was removed and DMSO was added to dissolve precipitates for 5 min. The optical density was measured spectrophotometrically at 570 nm using a microplate reader (SpectraMax M5).</p>", "<title>Cell Culture on 3D Printed Hybrid Scaffolds</title>", "<p id=\"P35\">Hybrid scaffolds (approximately 5 × 5 × 5 mm<sup>3</sup>) were manufactured and sterilized with 70% ethanol. Following washing with PBS, each sample was placed in serum-free <italic toggle=\"yes\">α-</italic> MEM for 30 min prior to cell seeding. MC3T3-E1 cells were harvested and suspended in basal <italic toggle=\"yes\">α</italic>-MEM at a concentration 1 × 107 cells mL<sup>-1</sup>. 10 μl of cell suspension was seeded onto each scaffold and incubated for 2 h with gent agitation every 20 min to ensure well distributed cell seeding. Each scaffold was then submerged in fresh basal <italic toggle=\"yes\">a</italic>-MEM and cultured for further 24 h.</p>", "<title>Visualization and Quantification of Cell Attachment</title>", "<p id=\"P36\">Vimentin and Actin staining: Cell-seeded samples were washed in PBS and fixed in 4% (w/v) paraformaldehyde for 24 h. Following permeabilisation with buffered 0.5% Triton X-100 in PBS (300 mM sucrose, 50 mM NaCl, 3 mM MgCl<sub>2</sub>, 20 mM Hepes and pH 7.2), immunostaining involved a 5 min incubation in 1% (w/v) BSA followed by hour-long incubation in anti-Vimentin antisera (1:500 dilution in 10 mg mL<sup>-1</sup> BSA in PBS, rabbit polyclonal, IgG, Ab- cam, Cambridge, UK) at 4 °C. Secondary anti-rabbit antibody (Alexa Fluor 488 conjugated, Abcam, Cambridge, UK) and Alexa Fluor 568-conjugated phalloidin (1:1000 dilution in 1% (w/v) BSA) was then added for 1 h incubation. DAPI (0.1μg ml<sup>-1</sup> in PBS, 10 min incubation) was used as a nuclear counter stain. No staining was observed in isotope primary antibody control groups. Samples were imaged under confocal microscopy (Leica SP5 Laser Scanning Confocal Microscope and software, Leica Microsystems, Wetzlar, Germany).</p>", "<p id=\"P37\">Total DNA quantification: The total DNA content of MC3T3-E1 cells on each scaffold was determined using fluorescent Hoechst stain. Cells were lysed by freeze-thaw cycles following the removal of culture media. Lysed cells were stored at -80 °C and thawed to room temperature on the day of assay. The lysate was incubated with molecular biology grade water for 1 h and Hoechst stain was added at a final concentration of 2 μg ml<sup>-1</sup>. The fluorescence of Hoechst dye was measured at 360 nm excitation wavelength and 460 nm emission wavelength. DNA and MC3T3-E1 cell standard curves were obtained from serial dilutions of known DNA (calf thymus, Sigma, UK) concentrations and cell densities respectively. The percentage of cells attached on the scaffold = Total DNA content from cell-scaffold construct / Total DNA of seeded cells × 100%.</p>", "<title>Animals and Surgical Procedure</title>", "<p id=\"P38\">All animals (SD-RAT, male, 8 weeks, 250 ± 15 g; Samtako, Korea) were cared for according to the methods approved by the Institutional Animal Care and Use Committee at Korea Institute of Science and Technology (2017-105). For the surgery, temporary anaesthesia was performed by intramuscular injection with a cocktail of zolazepam and tiletamine (0.3 mL kg<sup>-1</sup>, Zoletil, Vibrac, France) and xylazine (0.1 mL g<sup>-1</sup>, Rompun, Bayer, Germany). 8 mm round calvarial defects were created with a trephine bur (TPHB-B8, OSUNG, Korea) following the periosteum removal. The prepared samples were implanted (<italic toggle=\"yes\">n</italic> = 6) into the defect and the incision was closed with suture. For the control group, defects without scaffolds were used (<italic toggle=\"yes\">n</italic> = 6). Each implant was analyzed after 8 and 16 weeks by micro-computed tomography (CT) and histological staining analysis.</p>", "<title>Micro-CT Analysis</title>", "<p id=\"P39\">Quantitative 3D analysis of the calvarial defect samples was conducted using a Skyscan 1172 CT (Bruker, USA). At 8, 16 weeks following implantation, the animals were sacrificed and the bone defects were harvested. The specimens were fixed in 10% (v/v) buffered neutral formalin for 1 day. Samples were securely in a 5-mL conical tube and centered in the micro-CT machine. After calibration, samples were scanned using the following settings: 49 kV, 200 μA, and Al 0.5 mm filter. Raw data were collected and reconstructed using the NRecon software (Bruker, USA) and the volume of newly formed bone was calculated using the DataViewer (Bruker, USA) and CTAn software (Bruker, USA).</p>", "<title>Histological Analysis</title>", "<p id=\"P40\">The specimens from each group were decalcified in a decalcifying solution (Sigma, USA) for 6 h, followed by serial dehydration with a graded ethanol series (80-100%). Then, the samples were embedded in paraffin and sectioned at 10 μm thickness and then stained with hematoxylin and Eosin (H&amp;E) and Masson’s trichrome (MT) stains. The stained area was calculated with the MT-stained images (<italic toggle=\"yes\">n</italic> = 4) using the ImageJ (National Institutes of Mental Health, USA).</p>", "<title>Immunofluorescent Analysis</title>", "<p id=\"P41\">The central regions of S60 and control group defects were characterized. The micro-sectioned specimens were stained with CD68 (ab955, Abcam, UK) and CD206 (sc-34577, Santa Cruz Biotechnology, USA) antibodies. Alexa fluor 594 anti-mouse IgG and Alex fluor 488 anti-goat IgG were used as secondary antibodies, respectively. The nuclei were counterstained with DAPI (Molecular probes, USA), and the stained sections were observed using confocal laser scanning microscopy (LSM700, Zeiss, Germany). For evaluating the density of vascular cells in the defect region, endothelial cells (ECs) and vascular smooth muscle cells (SMCs) were stained by polyclonal rabbit anti-human von Wille-brand factor antibody (vWF, sc-365712 FITC, Santa Cruz Biotech, USA) and monoclonal mouse anti-human <italic toggle=\"yes\">a</italic>-smooth muscle actin antibody (<italic toggle=\"yes\">α</italic>-SMA, sc-53142 AF594, Santa Cruz Biotech, USA), respectively. vWF and <italic toggle=\"yes\">a</italic>-SMA- positive vessels in three fields were randomly selected and evaluated using ImageJ program (<italic toggle=\"yes\">n</italic> = 3 in each group). The maturation index was quantified as the ratio of smooth muscle cell-positive vessels to total vessel area. Furthermore, to verify the osteogenesis, the samples were stained with type I collagen (ab34710, Abcam, USA) and osteocalcin (sc-390877 FITC, Santa Cruz Biotech, USA) antibodies. For quantifying bone tissue regeneration in the defect region, type I collagen and osteocalcin positive regions in three fields were randomly selected and evaluated using ImageJ program (<italic toggle=\"yes\">n</italic> = 3 in each group). The type I collagen and osteocalcin positive areal density was quantified as the ratio of type I collagen and osteocalcin positive areas to total tissue area, respectively.</p>", "<title>Statistical Analysis</title>", "<p id=\"P42\">The statistical analysis was performed using one-way analysis of variance (ANOVA) with the Turkey’s significant difference post hoc test using SPSS software (IBM, USA). A value of <italic toggle=\"yes\">P &lt;</italic> 0.05 was considered as statistically significant.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>J.J.C. and J.Y. contributed equally to this work. The authors would like to acknowledge EPSRC (EP/M019950/1), National Research Foundation of Korea (NRF) grant (NRF-2020R1C1C1012881, 2021R1A2C2004634) funded by the Ministry of Science and ICT (MSIT), and KIST institutional program (2E30341, 2E31251). M.M.S. and J.R.J. were funded by a grant from the UK Regenerative Medicine Platform “Acellular / Smart Materials - 3D Architecture” (MR/R015651/1).</p>", "<title>Data Availability Statement</title>", "<p id=\"P43\">Research data are not shared.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>A) Side, and B) top photographs of 3D printed hybrid scaffolds of different inorganic:organic compositions (the numbers in S50, S60, and S70 refer to organic wt%). C) TGA curves and D) FTIR spectra of the hybrid scaffolds made with different organic contents, containing: absorption bands of organic polymer (i.e., C–H vibration, C=O, C–C–O asymmetric, and C–O–C symmetric stretch) and of the condensed silica network (i.e., Si–O–Si asymmetric and Si–OH stretch).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><title>SEM images of horizontal cross-sections of 3D printed hybrid scaffolds</title><p>A) S50, B) S60, and C) S70 (the numbers in S50, S60, and S70 refer to organic wt%). SEM images of vertical cross-sections of D) S50, E) S60, and F) S70. G) Representative 3D micro-CT image of the central region of S60 (scale bars: 500 μm). H) Representative uniaxial stress/strain curves of the 3D printed scaffolds. I) Cyclic compressive loading stress/strain curve of S60 (mechanical property region of trabecular bone<sup>[##UREF##0##1##,##REF##22311079##32##]</sup> is highlighted in grey).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>A) MTT metabolic activity assay performed in accordance to ISO 10993: MC3T3 cells were cultured with dissolution products of the hybrids and results were normalized against basal culture medium (Standard). PE refers to media conditioned with the non-toxic negative control material and Polyurethane (PU) was a positive control (for toxicity). Based on the ISO guidance, all test materials (S70, S60, and S50) in the current study can be deemed non-cytotoxic/biocompatible, as the viability of cells is &gt;70% of non-cytotoxic controls (marked by the dashed line). Immunohistochemical staining of MC3T3 cells on B) S70, C) S60, and D) S50. Images were produced by stacks of vimentin immunostain (green), f-actin labeling (red), and DAPI nuclear counter stain via confocal microscopy. E) Percentage of seeded MC3T3 cells attachment on the scaffolds determined using fluorescent Hoechst stain.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>A) Representative photograph of critical size calvarial defect <italic toggle=\"yes\">(φ</italic> 8 mm highlighted with yellow dotted line) model in a mouse, and <italic toggle=\"yes\">μCT</italic> images ofthe cranial bone defects: 3D and coronal (2D slice along the red line of 3D image) at 8 and 16 weeks. B) Representative photograph of implantation of S60 scaffold in the calvarial defect, and <italic toggle=\"yes\">μCT</italic> images of S60 implanted calvarial defects at 8 and 16 weeks (scale bars: 8 mm): 3D and coronal <italic toggle=\"yes\">μCT</italic> images. All the in vivo experiments were conducted on six animals. C) Morphometric analysis of the volume of newly formed bone volume (BV/TV) relative to total skull defect volume (8 mm-sized disk defect). D) Ratio of newly formed bone volume to central volume (5 mm-sized disk defect) (BV/CV) calculated by CTAn program (<italic toggle=\"yes\">n</italic> = 4, <italic toggle=\"yes\">**P &lt;</italic> 0.01).</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p>Histological evaluation of calvarial bone regeneration: A) Hematoxylin and eosin (H&amp;E) and B) Masson’s trichrome (MT) staining of calvarial bone defects in control and S60 groups at 8 and 16 weeks after implantation (black and red inset boxes in the left panels indicate the areas shown in the enlarged images of the edge and center of the defect, respectively. C: connective tissue; HB: host bone; NB: new bone). All the in vivo experiments were conducted on six animals. C) Quantitative analysis of the mature bone tissue formation based on MT staining images. The black and grey bars represent the area of bone matrix and collagenous regions, respectively (<italic toggle=\"yes\">n</italic> = 4, **<italic toggle=\"yes\">P &lt;</italic> 0.01).</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p>For sections through the center of the explanted scaffolds/defects: A) Representative immunofluorescence staining of control and S60 implanted calvarial defect at 8 and 16 weeks for angiogenic evaluations; vWF (green) and <italic toggle=\"yes\">α</italic>-SMA (red) co-immunolabeling was used to assess the maturation of vessels (scale bars: 100 μm). Quantitative analysis ofB) vWF positive vessel density, C) <italic toggle=\"yes\">α</italic>-SMA positive vessel density, and D) maturation index measured by ImageJ. The maturation index was based on comparison of the percentage of <italic toggle=\"yes\">α</italic>-SMA positive vessel area in the total vessel area <italic toggle=\"yes\">(n =</italic> 4, <italic toggle=\"yes\">*P &lt;</italic> 0.05, <italic toggle=\"yes\">***P &lt;</italic> 0.0005 and <italic toggle=\"yes\">****P &lt;</italic> 0.00005; †<italic toggle=\"yes\">P &lt;</italic> 0.05 and ††<italic toggle=\"yes\">P &lt;</italic> 0.005 compared with 8 weeks of the same group).</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p>For sections through the center of the explanted scaffolds/defects: A) Immunofluorescence staining of collagen Type I and osteocalcin in control and S60 implanted calvarial defect at 8 and 16 weeks; collagen type I (red) and osteocalcin (green) co-immunolabeling was used to assess bone tissue regeneration. White dotted lines indicate the scaffold region (scale bars: 100 μm). Quantitative analysis of B) collagen type I positive staining areal density, and C) osteocalcin positive staining areal density measured by ImageJ. (<italic toggle=\"yes\">n</italic> = 4, ***<italic toggle=\"yes\">P &lt;</italic> 0.0005 and <italic toggle=\"yes\">****P &lt;</italic> 0.00005; †<italic toggle=\"yes\">P &lt;</italic> 0.05 and ††††<italic toggle=\"yes\">P &lt;</italic> 0.00005 compared with 8 weeks of the same group)</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p>For sections through the center ofthe explanted scaffolds/ defects: A) Macrophage immunofluorescence staining in control and S60 implanted calvarial defect at 8 and 16 weeks. CD 68 (red) and CD 206 (green) co-immunolabeling was used to assess the phenotypic profiles of macrophages (scale bars: 200 μm). Quantitative analysis of B) CD 68 positive staining, and C) CD 206 to CD 68 ratio measured by ImageJ. (<italic toggle=\"yes\">n</italic> = 4, *<italic toggle=\"yes\">P &lt;</italic> 0.05)</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\" orientation=\"landscape\"><label>Table 1</label><caption><p>Mechanical properties of the 3D printed hybrid scaffolds made with different organic contents in wt%: 70%, (S70); 60%, (S60); 50% (S50) (<italic toggle=\"yes\">n</italic> = 4). Average horizontal and vertical cross-section pore sizes measured by SEM image analysis. Total DNA content of MC3T3-E1 cells on each scaffold determined by fluorescent Hoechst stain after 24 h of cell culture.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Scaffold</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Young’s Modulus [MPa]</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Ultimate stress [MPa]</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Failure strain [%]</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Horizontal cross-section pore size [μm]</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Vertical cross-section pore size [μm]</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Total DNA quantification [%]</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S70</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">101 ± 17<xref rid=\"TFN3\" ref-type=\"table-fn\">**</xref></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.9 ± 1.3<xref rid=\"TFN3\" ref-type=\"table-fn\">**</xref></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.4 ± 0.2<sup><xref rid=\"TFN2\" ref-type=\"table-fn\">*</xref></sup></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">190 ± 49</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">111 ± 27</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">36 ± 7<xref rid=\"TFN2\" ref-type=\"table-fn\">*</xref></td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">247 ± 12<xref rid=\"TFN3\" ref-type=\"table-fn\">**</xref></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.4 ± 2.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11.5 ± 2.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">191 ± 32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">151 ± 29<xref rid=\"TFN3\" ref-type=\"table-fn\">**</xref></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">56 ± 8</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S50</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">372 ± 25<xref rid=\"TFN3\" ref-type=\"table-fn\">**</xref></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">26.3 ± 0.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.9 ± 0.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">180 ± 29</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">121 ± 33</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">59 ± 7</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supplemental Video 1</label></supplementary-material>", "<supplementary-material id=\"SD2\" position=\"float\" content-type=\"local-data\"><label>Supporting Information</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P44\">\n<bold>Conflict of Interest</bold>\n</p><p id=\"P45\">The authors declare no conflict of interest.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><p id=\"P46\">Standard deviations are derived from the average values</p></fn><fn id=\"TFN2\"><label>*</label><p id=\"P47\"><italic toggle=\"yes\">P</italic>&lt; 0.05 (significant difference within the column values)</p></fn><fn id=\"TFN3\"><label>**</label><p id=\"P48\"><italic toggle=\"yes\">P</italic>&lt; 0.01 (significant difference within the column values)</p></fn></table-wrap-foot>" ]
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[{"label": ["[1]"], "person-group": ["\n"], "surname": ["Hench"], "given-names": ["LL"], "source": ["J Am Ceram Soc"], "year": ["1991"], "volume": ["74"], "fpage": ["1487"]}, {"label": ["[6]"], "person-group": ["\n"], "surname": ["Chung", "Sum", "Li", "Stevens", "Georgiou", "Jones"], "given-names": ["JJ", "BST", "SW", "MM", "TK", "JR"], "source": ["Macromol Rapid Commun"], "year": ["2017"], "volume": ["38"], "elocation-id": ["1700168"]}, {"label": ["[7]"], "person-group": ["\n"], "surname": ["Tallia", "Russo", "Li", "Orrin", "Shi", "Chen", "Steele", "Meille", "Chevalier", "Lee", "Stevens"], "given-names": ["F", "L", "SW", "ALH", "XM", "S", "JAM", "S", "J", "PD", "MM"], "source": ["Mater Horiz"], "year": ["2018"], "volume": ["5"], "fpage": ["849"]}, {"label": ["[8]"], "person-group": ["\n"], "surname": ["Novak"], "given-names": ["BM"], "source": ["Adv Mater"], "year": ["1993"], "volume": ["5"], "fpage": ["422"]}, {"label": ["[12]"], "person-group": ["\n"], "surname": ["Mahony", "Tsigkou", "Ionescu", "Minelli", "Ling", "Hanly", "Smith", "Stevens", "Jones"], "given-names": ["O", "O", "C", "C", "L", "R", "ME", "MM", "JR"], "source": ["Adv Funct Mater"], "year": ["2010"], "volume": ["20"], "fpage": ["3835"]}, {"label": ["[13]"], "person-group": ["\n"], "surname": ["Mahony", "Yue", "Turdean-Ionescu", "Hanna", "Smith", "Lee", "Jones"], "given-names": ["O", "S", "C", "JV", "ME", "PD", "JR"], "source": ["J Sol-Gel Sci Technol"], "year": ["2014"], "volume": ["69"], "fpage": ["288"]}, {"label": ["[14]"], "person-group": ["\n"], "surname": ["Gabrielli", "Connell", "Russo", "Jimenez-Barbero", "Nicotra", "Cipolla", "Jones"], "given-names": ["L", "L", "L", "J", "F", "L", "JR"], "source": ["RSC Adv"], "year": ["2014"], "volume": ["4"], "fpage": ["1841"]}, {"label": ["[19]", "a)", "b)", "c)"], "person-group": ["\n", "\n", "\n"], "surname": ["Sharp", "Michalczyk", "Sharp", "Manzano", "Arcos", "Delgado", "Ruiz", "Gil", "Vallet-Regi"], "given-names": ["KG", "MJ", "KG", "M", "D", "MR", "E", "FJ", "M"], "source": ["J Sol-Gel Sci Technol", "Adv Mater", "Chem Mater"], "year": ["1997", "1998", "2006"], "volume": ["8", "10", "78"], "fpage": ["541", "1243", "5696"]}, {"label": ["[20]"], "person-group": ["\n"], "surname": ["Chung", "Li", "Stevens", "Georgiou", "Jones"], "given-names": ["JJ", "S", "MM", "TK", "JR"], "source": ["Chem Mater"], "year": ["2016"], "volume": ["28"], "fpage": ["6127"]}, {"label": ["[23]"], "person-group": ["\n"], "surname": ["Poologasundarampillai", "Yu", "Jones", "Kasuga"], "given-names": ["G", "BB", "JR", "T"], "source": ["Soft Matter"], "year": ["2011"], "volume": ["7"], "elocation-id": ["10241"]}, {"label": ["[27]"], "person-group": ["\n"], "surname": ["Calvert", "O\u2019Kelly", "Souvignier"], "given-names": ["P", "J", "C"], "source": ["Mater Sci Eng, C"], "year": ["1998"], "volume": ["6"], "fpage": ["167"]}, {"label": ["[28]"], "person-group": ["\n"], "surname": ["Kashiwagi", "Inaba", "Brown", "Hatada", "Kitayama", "Masuda"], "given-names": ["T", "A", "JE", "K", "T", "E"], "source": ["Macromolecules"], "year": ["1986"], "volume": ["19"], "fpage": ["2160"]}, {"label": ["[29]"], "person-group": ["\n"], "surname": ["Gao", "Rahaman", "Gao", "Teramoto", "Abe"], "given-names": ["C", "MN", "Q", "A", "K"], "source": ["J Biomed Mater Res, Part A"], "year": ["2013"], "volume": ["101"], "fpage": ["2027"]}, {"label": ["[31]"], "person-group": ["\n"], "surname": ["Bose", "Vahabzadeh", "Bandyopadhyay"], "given-names": ["S", "S", "A"], "source": ["Mater Today"], "year": ["2013"], "volume": ["16"], "fpage": ["496"]}, {"label": ["[33]", "a)", "b)"], "person-group": ["\n", "\n"], "surname": ["Mammeri", "e Bourhis", "Rozes", "Sanchez", "Mammeri", "Rozes", "e Bourhis", "Sanchez"], "given-names": ["F", "EL", "L", "C", "F", "L", "EL", "C"], "source": ["J Eur Ceram Soc", "J Eur Ceram Soc"], "year": ["2006", "2006"], "volume": ["26", "26"], "fpage": ["259", "267"]}, {"label": ["[34]"], "person-group": ["\n"], "surname": ["Gao", "Rahaman", "Gao", "Teramoto", "Abe"], "given-names": ["CX", "MN", "Q", "A", "K"], "source": ["J Biomed Mater Res, Part A"], "year": ["2013"], "volume": ["101"], "fpage": ["2027"]}, {"label": ["[36]"], "collab": ["International Organization for Standardization"], "source": ["Biological Evaluation of Medical Devices Part 5: Tests for In Vitro Cytotoxicity"], "date-in-citation": ["accessed: March 2014"], "comment": ["\n"], "ext-link": ["https://www.iso.org/obp/ui%23iso:std:iso:10993:-5:ed-3:v1:en"]}, {"label": ["[43]"], "person-group": ["\n"], "surname": ["Gu", "Yang", "Shi"], "given-names": ["QL", "HL", "Q"], "source": ["J Orthop Transl"], "year": ["2017"], "volume": ["10"], "fpage": ["86"]}, {"label": ["[46]"], "collab": ["International Organization for Standardization"], "source": ["Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials"], "date-in-citation": ["accessed: March 2014"], "comment": ["\n"], "ext-link": ["https://www.iso.org/obp/ui/%23iso:std:iso:10993:-12:ed-5:v1:en"]}]
{ "acronym": [], "definition": [] }
46
CC BY
no
2024-01-13 00:07:37
Adv Healthc Mater. 2021 May 5; 10(12):e2100117
oa_package/c1/c4/PMC7615494.tar.gz
PMC7615498
37956646
[ "<title>Introduction</title>", "<p id=\"P9\">Sleep is a complex phenotype regulated by homeostatic and circadian processes [##UREF##0##1##] and characterised by multiple dimensions such as duration, quality and timing or chronotype [##REF##30956942##2##]. Studies have shown that these specific sleep traits are moderately heritable, with twin studies estimating that between 44-50% of their variability is genetically determined [##UREF##1##3##,##REF##33636423##4##], while SNP-based heritability studies have shown that heritability of self-reported traits ranges from 5 to 15% [##REF##33338765##5##–##REF##30804565##8##]. Moreover, these dimensions have been consistently associated with several adverse health outcomes. For example, inadequate sleep duration, poor quality, and inappropriate timing are associated with adverse health consequences [##REF##28579842##9##]. However, as most research has used observational epidemiology to study these associations, whether these links are causal remained elusive until very recently.</p>", "<p id=\"P10\">Mendelian randomisation (MR) is a method that uses genetic variants to assess causal relationships [##REF##12689998##10##]. The MR method addresses two questions: whether an observational association between an exposure and an outcome is causal alongside the magnitude of this effect [##REF##25939054##11##,##UREF##2##12##]. MR is increasingly used to overcome some limitations of traditional observational epidemiology, such as unmeasured confounding and reverse causality [##REF##12689998##10##], and the analysis is facilitated by MR packages, such as the widely used “MendelianRandomization” package for the R open-source software environment [##REF##28398548##13##] or the “mrrobust” Stata package [##UREF##3##14##]. Recently, numerous MR studies examined the causal relationship between genetic instruments of sleep traits and different health outcomes [##REF##25939054##11##–##REF##35141322##56##].</p>", "<p id=\"P11\">In the context of MR, a genetic variant can be considered an instrumental variable (IV) for a given exposure if it satisfies the following assumptions: i) it is associated robustly with the exposure of interest, ii) it does not influence the outcome through a pathway other than the exposure (horizontal pleiotropy) and iii) it is not associated with the outcome due to confounding [##UREF##2##12##]. Genetic variants used as IVs in MR are usually single-nucleotide polymorphisms (SNPs), a common variation at a single position of DNA sequence [##REF##12689998##10##].</p>", "<p id=\"P12\">MR studies have steadily grown as genetic variants reliably associated with different exposures have increased over the last decade, thanks to genome-wide association studies (GWAS) [##REF##25939054##11##]. GWA studies now test millions of genetic variants for their association with a given trait. Thus, finding genetic polymorphisms to use in an MR study is becoming more feasible. However, selecting optimal genetic instruments can be challenging [##REF##26961927##61##].</p>", "<p id=\"P13\">Although several guides exist for conducting MR studies [##UREF##2##12##,##REF##26961927##61##,##REF##27342221##62##], these are not widely adopted in the field of epidemiology of sleep; thus authors using genetic instruments for sleep traits have taken different approaches to the selection process. This review explores the criteria used for instrument selection in MR studies of sleep traits, discussing how this choice impacts analysis and some steps in the selection process that are often overlooked. We aim to demonstrate the importance of a careful selection of instruments to conduct an MR study. Nonetheless, it is worth mentioning that the selection process will always depend on the aim of the research and the specific exposure under study, and while we focus on MR studies of sleep traits, many of the issues discussed here apply to other behavioral phenotypes as well. In addition, even though MR has been particularly useful for understanding the causal role of sleep phenotypes on several health outcomes, other causal methods must also be used for replication and triangulation purposes. A summary of the main points to consider when selecting sleep genetic instruments in MR is presented in ##FIG##0##Figure 1##.</p>" ]
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[]
[]
[ "<title>Conclusions</title>", "<p id=\"P42\">In this article, we explored the criteria used for selecting genetic instruments for sleep traits in the context of MR, discussing how instrumental choice impacts analysis. We also presented GWAS of sleep phenotypes since 2016 and discussed MR studies using genetic sleep instruments to date. We are convinced that instrument selection is the most important decision when designing an MR study and that this is becoming even more important as the number of sleep genetic variants found in GWAS increases. We hope this review will aid researchers in designing robust MR studies and continue to elucidate our understanding of the causal role of sleep on health outcomes.</p>" ]
[ "<title>Summary</title>", "<p id=\"P1\">This review explores the criteria used for the selection of genetic instruments of sleep traits in the context of Mendelian randomisation studies. This work was motivated by the fact that instrument selection is the most important decision when designing a Mendelian randomisation study. As far as we are aware, no review has sought to address this to date, even though the number of these studies is growing rapidly. The review is divided into the following sections which are essential for genetic instrument selection: 1) Single-gene region vs polygenic analysis; 2) Polygenic analysis: biologically- vs statistically-driven approaches; 3) P-value; 4) Linkage disequilibrium clumping; 5) Sample overlap; 6) Type of exposure; 7) Total (R<sup>2</sup>) and average strength (F-statistic) metrics; 8) Number of single-nucleotide polymorphisms; 9) Minor allele frequency and palindromic variants; 10) Confounding. Our main aim is to discuss how instrumental choice impacts analysis and compare the strategies that Mendelian randomisation studies of sleep traits have used. We hope that our review will enable more researchers to take a more considered approach when selecting genetic instruments for sleep exposures.</p>" ]
[ "<title>Abbreviations</title>", "<title>Criteria used to select genetic instruments in MR for sleep traits</title>", "<title>Single-gene region vs. polygenic analysis</title>", "<p id=\"P14\">The first step in instrument selection is to decide whether the analysis will be performed using variants from a single gene region or multiple regions (a polygenic analysis). When a particular region has been reported to have a specific biological link to the exposure, the selection usually focuses on these variants [##UREF##2##12##]. This approach has the advantage of specificity, leading to a more plausible MR [##REF##24159078##63##]. However, for complex risk factors such as sleep, no single gene region encodes this risk factor [##REF##15737788##64##]. In fact, numerous genomic variants have been discovered by sleep GWAS in adults, indicating that sleep is a highly polygenic trait. For example, for insomnia, 554 risk loci have been reported in a recent study [##REF##35835914##65##]. Thus, a polygenic analysis is often used in MR studies of sleep traits.</p>", "<p id=\"P15\">A polygenic analysis supposes the inclusion of multiple variants [##UREF##2##12##]. If the variants are all valid instruments, power is maximised because each SNP contributes incrementally to affect levels of the biomarker [##REF##26961927##61##,##REF##24159078##63##]. In the case of sleep, as common individual genetic variants confer small effects, the polygenic approach will typically have greater power to detect a causal effect than the single gene region approach [##UREF##2##12##].</p>", "<title>Polygenic analysis: biologically-driven vs statistically-driven approach</title>", "<p id=\"P16\">For a polygenic analysis, one of two approaches may be chosen for selecting genetic variants: a biologically-driven or a statistically-driven approach [##UREF##2##12##]. The former implies selecting variants from regions with a highly plausible biological link with the exposure of interest [##UREF##2##12##,##REF##26961927##61##]. The advantage of this approach is that these instruments may be less susceptible to horizontal pleiotropy [##REF##26961927##61##]. However, biological understanding is rarely infallible [##UREF##2##12##], and the biological basis of sleep in humans is not fully understood [##REF##36028773##66##]. Thus, instrument selection is often performed using a statistically-driven approach [##UREF##11##67##] or a combination of both approaches [##UREF##2##12##].</p>", "<p id=\"P17\">The statistically-driven approach exploits the increasing availability of SNPs associated with specific exposures in GWAS [##REF##26961927##61##]. For this reason, authors tend to search for the latest and largest GWA study available and select SNPs robustly associated with the exposure of interest (MR assumption 1). However, it is important not to assume that the latest and largest study will always yield the best instruments. For example, most published GWAS of sleep traits have been performed in European samples and are also not sex-specific. Nonetheless, some GWAS have been performed in other ethnic groups, including Hispanic/Latino Americans [##REF##26977737##68##] and multi-ancestry samples [##REF##29077507##69##–##REF##37770476##74##]. Furthermore, some have employed sex-stratified analyses for obstructive sleep apnea and insomnia, which display marked sexual dimorphism in disease prevalence [##REF##29077507##69##,##REF##28604731##75##]. However, further work is needed to better understand sex-related sleep differences, which have been associated with the influence of sex hormones on sleep regulation but have been understudied [##UREF##12##76##].</p>", "<p id=\"P18\">When using a statistically-driven approach, it is crucial to evaluate the reported SNPs carefully. Briefly, as described more thoroughly in the review, some important criteria for instrument selection include: 1) evaluating the number of variants to incorporate and their p-value, minor allele frequency (MAF) and whether they are palindromic; 2) selecting independent variants; 3) avoiding sample overlap between the discovery GWAS and the data under study (where possible); 4) prioritisation of GWAS with well-measured/defined phenotypes and determine whether to use a continuous or a binary exposure; 5) choosing variants based on their total and average strength and; 6) taking into account confounders of the genetic instrument-outcome relationship. If the instruments are not suitable, they could be selected from a different GWA study, which could even mean choosing them from an older one. In addition, combining the SNPs into a single instrument is another option if various studies report adequate variants. Where possible, it is best practice to choose SNPs found both in the discovery dataset and in a replication cohort, as these are likely to be more reliable.</p>", "<p id=\"P19\">In ##TAB##0##Table 1##, we present the latest GWAS of sleep phenotypes (for a detailed list of SNPs reported in the GWAS see ##SUPPL##0##Supplementary Table 1##). Of note is that some sleep traits are still lacking robust instruments. This is the case of sleep quality and multidimensional sleep (whereby rather than a series of single separate characteristics, sleep is thought of as a multidimensional construct with domains including regularity, satisfaction, alertness/sleepiness, timing, efficiency, and duration, among others) [##REF##24470692##77##,##UREF##13##78##].</p>", "<title>P-value</title>", "<p id=\"P20\">A common statistical approach, and usually the first step in MR studies, is to evaluate the level of statistical significance of the genetic variants associated with the exposure of interest and to include all variants at a given level of significance. The conventional threshold is p&lt;5×10<sup>−8</sup> [##UREF##2##12##]. This threshold is the equivalent of p&lt;0.05 when corrected for the multiple testing based on performing a Bonferroni correction for all the independent common SNPs across the human genome and thus, it is referred to as “genome-wide level of significance”. Using this threshold has been shown to lead to robust results [##UREF##14##79##]. Nonetheless, given the more recent mega-GWAS because of access to large biobanks, there have been proposals to change it to p&lt;5×10<sup>−9</sup> to decrease the chances of false-positive associations. However, the latest MR studies of sleep traits have selected variants using the traditional threshold [##REF##35349659##30##,##REF##35034128##44##,##REF##35403707##51##,##UREF##10##52##].</p>", "<p id=\"P21\">In the field of sleep, some of the first GWAS published discovered genetic variants for restless leg syndrome, a neurological disorder that causes involuntary leg movements during sleep [##REF##17634447##80##,##REF##17637780##81##]. Recently, a large GWAS reported that some of the variants previously associated with restless leg syndrome did not reach genome-wide significance, emphasising the need for stringent thresholds [##REF##35486972##82##]. Even though it is important to consider the p-value threshold, this is not the only factor to consider when selecting variants for an MR study. The following sections will discuss other steps to assess whether variants are valid genetic instruments.</p>", "<title>Linkage disequilibrium clumping</title>", "<p id=\"P22\">Linkage disequilibrium refers to the correlation between SNPs at different positions. This phenomenon occurs because of the physical proximity of variants on the chromosome [##REF##12689998##10##]. In GWAS, the reported variants are often ‘clumped’ to near independence using distance-based or correlation-based thresholds [##UREF##11##67##].</p>", "<p id=\"P23\">The distance-based approach consists of pruning the variants to include those separated by a certain distance (usually 500.000 base pairs = 500 kilobases). In the correlation-based approach, only variants that are correlated at a certain threshold (usually r<sup>2</sup>&lt;0.01, 0.1 or 0.2) are included [##UREF##11##67##]. Implementing the correlation-based approach, Broberg et al. (2021) [##REF##33608734##19##], in their study about the association between insomnia and pain, decided to use an r<sup>2</sup>=0.6 as their primary threshold and an r<sup>2</sup>=0.1 as their secondary threshold. Cullel et al. (2021) [##UREF##5##24##] and Zhou et al. (2021) [##REF##34544013##42##] clumped genetic variants considering both approaches, an r<sup>2</sup>&lt;0.001 and a kb=10,000 distance, which is more conservative.</p>", "<p id=\"P24\">It is important to consider LD when selecting the variants as it could violate core MR assumptions. Genetic variants that are correlated with the variants used may have effects on competing risk factors. The LDkit (a graphical user interface software) or PLINK (open-source C/C++ toolset) could be used for calculating linkage disequilibrium [##REF##25722852##83##,##REF##33066733##84##]. Testing the association of potential confounders of the variants could reduce concerns about making invalid inferences due to LD [##UREF##11##67##].</p>", "<title>Sample overlap</title>", "<p id=\"P25\">When selecting a GWA study, it is essential to understand in detail the sample studied. This is because when using genetic variants discovered in the analytical sample, a bias called “winner’s curse” may occur. This bias implies overestimating the strongest variant in the data under analysis [##REF##24388127##85##]. An overestimation will generally occur when the associations with confounders are stronger than expected by chance. Thus, an overlap between the genetic variant discovery dataset and the data under analysis may overestimate the variant–outcome associations and lead to false-positive results [##UREF##11##67##]. To overcome this issue, Liu et al. (2022) [##REF##35034128##44##] excluded data from participants in the UKB from their COVID-19 outcome dataset since their exposures (sleep and circadian phenotypes) were derived from this biobank.</p>", "<p id=\"P26\">The ideal situation to avoid this bias is having two non-overlapping datasets, what is called “Two-sample MR” [##UREF##2##12##]. MR-Base, a platform that integrates a database of GWAS results with an application programming interface, a web application and R packages, allows the automation of two-sample MR [##REF##29846171##86##]. However, different datasets are not always available with the data or sample size necessary to perform the analysis. To mitigate potential issues with sample overlap, there are several alternatives thought to balance the risk of an imprecise estimation [##UREF##11##67##]. One option is to calculate the bias due to sample overlap, which can be done with the formulae from Burgess, Davies &amp; Thompson (2016) [##REF##27625185##87##]. Henry et al. (2019) [##REF##31062029##45##] did this in their MR study about the impact of sleep duration on cognitive outcomes. In their study about the association of insomnia with depressive symptoms and subjective well-being, Zhou et al. (2021) [##REF##34544013##42##] also calculated sample overlap finding a bias ranging from 3% to 14%.</p>", "<p id=\"P27\">Another possible solution is to perform the MR analysis using a reduced genetic instrument replicated in an independent cohort, which could be a good option as a sensitivity analysis for studies that are unable to bypass sample overlap. In our own MR study, which examined the association between genetically-instrumented habitual daytime napping (using 92 SNPs) and cognitive function and brain volume, we replicated our findings using a reduced instrument consisting of 17 SNPs that were replicated in an independent cohort (23andMe) with no sample overlap with UKB (our analytical sample) [##UREF##15##88##]. Additional analyses with this reduced instrument were largely consistent with our main findings. We are unaware of other studies using genetic instruments of sleep traits taking this approach. However, a study which investigated the relationship between glycaemia and cognitive function, brain structure and incident dementia, used a reduced genetic instrument for diabetes to avoid the “Winner’s curse” bias [##REF##33632741##89##].</p>", "<title>Type of exposure</title>", "<p id=\"P28\">When deciding which GWA study to select, it is important to prioritise well-measured/defined phenotypes used for identifying the genetic instruments. One aspect to consider is whether the phenotype was measured using self-reported data or an objective method (e.g. accelerometer-derived data). Many of the GWAS of sleep traits available are based on self-report questions, but some used and/or have been replicated with accelerometer-derived data, polysomnography or electronic medical records [##REF##29077507##69##,##REF##32332799##90##,##REF##30531941##91##]. Moreover, those using self-reported data sometimes have support from objective measures. For example, Dashti et al. (2019) [##REF##30846698##92##] tested whether the 78 loci found for self-reported habitual sleep duration (using a question on hours of sleep) in their GWA study were also associated with accelerometer-derived sleep estimates. Another study by Dashti et al. (2019) [##UREF##6##27##] found that the variants were also valid when sleep duration was determined by bed and wake times. Ideally, genetic instruments discovered and replicated based on objective data should be selected.</p>", "<p id=\"P29\">Moreover, it is essential to understand how the exposure was analysed. For example, Lane et al. (2019) [##REF##30804566##93##] performed two parallel GWAS for frequent and any insomnia symptoms based on participants’ responses to the question “Do you have trouble falling asleep at night, or do you wake up in the middle of the night?”. For frequent insomnia, they considered participants who responded “usually” as cases and “never/rarely” as controls, with those reporting “sometimes” being excluded. For any insomnia, they considered participants who responded “sometimes” or “usually” as cases and “never/rarely” as controls. On the contrary, a GWA study by Jansen et al. (2019) [##REF##30804565##8##], using the same question, defined insomnia cases as participants who answered “usually”, while participants who answered “never/rarely” or “sometimes” were defined as controls. In this example, insomnia symptoms are analysed in three different ways using the same underlying question. Understanding how the exposure was measured is crucial for adequately interpreting results.</p>", "<p id=\"P30\">Another crucial aspect of genetic instrument selection is whether the exposure is continuous or binary. It is well-established that continuous measures should be used where possible in MR [##REF##30039250##94##]. However, using continuous exposures has the caveat that sometimes MR studies aim to test whether a particular disease status (e.g., insomnia) might be causally related to a specific outcome. Furthermore, some sleep traits are often considered binary: chronotype (evening vs. morning types), napping (frequent vs. infrequent nappers), and duration (longer vs. shorter sleepers), amongst others. In the case of using a binary exposure, it is important to be aware of its limitations. Burgess and Labrecque’s paper (2018) [##REF##30039250##94##] explained that the problem arises when using a binary exposure that dichotomises a continuous variable (e.g. short/long sleep arises from dichotomised sleep duration). In the cases of MR studies using these types of exposures, the results should be conceptualised in terms of the underlying continuous risk factor.</p>", "<title>Total (R<sup>2</sup>) and average strength (F-statistic) metrics</title>", "<p id=\"P31\">Selection of genetic instruments is often conducted by considering each variant’s effect size to avoid weak-instrument bias. This bias can occur when the genetic instruments explain a small proportion of the variance in the exposure. Weak instruments may lead to non-robust results and bias the estimates towards the confounded observational estimate [##REF##21414999##95##].</p>", "<p id=\"P32\">Some of the most commonly used effect indicators are the proportion of the phenotypic variance explained by all of the genome-wide significant SNPs (R<sup>2</sup>) and the F-statistic obtained from regressing the exposure on the genetic instrument (in a multivariable linear regression) [##REF##27342221##62##]. The R2 provides information about the total strength of the genetic variant, and usually, the larger, the better. Swerdlow et al. (2016) [##REF##27342221##62##] argue that the R2 is the most useful effect metric when selecting genetic instruments for MR analysis. However, the F-statistic provides information about the average strength of the instrument, with an F&gt;10 indicating that substantial weak instrument bias is unlikely [##REF##21414999##95##].</p>", "<p id=\"P33\">Several options for obtaining F-statistics are available. If individual-level data are available for the exposure, the ‘Individual-level data regression’ approach can be performed. However, if individual-level data are not available and the R<sup>2</sup> from the exposure GWA study is obtainable, the Cragg-Donald F-statistic method may be used [##REF##21414999##95##]. This method uses the R<sup>2</sup>, sample size (n), and the number of instruments (k) to calculate the statistic (F=(n−k−1/k) (R<sup>2</sup>/1−R<sup>2</sup>)) [##UREF##11##67##]. Liu et al. (2021) [##REF##34315237##31##] used this formula reporting a F-statistic of 143.24 in their study about the association between genetically-instrumented insomnia and cardiovascular diseases. When the R<sup>2</sup> is unknown, the ‘t-statistic’ summary-level method can be used (F=ß<sup>2</sup>/SE<sup>2</sup>). In this case, the F-statistic will be an approximation because it uses the sample size for the discovery GWA study, not the one from the data under analysis. Finally, the “MendelianRandomization” R package allows the calculation of the F-statistic [##REF##28398548##13##].</p>", "<title>Number of SNPs</title>", "<p id=\"P34\">MR studies including large numbers of genetic variants are rapidly increasing. This growth is related to the proliferation of GWAS and the desire to obtain more precise estimates. However, as previously discussed, not all variants are valid IVs [##REF##26050253##96##], and an enlarged set of genetic instruments is not always better [##UREF##2##12##]. Selecting a large number of variants could lead to a larger R<sup>2</sup> but a weaker F-statistic and greater chances of including pleiotropic variants, violating a core MR assumption. Including more variants also allows the use of more robust methods, including common sensitivity analyses such as the MR-Egger test. On the contrary, fewer variants will lead to a lower R<sup>2</sup> but potentially a greater F-statistic, which could lead to an instrument with insufficient power [##REF##26050253##96##].</p>", "<p id=\"P35\">To understand how the strength of the instrument depends on the number of SNPs, we present Liu et al. (2022) [##REF##35349659##30##] study on the relationship between sleep traits and glycated haemoglobin -HbA1c- (see ##TAB##1##Table 2##). In this study, the F-statistic for all exposures was higher than 10 (which indicates an appropriate average strength), while the R<sup>2</sup> ranged between 0,06 and 2,09%. In the case of long sleep duration, including fewer variants (five) lead to a low total strength (R<sup>2</sup>=0,06%) and a good average strength (F-statistic=41).</p>", "<title>Minor allele frequency and palindromic variants</title>", "<p id=\"P36\">MAF is the proportion of minor alleles for a specific SNP in a given population [##UREF##11##67##]. In other words, it is the frequency at which the second most common allele occurs. Usually, GWAS identify common variants [##UREF##16##97##]; however, SNPs with a wide distribution of MAFs can sometimes be included. Some MR studies exclude variants with a low MAF because causal estimates from those variants may have low precision [##REF##26050253##96##,##REF##20018033##98##]. For example, Chen et al. (2021) (Chen et al., 2021) decided to remove variants with a MAF&lt;1% in their study about the association between sleep traits and low bone mineral density. However, excluding variants with low MAF could mean removing variants associated with the exposure of interest. For example, low-frequency variants in <italic toggle=\"yes\">PERIOD3</italic> have been associated with chronotype [##REF##30696823##7##] and familial advanced sleep phase syndrome [##UREF##17##99##].</p>", "<p id=\"P37\">Another potential problem is palindromic variants because they can introduce ambiguity into the identification of the effect allele. A palindromic SNP occurs when the two possible alleles are complementary base pairs [##REF##29846171##86##]. Additional care should be taken with palindromic variants because studies might report effects of the same SNP using different strands (e.g. a study reports an SNP with A/G alleles and another with T/C alleles). In those cases, the ambiguity can be identified if the effect allele frequency is reported and the MAF is substantially below 50% [##REF##28338968##100##]. For example, if a specific SNP has alleles A/T, with allele A frequency being 0.11 in the GWA study and 0.91 in the data under study (both coding this allele as the effect allele) and both studies have the same ethnic origin, this means that authors used different reference strands. In this case, it is necessary to switch the direction of the effect in either the discovery GWA study or the analytical sample, a procedure called variant harmonisation [##REF##29846171##86##].</p>", "<p id=\"P38\">However, if it is not possible to verify that alleles are correctly orientated, it may be necessary to take some precautions [##UREF##11##67##]. There are options to deal with this problem: replace the variants with suitable, non-palindromic linkage disequilibrium proxies, perform sensitivity analyses to evaluate the impact of these variants on the results or exclude them [##REF##28338968##100##]. For example, in a study by Alimenti et al. (2021) [##REF##34095735##16##] about causal links between habitual sleep duration/napping and macronutrient composition palindromic SNPs with MAF close to 0.50 were excluded and the remaining palindromic instruments were aligned based on their MAF.</p>", "<title>Confounding</title>", "<p id=\"P39\">The third MR assumption states that the genetic variant-outcome association is unconfounded [##REF##30002074##101##]. Violations of this assumption could be due to at least two different types of confounding. One is confounding by ancestry (e.g., if SNPs associated with sleep duration have higher/lower frequencies in different ancestry groups in the sample under study and additionally, cultural differences have an impact on the outcome under study), which could be controlled by restricting the sample to a single ancestry group, and/or adjusting for principal components of ancestry. A second source of confounding occurs if SNPs associated with the exposure of interest are also associated with common confounders of the relationship under study. One of the advantages of MR is that it exploits the fact that genotypes are not generally associated with confounders. However, such associations may occur, especially when using weak instruments or small samples [##UREF##11##67##]. Thus, it is important to test whether the genetic instruments are associated with confounders of the exposure outcome relationship [##REF##12689998##10##].</p>", "<p id=\"P40\">To address this issue, authors must first identify common confounders previously reported between their exposures and outcomes. For example, in the case of the association between obstructive sleep apnea and hypertension, weight and age are proposed as two of the main confounding factors in this putative relationship [##REF##19567173##102##]. For the long sleep-mortality association, some authors have argued that depression is most likely to confound this relationship [##REF##17625932##103##]. Therefore, it is essential that, regardless of the exposure of interest, a literature review is carried out to identify the confounders to be considered.</p>", "<p id=\"P41\">Then, authors often statistically test associations between their genetic instrument and variables reported in the literature as potential confounders in the exposure-outcome association. This is crucial as MR aims to give causal estimates that are not biased due to confounding [##UREF##11##67##]. In the MR study performed by Henry et al. (2019) [##REF##31062029##45##], the authors explored the validity of their instruments by testing associations of potential confounders (such as sex, age, educational attainment and use of sleep-inducing medication) with their sleep duration genetic score.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>Valentina Paz was supported by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, MEC-UdelaR, Uruguay), Agencia Nacional de Investigación e Innovación (ANII, Uruguay) [grant number MOV_CA_2020_1_163153], Comisión Sectorial de Investigación Científica (CSIC, UdelaR, Uruguay), Comisión Académica de Posgrados (CAP, UdelaR, Uruguay). Hassan S. Dashti was supported by the National Heart, Lung, and Blood Institute (NHLBI) [grant number K99HL153795]. Stephen Burgess was supported by the United Kingdom Research and Innovation Medical Research Council [grant number MC_UU_00002/7] and the National Institute for Health Research Cambridge Biomedical Research Centre [grant number BRC-1215-20014]. Victoria Garfield was supported by the Professor David Matthews Non-Clinical Fellowship from the Diabetes Research and Wellness Foundation [grant number SCA/01/NCF/22], and a grant from Diabetes UK [grant number 15/0005250] and the British Heart Foundation [grant number SP/16/6/32726]. The views expressed are those of the authors and not necessarily those of the National Institute for Health Research or the Department of Health and Social Care.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><title>Flowchart with the main points to consider when selecting sleep genetic instruments in MR.</title></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Genome-wide association studies of sleep phenotypes since 2016</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Author (year<sup><xref rid=\"TFN1\" ref-type=\"table-fn\">1</xref></sup>) [reference]</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Phenotype</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Phenotype measurement</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Ancestry</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Number of novel SNPs</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Number of replicated SNPs from previous studies</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Examples of health outcomes studied using these instruments</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Cade et al. (2016) [##REF##26977737##68##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Obstructive sleep apnea</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Apnea monitors and polysomnogra phy</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12,558</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Hispanic/La tino Americans</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 + 1 suggestive (P&lt;5×10<sup>−7</sup>) (Apnea-hypopnea index) / 1 + 4 suggestive (Respiratory event duration) / 2 suggestive (Sleep Sp O2)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Cancer [##REF##32112550##28##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Hu et al. (2016) [##REF##26835600##104##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Morningness</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sel-freported questions</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">89,283</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Inflammatory bowel disease [##REF##34916940##22##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Jones et al. (2016) [##REF##27494321##105##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Morningness and sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">128,266</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European (validation in Koreans)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10 (morningness) + 2 suggestive (P&lt;1x10-4) / 3 (sleep duration)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3 + 1 suggestive from Lane et al. (2016) (morningness)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Caffeine consumption [##REF##29682839##60##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Lane et al. (2016) [##REF##26955885##106##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Chronotype</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">100,420</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Hammersch ag et al. (2017) [##REF##28604731##75##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>l</bold> Insomnia symptoms</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question validated with questionnaires and a structured interview</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">113,006</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11 + 2 suggestive (P&lt;2×10<sup>−3</sup>)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 + 1 suggestive from Lane et al. (2017)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Caffeine consumption [##REF##29682839##60##]; Peptic ulcer disease [##UREF##7##40##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Lane et al. (2017) [##REF##27992416##107##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep duration, insomnia symptoms, excessive daytime sleepiness &amp; composite sleep trait<sup><xref rid=\"TFN2\" ref-type=\"table-fn\">2</xref></sup></td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported questions</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">112,586</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 + 3 suggestive (P&lt;5×10<sup>−7</sup>) (sleep duration) / 5 + 3 suggestive (insomnia symptoms) / 3 + 7 suggestive (excessive daytime sleepiness) / 3 (composite sleep trait)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 suggestive from Jones et al. (2016) (sleep duration) / 3 from Jones et al. (2016) &amp; Lane et al. (2016) (composite sleep trait)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Schormair et al. (2017) [##REF##29029846##108##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Restless legs syndrome</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Interviews and self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">110,851</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 from Lane et al. (2017)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Parkinson’s disease [##REF##33974305##53##]; Essential tremor [##REF##30366831##109##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Chen et al. (2018)[##REF##29077507##69##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Obstructive sleep apnea</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Polysomnogra phy</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19,733</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Multiancestry</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23 suggestive (P&lt;1.0×10<sup>−6</sup>) (Apnea-Hypopnea Index total) / 10 suggestive (Apnea-Hypopnea Index-Non-Rapid Eye Movement) / 7 suggestive (Apnea-Hypopnea Index-Rapid Eye Movement).</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 suggestive from Cade et al. (2016) (Apnea-Hypopnea Index total)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Amyotrophic lateral sclerosis [##REF##35594779##57##]; Neurodegenerative diseases [##UREF##5##24##]; Parkinson’s disease [##REF##35594779##57##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Doherty et al. (2018) [##REF##30531941##91##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Accelerometer data</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91,105</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 from Lane et al. (2017)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Ferguson et al. (2018) [##REF##30120083##110##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Low relative amplitude</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Accelerometer data</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">71,500</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">‘</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Stein et al. (2018)[##REF##29520036##73##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Insomnia disorder</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Questionnaire</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">17,651</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Multiancestry</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4 + 8 suggestive (P&lt;1×10<sup>−6</sup>)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Dashti et al. (2019) [##REF##30846698##6##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question supported by accelerometer data</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">446,118</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">77</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 from Jones et al. (2019)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Alzheimer’s disease [##REF##33150399##17##,##REF##32817390##111##]; Amyotrophic lateral sclerosis [##REF##35594779##57##]; Atrial fibrillation [##REF##33719330##41##]; Cancer [##REF##32895918##59##,##UREF##18##112##]; Cardiovascular diseases [##REF##33822910##15##]; COVID-19 [##REF##35034128##44##]; Dietary intake [##REF##34095735##16##]; Fracture [##REF##34837057##58##]; Glycated hemoglobin [##REF##35349659##30##]; Heart failure [##REF##33719330##41##]; Heel bone mineral density [##REF##34184784##20##]; Intracranial aneurysm and Aneurysmal subarachnoid hemorrhage [##REF##34729997##55##]; Ischemic stroke [##REF##33117014##113##]; Longevity [##REF##33107078##50##]; Major depressive disorder [##REF##32817390##111##]; Metabolomic traits [##REF##33731105##18##]; Migraine [##REF##33125193##114##]; Osteoarthritis [##REF##34890811##33##]; Parkinson’s disease [##REF##35594779##57##]; Periodontal disease [##REF##34109656##43##]; Renal function [##REF##32970283##32##]; Stroke [##REF##34596745##37##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Jansen et al. (2019) [##REF##30804565##8##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Insomnia symptoms</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,331,0 10</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">243</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5 from Jones et al. (2016, 2019), Lane et al. (2017) &amp; Doherty et al. (2018)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Asthma [##REF##34750467##29##]; Body composition [##UREF##10##52##]; Cancer [##REF##34925448##35##,##REF##35403707##51##]; Cardiovascular conditions [##REF##34315237##31##,##REF##33712906##39##]; Depressive symptoms [##REF##34544013##42##]; Fracture [##REF##34837057##58##]; Hypertension [##REF##33131310##115##]; Intracranial aneurysm and Aneurysmal subarachnoid hemorrhage [##REF##34729997##55##]; Longevity [##REF##33107078##50##]; Major depressive disorder [##REF##33122081##116##]; Metabolomic traits [##REF##33731105##18##]; Migraine [##UREF##4##23##]; Osteoarthritis [##REF##34890811##33##]; Pain diagnoses [##REF##33608734##19##]; Subjective well-being [##REF##34544013##42##]; Suicidal behavior [##UREF##8##46##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Jones et al. (2019) [##REF##30696823##7##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Morningness</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">697,828</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">344</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7 from Hu et al. (2016), Lane et al. (2016, 2017) &amp; Jones et al. (2016)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Alzheimer’s disease [##REF##33150399##17##,##REF##32817390##111##]; Amyotrophic lateral sclerosis [##REF##35594779##57##]; Cancer [##REF##33878190##36##,##REF##35403707##51##,##UREF##18##112##]; COVID-19 [##REF##35034128##44##]; Depressive symtoms [##REF##34099873##34##]; Food intake [##REF##32860398##26##]; Fracture [##REF##34837057##58##]; General anxiety disorder [##REF##34099873##34##]; General wellbeing [##REF##34099873##34##]; Glycated hemoglobin [##REF##35349659##30##]; Heel bone mineral density [##REF##34184784##20##]; Inflammatory bowel disease [##REF##34916940##22##]; Ischemic stroke [##REF##33117014##113##]; Major depressive disorder [##REF##34037671##25##,##REF##34099873##34##,##REF##32817390##111##]; Metabolomic traits [##REF##33731105##18##]; Migraine [##REF##33125193##114##]; Neurodegenerative diseases [##UREF##5##24##]; Parkinson’s disease [##REF##35594779##57##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Jones et al. (2019) [##REF##30952852##117##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep traits derived by acceleromete r data</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Accelerometer data</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">85,670</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9 (sleep duration) / 1 (sleep midpoint) / 4 (sleep eficiency) / 20 (number of sleep episodes).</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 from Lane et al. (2017) &amp; Doherty et al. (2018) (sleep duration) / 1 from Lane et al. (2017) (sleep eficiency) / 1 from Jansen et al. (2019) (number of sleep episodes).</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Alzheimer’s disease [##REF##33150399##17##,##REF##32817390##111##]; Amyotrophic lateral sclerosis [##REF##35594779##57##]; Cancer [##REF##33878190##36##]; Major depressive disorder [##REF##32817390##111##]; Neurodegenerative diseases [##UREF##5##24##]; Parkinson’s disease [##REF##35594779##57##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Lane et al. (2019) [##REF##30804566##93##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Insomnia symptoms</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">453,379</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6 from Lane et al. (2017), Doherty et al. (2018)&amp; Jansen et al. (2019)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Alzheimer’s disease [##REF##33150399##17##,##REF##32817390##111##]; Amyotrophic lateral sclerosis [##REF##35594779##57##]; Cancer [##UREF##18##112##]; COVID-19 [##REF##35034128##44##]; Heel bone mineral density [##REF##34184784##20##]; Inflammatory bowel disease [##REF##34916940##22##]; Ischemic stroke [##REF##33117014##113##]; Major depressive disorder [##REF##32817390##111##]; Migraine [##REF##33125193##114##]; Neurodegenerative diseases [##UREF##5##24##]; Pain diagnoses [##REF##33608734##19##]; Parkinson’s disease [##REF##35594779##57##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Wang et al. (2019) [##REF##31409809##118##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Daytime sleepiness</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">452,071</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 from Jones et al. (2016) &amp; Jansen et al. (2019)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Alzheimer’s disease [##REF##33150399##17##]; Amyotrophic lateral sclerosis [##REF##35594779##57##]; COVID-19 [##REF##35034128##44##]; Glycated hemoglobin [##REF##35349659##30##]; Heel bone mineral density [##REF##34184784##20##]; Inflammatory bowel disease [##REF##34916940##22##]; Migraine [##REF##33125193##114##]; Neurodegenerative diseases [##UREF##5##24##]; Parkinson’s disease [##REF##35594779##57##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Campos et al. (2020) [##REF##32060260##119##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Snoring</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">408,000</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Amyotrophic lateral sclerosis [##REF##35594779##57##]; Atrial fibrillation [##REF##35052444##54##]; Body mass index [##REF##35052444##54##]; Fracture [##REF##34837057##58##]; Major depressive disorder [##REF##35141322##56##]; Parkinson’s disease [##REF##35594779##57##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Didriksen et al. (2020) [##REF##33239738##120##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Restless legs syndrome</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Clinical diagnosis and questionnaire</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">480,982</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3 + 2 suggestive at (P&lt;7×10<sup>−7</sup>)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20 from Lane et al. (2017) &amp; Schormair et al. (2017)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Farias Tempaku et al. (2020) [##REF##31786426##70##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Obstructive sleep apnea</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Polysomnogra phy</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1074</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Multiancestry</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 + 21 suggestive (P&lt;5×10<sup>−6</sup>)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Song et al. (2020) [##REF##32332799##90##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Insomnia disorder</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Electronic health records</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">18,055</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Dashti et al. (2021) [##REF##33568662##121##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Daytime napping</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported question</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">452,633</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">119</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4 from Jones et al. (2016, 2019) &amp; Jansen et al. (2019)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">COVID-19<bold> [##REF##35034128##44##];</bold> Dietary intake [##REF##34095735##16##]; Glycated hemoglobin [##REF##35349659##30##]; Inflammatory bowel disease [##REF##34916940##22##]; Migraine [##REF##33125193##114##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>Khoury et al. (2021)</bold> [##REF##33034629##71##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep quality</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Questionnaire</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2868</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Multiancestry</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3 + 11 suggestive (P≤5×10<sup>−7</sup>)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Strausz et al. (2021) [##REF##33243845##122##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Obstructive sleep apnea</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Electronic health records</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">217,955</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Atrial fibrillation [##REF##35172829##21##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Yao et al. (2022) [##REF##35023977##123##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep health score<sup><xref rid=\"TFN3\" ref-type=\"table-fn\">3</xref></sup></td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported questions</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">336,463</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 from Lane et al. (2017)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Watanabe et al. (2022) [##REF##35835914##65##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Insomnia symptoms</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported questions</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,365,0 10</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">European</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">364</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">190 from Jansen et al. (2019), Lane et al. (2017, 2019) &amp; Hammerschlag et al. (2017)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sepsis [##REF##37556136##124##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Austin-Zimmerman et al. (2023) [##REF##37770476##74##]</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Self-reported questions</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">493,142</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Multi-ancestry</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">71 (short sleep duration)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13 (short sleep duration) + 1 (long sleep duration) from Gottlieb et al. (2015) &amp; Song et al. (2020)</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Instrument strength metrics from Liu et al. (2022)</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"6\"/></colgroup><thead><tr><th align=\"left\" valign=\"middle\" colspan=\"6\" rowspan=\"1\">Information of the discovery GWAS</th><th align=\"left\" valign=\"middle\" colspan=\"3\" rowspan=\"1\">Two-sample MR</th></tr><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Author (year) [reference]</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Trait</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Cohort</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">nSNPs identified</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">R<sup>2</sup></th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">nSNPs merged</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">R<sup>2</sup><xref rid=\"TFN5\" ref-type=\"table-fn\">*</xref></th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">F-statistics<xref rid=\"TFN6\" ref-type=\"table-fn\">**</xref></th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Jansen et al. (2019) [##REF##30804565##8##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Insomnia symptoms</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.331.010</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB/23andMe</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">248</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,60%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">179</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,55%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Dashti et al. (2019) [##REF##30846698##92##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">446.118</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,69%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,49%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Dashti et al. (2019) [##REF##30846698##92##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Short sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">411.934</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,13%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Dashti et al. (2019) [##REF##30846698##92##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Long sleep duration</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">339.926</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,06%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Wang et al. (2019) [##REF##31409809##118##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Daytime sleepiness</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">452.071</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,25%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">44</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Dashti et al. (2021) [##REF##33568662##121##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Daytime napping</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">993.966</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB/23andMe</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">108</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,10%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0,56%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">79</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Jones et al. (2019) [##REF##30696823##7##]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Chronotype</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">697.828</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">UKB/23andMe</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">351</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">250</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,09%</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">60</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Table S1</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P43\">\n<bold>Conflict of interest</bold>\n</p><p id=\"P44\">No authors have any conflict of interest to declare.</p></fn><fn id=\"FN2\" fn-type=\"con\"><p id=\"P45\">\n<bold>Author contributorship</bold>\n</p><p id=\"P46\">VP wrote the review, and HSD, SB, and VG made suggestions and additions to the text. All authors approved the final version of the manuscript.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><label>1</label><p id=\"P47\">We reported studies since 2016 because at that time there was a proliferation of GWAS of sleep phenotypes (previously most studies were done on Restless legs syndrome).</p></fn><fn id=\"TFN2\"><label>2</label><p id=\"P48\">Composite trait of sleep duration, insomnia symptoms, excessive daytime sleepiness and chronotype.</p></fn><fn id=\"TFN3\"><label>3</label><p id=\"P49\">Overall assessment of sleep duration, snoring, insomnia symptoms, chronotype, and daytime dozing.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN4\"><p id=\"P50\">GWAS: Genome-wide association studies; nSNPs: number of single-nucleotide polymorphism; UKB: UK Biobank.</p></fn><fn id=\"TFN5\"><label>*</label><p id=\"P51\">R<sup>2</sup> was calculated via Sum(R2i==K*Fi/(N--K--1+K*Fi), K=1, Fi=BetaXGi^2/seBetaXGi^2 (BetaXGi and seBetaXGi were obtained from the discovery GWAS).</p></fn><fn id=\"TFN6\"><label>**</label><p id=\"P52\">F statistic was calculated via Cragg-Donald method.</p></fn></table-wrap-foot>" ]
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[{"label": ["[1]"], "person-group": ["\n"], "surname": ["Borbely"], "given-names": ["AA"], "article-title": ["A Two Process Model of Sleep Regulation"], "year": ["1982"], "fpage": ["11"]}, {"label": ["[3]"], "person-group": ["\n"], "surname": ["Kalmbach", "Schneider", "Cheung", "Bertrand", "Kariharan", "Pack"], "given-names": ["DA", "LD", "J", "SJ", "T", "AI"], "article-title": ["Genetic Basis of Chronotype in Humans: Insights From Three Landmark GWAS"], "source": ["Sleep"], "year": ["2017"], "volume": ["40"], "pub-id": ["10.1093/sleep/zsw048"]}, {"label": ["[12]"], "person-group": ["\n"], "surname": ["Burgess", "Davey Smith", "Davies", "Dudbridge", "Gill", "Glymour"], "given-names": ["S", "G", "NM", "F", "D", "MM"], "article-title": ["Guidelines for performing Mendelian randomization investigations"], "source": ["Wellcome Open Res"], "year": ["2020"], "volume": ["4"], "fpage": ["186"], "pub-id": ["10.12688/wellcomeopenres.15555.2"]}, {"label": ["[14]"], "person-group": ["\n"], "surname": ["Spiller", "Davies", "Palmer"], "given-names": ["W", "NM", "TM"], "article-title": ["Software application profile: mrrobust \u2014 a tool for performing two-sample summary Mendelian randomization analyses"], "source": ["Int J Epidemiol"], "year": ["2019"], "volume": ["48"], "fpage": ["684"], "lpage": ["90"], "pub-id": ["10.1093/ije/dyy195"]}, {"label": ["[23]"], "person-group": ["\n"], "surname": ["Chu", "Wu", "Wu", "Wu", "Qian"], "given-names": ["S", "Z", "Z", "J", "Y"], "article-title": ["Association Between Insomnia and Migraine Risk: A Case\u2013Control and Bidirectional Mendelian Randomization Study"], "source": ["Pharmacogenomics Pers Med"], "year": ["2021"], "volume": ["14"], "fpage": ["971"], "lpage": ["6"], "pub-id": ["10.2147/PGPM.S305780"]}, {"label": ["[24]"], "person-group": ["\n"], "surname": ["Cullell", "C\u00e1rcel-M\u00e1rquez", "Gallego-F\u00e1brega", "Mui\u00f1o", "Lluci\u00e0-Carol", "Lled\u00f3s"], "given-names": ["N", "J", "C", "E", "L", "M"], "article-title": ["Sleep/wake cycle alterations as a cause of neurodegenerative diseases: A Mendelian randomization study"], "source": ["Neurobiol Aging"], "year": ["2021"], "volume": ["106"], "fpage": ["320.e1"], "lpage": ["320.e12"], "pub-id": ["10.1016/j.neurobiolaging.2021.05.008"]}, {"label": ["[27]"], "person-group": ["\n"], "surname": ["Dashti", "Redline", "Saxena"], "given-names": ["HS", "S", "R"], "article-title": ["Polygenic risk score identifies associations between sleep duration and diseases determined from an electronic medical record biobank"], "source": ["Sleep"], "year": ["2019"], "volume": ["42"], "pub-id": ["10.1093/sleep/zsy247"]}, {"label": ["[40]"], "person-group": ["\n"], "surname": ["Zha", "Dong", "Wang", "Chen", "Wu", "Zhou"], "given-names": ["L-F", "J-T", "J-L", "Q-W", "J-F", "Y-C"], "article-title": ["Effects of Insomnia on Peptic Ulcer Disease Using Mendelian Randomization"], "source": ["Oxid Med Cell Longev"], "year": ["2021"], "volume": ["2021"], "fpage": ["1"], "lpage": ["11"], "pub-id": ["10.1155/2021/2216314"]}, {"label": ["[46]"], "person-group": ["\n"], "surname": ["Nassan", "Daghlas", "Winkelman", "Dashti", "Saxena"], "given-names": ["M", "I", "JW", "HS", "R"], "collab": ["International Suicide Genetics Consortium"], "article-title": ["Genetic evidence for a potential causal relationship between insomnia symptoms and suicidal behavior: a Mendelian randomization study"], "source": ["Neuropsychopharmacology"], "year": ["2022"], "pub-id": ["10.1038/s41386-022-01319-z"]}, {"label": ["[47]"], "person-group": ["\n"], "surname": ["Paz", "Dashti", "Garfield"], "given-names": ["V", "HS", "V"], "article-title": ["Is there an association between daytime napping, cognitive function and brain volume? A Mendelian randomisation study in the UK Biobank"], "source": ["Prepr MedRxiv"], "year": ["2022"], "pub-id": ["10.1101/2021.09.28.21264215"]}, {"label": ["[52]"], "person-group": ["\n"], "surname": ["Chen", "Li", "Cheng", "Pan", "Zhang", "Zhang"], "given-names": ["Y", "C", "S", "C", "H", "J"], "article-title": ["The Causal Relationships Between Sleep-related Phenotypes and Body Composition: A Mendelian Randomized Study"], "source": ["J Clin Endocrinol Metab"], "year": ["2022"], "elocation-id": ["dgac234"], "pub-id": ["10.1210/clinem/dgac234"]}, {"label": ["[67]"], "person-group": ["\n"], "surname": ["Burgess", "Thompson"], "given-names": ["S", "SG"], "article-title": ["Mendelian Randomization: Methods for Causal Inference Using Genetic Variants"], "source": ["CRC Press"], "year": ["2021"]}, {"label": ["[76]"], "person-group": ["\n"], "surname": ["Mong", "Cusmano"], "given-names": ["JA", "DM"], "article-title": ["Sex differences in sleep: impact of biological sex and sex steroids"], "source": ["Philos Trans R Soc B Biol Sci"], "year": ["2016"], "volume": ["371"], "elocation-id": ["20150110"], "pub-id": ["10.1098/rstb.2015.0110"]}, {"label": ["[78]"], "person-group": ["\n"], "surname": ["Wallace", "Buysse", "Redline", "Stone", "Ensrud", "Leng"], "given-names": ["ML", "DJ", "S", "KL", "K", "Y"], "article-title": ["Multidimensional Sleep and Mortality in Older Adults: A Machine-Learning Comparison With Other Risk Factors"], "source": ["J Gerontol Ser A"], "year": ["2019"], "volume": ["74"], "fpage": ["1903"], "lpage": ["9"], "pub-id": ["10.1093/gerona/glz044"]}, {"label": ["[79]"], "person-group": ["\n"], "surname": ["Chen", "Boehnke", "Wen", "Mukherjee"], "given-names": ["Z", "M", "X", "B"], "article-title": ["Revisiting the genome-wide significance threshold for common variant GWAS"], "year": ["2021"]}, {"label": ["[88]"], "person-group": ["\n"], "surname": ["Paz", "Dashti", "Garfield"], "given-names": ["V", "HS", "V"], "article-title": ["Is there an association between daytime napping, cognitive function, and brain volume? A Mendelian randomization study in the UK Biobank"], "source": ["Sleep Health"], "year": ["2023"], "elocation-id": ["S235272182300089X"], "pub-id": ["10.1016/j.sleh.2023.05.002"]}, {"label": ["[97]"], "person-group": ["\n", "\n"], "surname": ["Dehghan", "Evangelou"], "given-names": ["A", "E"], "part-title": ["Genome-Wide Association Studies"], "source": ["Genet Epidemiol"], "publisher-name": ["Springer New York"], "publisher-loc": ["New York, NY"], "year": ["2018"], "volume": ["1793"], "fpage": ["37"], "lpage": ["49"], "pub-id": ["10.1007/978-1-4939-7868-7_4"]}, {"label": ["[99]"], "person-group": ["\n"], "surname": ["Zhang", "Hirano", "Hsu", "Jones", "Sakai", "Okuro"], "given-names": ["L", "A", "P-K", "CR", "N", "M"], "article-title": ["A "], "italic": ["PERIOD3"], "source": ["Proc Natl Acad Sci"], "year": ["2016"], "volume": ["113"], "pub-id": ["10.1073/pnas.1600039113"]}, {"label": ["[112]"], "person-group": ["\n"], "surname": ["Richmond", "Anderson", "Dashti", "Jones", "Lane", "Strand"], "given-names": ["RC", "EL", "HS", "SE", "JM", "LB"], "article-title": ["Investigating causal relations between sleep traits and risk of breast cancer in women: mendelian randomisation study"], "source": ["BMJ"], "year": ["2019"], "elocation-id": ["12327"], "pub-id": ["10.1136/bmj.l2327"]}]
{ "acronym": [ "GWAS", "IV", "LD", "MAF", "MR", "SNP", "UKB" ], "definition": [ "Genome-wide association studies", "Instrumental variable", "Linkage disequilibrium", "Minor allele frequency", "Mendelian randomisation", "Single-nucleotide polymorphism", "UK Biobank" ] }
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CC BY
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2024-01-13 00:07:36
Sleep Med. 2023 Dec 1; 112:342-351
oa_package/00/59/PMC7615498.tar.gz
PMC7615502
37827864
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[ "<p id=\"P1\">The therapeutic potential of interleukin (IL)-2 in cancer treatment has been known for decades, yet its widespread adoption in clinical practice remains limited. Recently, chimeric proteins of an anti-PD-1 antibody and suboptimal IL-2 variants were shown to stimulate potent antitumor and antiviral immunity by inducing unique effector CD8<sup>+</sup> T cells in mice. A similar subset of cytotoxic T cells is induced by depletion of regulatory T cells (Tregs), suggesting IL-2 sequestration as a major mechanism through which regulatory T cells suppress activated CD8<sup>+</sup> T cells. Here, we present our view of how IL-2-based biologicals can boost the antitumor response at a cellular level, and propose that the role of Tregs following such treatments may have been previously overestimated.</p>" ]
[ "<title>IL-2 in T cell biology and immunotherapy</title>", "<p id=\"P2\">IL-2 is a key signaling glycoprotein produced by antigen-activated T cells, which promotes T cell survival, proliferation, and differentiation. T cell antigen receptor (TCR) as well as <xref rid=\"G10\" ref-type=\"other\">IL-2 receptor</xref> (IL-2R; see ##BOX##3##Glossary##) signaling induces the expression of IL-2Rα (CD25), which is a part of the high-affinity trimeric IL-2 receptor [##REF##20732639##1##]. This constitutes a potent amplification of the IL-2-mediated boost, which is more important for cytotoxic CD8<sup>+</sup> T cells than for helper CD4<sup>+</sup> T cells [##REF##28159902##2##–##REF##27595233##4##]. Due to the unique biological properties of IL-2, various <xref rid=\"G6\" ref-type=\"other\">IL-2-based immunotherapies</xref> have been considered for treating cancer and autoimmune diseases (##BOX##2##Box 1##) [##REF##35217787##5##,##REF##36350987##6##].</p>", "<p id=\"P3\">Recently, several studies combined IL-2 agonists with immune checkpoint therapies to treat cancer and chronic infections in preclinical studies [##REF##35104810##7##–##REF##36171288##11##]. Here, we focus on these emerging findings to present our view whereby we argue that a major mechanism of IL-2-mediated immunotherapy is the alleviation of Treg-mediated suppression and the induction of superior cytotoxic CD8<sup>+</sup> T cells.</p>", "<title>Targeting intermediate-affinity IL-2 on PD-1+ T cells has a potent antitumor effect</title>", "<p id=\"P4\">Clinical usage of IL-2 is limited by not only severe adverse effects caused by off-target stimulation of endothelial cells in lungs, brain, and liver [##REF##27601468##12##–##REF##8564962##14##], but also its short <italic toggle=\"yes\">in vivo</italic> half-life [##REF##2317789##15##]. Moreover, until recently, the paradigm in the field proposed that IL-2Rα-binding IL-2-based molecules, such as wild-type (WT) IL-2, predominantly stimulate Tregs and, thus, are largely tolerogenic, whereas IL-2 variants with disabled/limited IL-2Rα interaction preferentially stimulate cytotoxic CD8<sup>+</sup> T cells and natural killer (NK) cells (##FIG##0##Figure 1A,B##). Accordingly, multiple engineered variants of IL-2-based therapeutics, such as <xref rid=\"G7\" ref-type=\"other\">IL-2 immunocomplexes</xref> (IL2ICx), <xref rid=\"G8\" ref-type=\"other\">IL-2 immunocytokines</xref>, or <xref rid=\"G9\" ref-type=\"other\">IL-2 muteins</xref>, were developed to avoid the adverse effects of IL-2 and to target only one arm of the immune system (i.e., cytotoxic CD8<sup>+</sup> T and NK cells for anti-cancer treatment or Tregs in the treatment of autoimmune diseases) (reviewed in [##REF##37004361##16##]).</p>", "<p id=\"P5\"><xref rid=\"G12\" ref-type=\"other\">Immune checkpoint inhibitors</xref> represent a major advance in cancer immunotherapy [##REF##32433532##17##]. However, because ICIs are efficient only in a subset of patients, combination therapies are being tested, including the potential synergy of PD-1 blockade and IL-2R agonists [##REF##35058325##18##–##REF##34255535##20##]. Several recent studies showed the robust antitumor effects of <xref rid=\"G11\" ref-type=\"other\">IL-2 variant (IL-2v)</xref>, an engineered variant of IL-2 not interacting with IL-2Rα, which is fused to monovalent or bivalent anti-PD-1 antibody (<xref rid=\"G19\" ref-type=\"other\">PD1-IL2v</xref>) in various preclinical tumor models (orthotopic pancreatic adenocarcinoma Panc02-H7-Fluc [##REF##36171284##8##] or PK5L1940 [##REF##37116489##10##] in C57/BL6 mice; spontaneous pancreatic tumors in RIP1-Tag5 transgenic C57BL/6 mice [##REF##36630914##9##,##REF##37116489##10##]; subcutaneous A20 lymphoma and Renca adenocarcinoma in BALB/C mice; and MC38 carcinoma in C57BL/6 mice [##REF##35104810##7##], as well as orthotopic GL261 glioma in C57BL/6 mice [##REF##36630914##9##]) (##FIG##0##Figure 1C,D##).</p>", "<p id=\"P6\">Blockade of PD-L1, together with WT IL-2 or PD1-IL2v, but not with nontargeted IL-2v, reduced the titers of lymphocytic choriomeningitis virus (LCMV) clone 13 in the spleen and lungs of infected mice during the chronic phase of the disease, relative to no treatment or PD-L1 blockade only [##REF##36171284##8##,##REF##36171288##11##]. Blockade of IL-2Rα disrupted the effect of WT IL-2 in this model [##REF##36171288##11##]. These experiments showed that the binding of IL-2v to the dimeric receptor IL-2Rβγ was sufficient to induce a therapeutic effect only if it was anchored to target T cells in <xref rid=\"G4\" ref-type=\"other\"><italic toggle=\"yes\">cis</italic></xref>, and argued for the importance of the trimeric engagement of IL-2R. It is not clear whether IL-2R anchoring needs to be via PD-1 or whether other T cell surface proteins would work in a similar manner.</p>", "<p id=\"P7\">Based on the above-mentioned studies [##REF##35104810##7##–##REF##36171288##11##], which used various mouse models of cancer and chronic infection, we propose that PD1-IL2v has multiple potential molecular mechanisms of action: (i) targeting of IL-2v to PD-1<sup>+</sup> tumor- or virus-specific T cells; (ii) strong IL-2Rα-independent binding to IL-2R via its anti-PD-1 antibody-mediated targeting to the cell surface in <italic toggle=\"yes\">cis;</italic> (iii) prolonged interaction with IL-2R via anchoring to PD-1 and slow internalization of the whole complex leading to the removal of PD-1 from the cell surface, as shown in human CD4<sup>+</sup> T cells [##REF##36171284##8##]; and (iv) PD-1 blockade. The inhibition of PD-1 signaling via PD1-IL2v appears to be only partial, because the addition of a blocking antibody to PD-L1 on top of PD1-IL2v further increases the antitumor effect in pancreatic adenocarcinoma in mice [##REF##35104810##7##,##REF##36630914##9##], as well as the antiviral effect in chronic LCMV infection in mice [##REF##36171284##8##].</p>", "<p id=\"P8\">Collectively, these studies attribute a candidate therapeutic potential for IL-2-based drugs largely to the stimulatory effects of this cytokine on cytotoxic CD8<sup>+</sup> T cells, particularly when combined with checkpoint blockade [##REF##35104810##7##–##REF##36171288##11##], although further preclinical and clinical testing is warranted.</p>", "<title>IL-2-based therapy can induce ‘better effectors’: a unique subset of cytotoxic T cells</title>", "<p id=\"P9\">IL-2 enhances differentiation and cytotoxic effector cell formation in the CD8<sup>+</sup> T cell compartment [##REF##30089912##21##]. However, <xref rid=\"G20\" ref-type=\"other\">single cell RNA sequencing (scRNAseq)</xref> of tumor-infiltrating lymphocytes from murine subcutaneous Panc02-H7-Fluc tumors revealed that PD1-IL2v treatment also increases the frequency of a population of ‘<xref rid=\"G1\" ref-type=\"other\">better effector</xref>’ T cells (CD8<sup>+</sup> GZMB<sup>+</sup> TIM-3<sup>−</sup> PD-1<sup>+</sup> TCF7<sup>low/−</sup>) [##REF##36171284##8##]. Similarly, PD1-IL2v treatment increased the frequency of better effector T cells in another tumor model using RIP1-Tag5 transgenic mice, which spontaneously develop solid tumors resistant to immune checkpoint blockade [##REF##36630914##9##]. Taken together, better effector T cells canexpand upon treatment with PD1-IL2v [##REF##36171284##8##,##REF##36630914##9##] (##FIG##1##Figure 2A,B##, data shown for illustrative purposes only). Accordingly, the combination of anti-PD-1 antibody and IL-2 treatment in chronic LCMV infection induced the formation of better effectors in splenic CD8<sup>+</sup> T cells [##REF##36171288##11##] (##FIG##1##Figure 2C##, data shown for illustrative purposes only). These findings suggest that PD1-IL2v induces a unique gene expression program in CD8<sup>+</sup> T cells, which leads to the formation of a subset with superior effector functionality in certain cancers and chronic infection.</p>", "<p id=\"P10\">The gene expression profile induced in murine intratumoral and splenic CD8<sup>+</sup> T cells upon IL-2 treatment in the above-mentioned models of cancer and chronic infection [##REF##35104810##7##–##REF##36630914##9##,##REF##36171288##11##] is characterized by increased expression of genes encoding cytotoxic molecules (e.g., granzymes or cathepsins), adhesion molecules, receptors for proinflammatory cytokines and chemokines (IL-18R, IFNGR, and CCR5), transcription factors (e.g., TBET/<italic toggle=\"yes\">Tbx21</italic>), interferon-response genes, NK receptors (<xref rid=\"G14\" ref-type=\"other\">killer cell lectin like receptor K1; KLRK1/NKG2D</xref>), and proinflammatory S100 proteins [##REF##29379499##22##] (##FIG##2##Figure 3A##, data shown for illustrative purposes only). Collectively, these genes are associated with a strong cytotoxic response, suggesting that induction of this gene expression profile provides superior antitumor and antiviral killing properties to these better effector cells. Although it remains to be rigorously demonstrated, we propose that the formation of better effectors is a key mechanism mediating the potent antitumor effect of IL-2-based cancer immunotherapies in preclinical mouse models.</p>", "<p id=\"P11\">Although a better effector signature has been reported by three studies [##REF##36171284##8##,##REF##36630914##9##,##REF##36171288##11##], in another study, PD1-IL2v treatment did not induce the better effector signature (NK receptors and cytotoxic molecules) in A20 lymphomas in BALB/C mice [##REF##35104810##7##]. Although the differences among gene expression signatures upon various IL-2 treatments need to be explained by further studies, we suggest that the difference is caused by the monovalency of PD1-IL2v used in the latter study [##REF##35104810##7##] (##FIG##0##Figure 1C,D##), indicating a low-avidity interaction of this molecule with target T cells. Moreover, the actual topology of the relative orientation of the IL-2v and anti-PD-1 antibody in the chimeric molecule might be important for the spatial assembly of IL-2v with the IL-2Rβγ on the cell surface, which also remains to be further tested.</p>", "<p id=\"P12\">The transcriptional profiles of CD8<sup>+</sup> T cells from chronic LCMV infection in mice treated with WT IL-2 or with WT IL-2 plus anti-PD-1 antibody are largely similar, which suggests that the differentiation of CD8<sup>+</sup> T cells into better effectors is induced by the IL-2 signal rather than by PD-1 blockade [##REF##36171288##11##] (##FIG##1##Figure 2C##). Accordingly, treatment of C57BL/6 mice bearing B16F10 tumors with IL-2ICx selective for trimeric IL-2Rαβγ increased the frequency of GZMB<sup>+</sup> and KLRK1/NGK2D<sup>+</sup> cells (corresponding to the better effector T cells) among splenic and tumor-infiltrating CD8<sup>+</sup> T cells evaluated by flow cytometry [##REF##36705564##3##].</p>", "<title>Better effector T cells are clonally expanded antigen-specific cells</title>", "<p id=\"P13\">Only some CD8<sup>+</sup> T cells differentiate into better effectors upon PD1-IL2v treatment (##FIG##1##Figure 2A–C##). Indeed, the preferential expansion of T cells specific to viral or tumor antigens, revealed as increased frequencies of CD8<sup>+</sup> T cells binding the LCMV-specific or tumor-specific MHCI-tetramers in mouse models of chronic infections and cancer [##REF##35104810##7##–##REF##36630914##9##,##REF##36171288##11##], has indicated that only antigen-stimulated CD8<sup>+</sup> T cells form better effectors. Accordingly, better effectors are highly enriched in clonally expanded CD8<sup>+</sup> T cells, as shown by the presence of multiple T cells with the same TCR in the better effector subset isolated from pancreatic tumors of PD1-IL2v-treated mice [##REF##36171284##8##,##REF##36630914##9##]. In some mouse tumor models, the therapeutic effects of IL-2-based treatment only manifest in combination with immunogenic chemotherapy (B16F10 melanoma in C57BL/6 mice and BCL1 leukemia in BALB/C mice) [##REF##36705564##3##] or irradiation (K5L1940 adenocarcinoma in C57BL/6 mice) [##REF##37116489##10##]. Most likely, the additional treatment might trigger the release and subsequent presentation of cancer antigens, which is required for better effector formation from tumor-specific T cells, although this remains conjectural.</p>", "<title>Strong IL-2 signals are required for the formation of better effectors</title>", "<p id=\"P14\">While treatment with WT IL-2 or PD1-IL2v increased the frequencies of better effectors in chronic LCMV infection or in Panc02-Fluc pancreatic adenocarcinoma, this effect was not observed upon treatment with IL-2v, which was not targeted to T cells [##REF##36171284##8##,##REF##36171288##11##] (##FIG##1##Figure 2B##). Hence, we propose that strong binding of IL-2 to its receptor, which is mediated via intact IL-2:IL-2Rα interaction or via anchoring of IL-2v to PD-1 in <italic toggle=\"yes\">cis</italic>, is required to make better effector T cells. Accordingly, ‘<xref rid=\"G22\" ref-type=\"other\">synthetic effector</xref>’ T cells [i.e., genetically engineered chicken ovalbumin (OVA)-specific <xref rid=\"G7\" ref-type=\"other\">OT-I</xref> CD8<sup>+</sup> T cells secreting nontargeted IL-2v and IL-33 alarmin] have potent antitumor activity in mice with implanted OVA-expressing B10 melanoma, but do not form better effectors [##REF##37081150##23##]. This suggests that ‘synthetic effector’ T cells work via a different mechanism compared with PD1-IL2v treatment, perhaps based on the combination of stimulation of IL-2Rβγ by IL-2v, and activation of tumor-associated dendritic cells by IL-33:IL-33R interactions, although this remains to be further tested.</p>", "<p id=\"P15\">The better effector signature genes [##REF##36171284##8##] appear to overlap with the profile of human and murine <xref rid=\"G2\" ref-type=\"other\">bystander activated CD8<sup>+</sup> T cells</xref> stimulated by IL-15 (reviewed in [##REF##36913776##24##]), a cytokine that binds to IL-2Rβγ and uses the identical signaling pathway to IL-2. IL-15-based biologicals represent another promising direction of experimental anticancer therapy (reviewed in [##REF##37251333##25##]). Recently, modified IL-15 conjugated to an anti-PD-1 monoclonal antibody (<xref rid=\"G18\" ref-type=\"other\">PD1-IL15m</xref>) was shown to inhibit the growth of B16F10 or MC38 tumors in C57BL/6 mice in a dose-dependent manner by inducing proliferation (expression of <italic toggle=\"yes\">Mki67</italic>) and cytotoxic effector gene expression (<italic toggle=\"yes\">Gzmb</italic>, <italic toggle=\"yes\">Tbx21</italic>, and <italic toggle=\"yes\">Ifng</italic>) in CD8<sup>+</sup> T cells [##REF##34376502##26##]; the results showed a striking analogy between IL-2 and IL-15 targeted to PD-1<sup>+</sup> T cells. Overall, IL-2 and IL-15 antitumor therapies might have a similar mode of action, which includes the formation of better effectors, although this remains to be further tested.</p>", "<title>T cell priming in the absence of Tregs can induce a similar gene expression program to IL-2-based therapy</title>", "<p id=\"P16\">It is well established that Tregs suppress effector T cell responses using multiple mechanisms (reviewed in [##REF##22566933##27##]), mostly based on experiments in which Tregs suppressed activated CD4<sup>+</sup> T cells. However, we argue that Tregs use IL-2 depletion as a dominant mechanism for the suppression of CD8<sup>+</sup> T cells, as shown in several studies in mice [##REF##36705564##3##,##REF##27595233##4##,##REF##21849683##28##,##REF##21502514##29##]. These studies provide multiple layers of evidence for such a conclusion. First, IL-2 serum concentrations are increased in the absence of Tregs in mice [##REF##21502514##29##]. Second, depletion of Tregs upregulates IL-2 signaling in activated CD8<sup>+</sup> T cells in mice [##REF##36705564##3##]. Third, although a high dose of IL-2 therapy causes Treg expansion in mice, it also induces effector CD8<sup>+</sup> T cell differentiation and renders mice susceptible to CD8<sup>+</sup> T cell-mediated experimental autoimmune diabetes to a similar extent to Treg depletion [##REF##36705564##3##]. This would not be expected if Tregs used predominantly IL-2-independent mechanism(s) for CD8<sup>+</sup> T cell suppression. Fourth, <italic toggle=\"yes\">Foxp3</italic><sup>Cre</sup><italic toggle=\"yes\">Il2ra</italic><sup>fl/fl</sup><italic toggle=\"yes\">Rosa26</italic><sup>Stat5bCA</sup> mice with IL-2Rα-deficient Tregs (rescued by constitutive intracellular IL-2R signaling) develop hyperproliferation of CD8<sup>+</sup>, but not CD4<sup>+</sup> T cells in lymph nodes [##REF##27595233##4##]. However, additional mechanisms of Treg-mediated suppression of CD8<sup>+</sup> T cells might also be important in particular contexts, warranting further investigation.</p>", "<p id=\"P17\">In the absence of Tregs, OVA-specific OT-I CD8<sup>+</sup> T cells form unusual effector KLRK1<sup>+</sup>IL-7Rα<sup>+</sup> (KILR) CD8<sup>+</sup> T cells after activation with their cognate antigen (intravenous injection of bone marrow-derived dendritic cells pulsed with OVA peptide) in C57BL/6 mice, as revealed by scRNAseq and flow cytometry [##REF##36705564##3##]. Of note, <xref rid=\"G13\" ref-type=\"other\">KILR T cells</xref> resemble better effectors, as documented by their upregulation of better effector signature genes (##FIG##2##Figure 3B##) and, reciprocally, by the upregulation of KILR signature genes, such as <italic toggle=\"yes\">Klrk1</italic>, <italic toggle=\"yes\">Ifitm1-3</italic>, <italic toggle=\"yes\">Cd7</italic>, and <italic toggle=\"yes\">Nkg7</italic> in CD8<sup>+</sup> T cells upon IL-2-based treatment (##FIG##2##Figure 3C##) [##REF##36705564##3##,##REF##35104810##7##–##REF##36630914##9##,##REF##36171288##11##]. Moreover, KILR T cells showed superior cytotoxic activity against adoptively co-transferred splenocytes loaded with cognate antigen in C57BL/6 mice [##REF##36705564##3##]. Putative strong cytotoxicity was also proposed as a feature of better effectors [##REF##36171284##8##]. Based on gene expression similarity and IL-2 dependency between KILR T cells and better effectors, we propose that these subsets may be related (##FIG##3##Figure 4, Key figure##) or even represent an identical subset, although this remains speculative.</p>", "<title>Stem-like cells are putative precursors of KILR and better effector T cells</title>", "<p id=\"P18\">KILR and better effector CD8<sup>+</sup> T cells express IL-7Rα, the receptor for the prosurvival cytokine IL-7 [##REF##36705564##3##,##REF##36171284##8##,##REF##36171288##11##]. This is paradoxical since: (i) IL-2 treatment <italic toggle=\"yes\">ex vivo</italic> [##REF##12354940##30##] or without antigenic activation <italic toggle=\"yes\">in vivo</italic> [##REF##36705564##3##] decreases the expression of IL-7Rα; and (ii) the expression of IL-7Rα is typical for memory, but not for effector T cells [##REF##14625547##31##]. A possible explanation of IL-7Rα expression in KILR T cells is their putative origin from memory precursors rather than from effector T cells. Accordingly, the formation of KILR T cells by the above-described OT-I T cell priming in the absence of Tregs was accompanied by a decreased frequency of conventional <xref rid=\"G23\" ref-type=\"other\">TCF7<sup>+</sup></xref> stem-like memory precursor T cells, but not classical effector T cells, suggesting that TCF7<sup>+</sup> stem-like precursor T cells are precursors of KILR T cells [##REF##36705564##3##].</p>", "<p id=\"P19\">One study described intratumoral CD8<sup>+</sup> T cells as the major target of PD1-IL2v therapy, because the inhibition of the T cell egress from the lymphoid tissues by <xref rid=\"G5\" ref-type=\"other\">FTY720 (fingolimod)</xref> did not impact the treatment efficacy of PD1-IL2v in a renal adenocarcinoma mouse model [##REF##35104810##7##]. Similarly, a negligible effect of FTY720 administration was observed in a mouse model of B16F10 melanoma, when treated with PD1-IL15m [##REF##34376502##26##]. Two studies proposed that better effectors were derived from CD8<sup>+</sup> PD-1<sup>+</sup> TCF7<sup>+</sup>\n<xref rid=\"G21\" ref-type=\"other\">stem-like T cells</xref>, based on observations that PD1-IL2v expands CD8<sup>+</sup> PD-1<sup>+</sup> TCF7<sup>+</sup> T cells in Panc02-H7-Fluc adenocarcinoma [##REF##36171284##8##], in spontaneous pancreatic tumors of RIP1-Tag5 mice [##REF##36630914##9##], and in mouse GL261 gliomas [##REF##36630914##9##], validating this T cell subset as the putative target of this therapy. Overall, current evidence suggests that PD1-IL2v therapy induces the differentiation of better effectors from intratumoral TCF7<sup>+</sup> stem-like T cells, which parallels the putative formation of KILR T cells from stem-like memory precursors in the spleen.</p>", "<title>The ‘exaggerated’ role of Tregs in IL-2-based cancer immunotherapy</title>", "<p id=\"P20\">The development of IL-2-based therapeutics has been accompanied by significant concern regarding its dual impact on tumor-specific T cells and immunosuppressive Tregs. Thus, IL-2v modifications have been developed to avoid/lower binding to the IL-2Rα subunit constitutively expressed on Tregs. However, clinical trials with these variants have not been successful, yet [##REF##37004361##16##], perhaps because of their weak binding to the high-affinity IL-2Rαβγ expressed on activated CD8<sup>+</sup> T cells.</p>", "<p id=\"P21\">Based on the above-mentioned model that Tregs might suppress CD8<sup>+</sup> T cells via sequestering IL-2, we hypothesize that the concurrent stimulation of Tregs by IL-2-based biologicals does not pose a significant complication, since Tregs would not be able to suppress CD8<sup>+</sup> T cells in the excess of exogenous IL-2R agonists. This is supported by experiments with mouse tumor models indicating that the administration of IL-2Rα-biased IL2ICx [##REF##25992858##32##] or IL-2-IL-2Rα fusion protein preferentially stimulating IL-2Rα<sup>+</sup> cells [##REF##30282751##33##] can induce the potent antitumor activity of CD8<sup>+</sup> T cells (B16F10 melanoma in C57BL/6 mice; and BCL1 leukemia and CT26 colon carcinoma in BALB/C mice) [##REF##36705564##3##,##REF##37270181##34##]. Moreover, antibody-mediated depletion of Tregs by anti-IL-2Rα antibody did not improve the survival of C57BL/6 mice bearing PK5L1940 adenocarcinoma that were treated with irradiation and PD1-IL2v [##REF##37116489##10##], suggesting that Tregs did not efficiently suppress antitumor CD8<sup>+</sup> T cells in response to the excess of exogenous IL-2R agonist. These observations are paradigm changing, since they challenge the scenario that IL-2-based biologicals targeting IL-2Rα<sup>+</sup> T cells are immunosuppressive by stimulating Treg cells [##REF##16484453##35##–##REF##29942088##41##].</p>", "<p id=\"P22\">A large proportion of Tregs express PD-1, especially in tumors, such as human gastric cancer and nonsmall cell lung cancer [##REF##35090594##42##,##REF##32868929##43##]. Thus, PD1-IL2v might induce proliferation and boost a suppressive phenotype in intratumoral Tregs, although this has not been demonstrated. However, CD8<sup>+</sup> T cells outnumbered Tregs in the tumor of PD1-IL2v-treated mice, as shown in the above-mentioned studies using pancreatic cancer and lymphoma models [##REF##35104810##7##–##REF##37116489##10##]; the resulting anti-cancer effect was evidenced by the prolonged survival and/or reduced tumor burden of mice. We propose a possible explanation for this phenomenon, whereby the intrinsic effect of PD1-IL2v on CD8<sup>+</sup> T cells would be higher than that on Tregs, perhaps because of lower expression of PD-1 and/or or IL-2Rβ on Tregs, although this remains conjectural. A second hypothetical reason might be the resistance of CD8<sup>+</sup> T cells to Treg-mediated inhibition upon IL-2 based therapy. In this scenario, Tregs might still use other mechanisms of suppression to regulate other cell types, such as effector CD4<sup>+</sup> T cells [##REF##27595233##4##], which can also contribute to tumor clearance [##REF##34619457##44##,##REF##37400675##45##].</p>", "<p id=\"P23\">Moreover, the effect of PD-1 blockade on Tregs upon PD1-IL2v treatment is unclear, since there is controversy over whether PD-1 signaling is a positive [##REF##33045061##46##] or negative [##REF##32868929##43##,##REF##31028147##47##] regulator of Treg-mediated suppression, which further complicates the elucidation of the potential role of Tregs during PD1-IL2v therapy.</p>", "<p id=\"P24\">Overall, we argue that the significance of off-target stimulation of Tregs upon IL-2-based immunotherapy is likely not as serious an issue as conventionally believed, which would open new avenues for the development of novel candidate IL-2-based antitumor treatments. Certainly, this warrants robust investigation.</p>", "<title>Concluding remarks</title>", "<p id=\"P25\">Although the original idea of using IL-2 for therapeutic purposes is not new, we are currently experiencing a boom of different strategies using IL-2 for antitumor and antiviral therapies. In particular, these include the recent utilization of chimeric molecules of IL-2 and anti-PD-1 antibody, showing excellent efficacy in preclinical mouse models of cancer and chronic viral infection [##REF##35104810##7##–##REF##36171288##11##]. Single cell transcriptomics has revealed that strong IL-2R agonists can not only promote the differentiation of effector T cells, but also induce a unique gene expression profile in CD8<sup>+</sup> T cells, which aligns with superior cytotoxic properties [##REF##36171284##8##,##REF##36630914##9##,##REF##36171288##11##]. However, one of the biggest concerns in the design of IL-2-derived biologicals is avoidance of the concomitant stimulation of Tregs. This has motivated the design of IL-2 variants that do not act on Tregs [##REF##23677467##48##–##REF##35817480##51##], but suffer from low efficacy on CD8<sup>+</sup> T cells [##REF##36171284##8##,##REF##36171288##11##]. Based on recent data, we propose that Tregs might not be able to suppress CD8<sup>+</sup> T cell responses in the presence of strong exogenous IL-2R agonists and, thus, might not substantially mitigate the effects of IL-2-based therapy. However, their role might depend on particular immunological context, especially based on disease and tumor type, which is not fully understood currently. Another potential limitation of our proposed model is that the most of the underlying evidence has been generated in preclinical mouse models and it is not clear to what extent they apply to humans. Therefore, these and other open questions (see ##BOX##4##Outstanding questions##) need to be resolved to bring optimal IL-2-derived treatments into the clinic, representing a fruitful area of future investigation.</p>" ]
[ "<title>Acknowledgments</title>", "<p>This study was supported by a Czech Science Foundation grants (22-21356S to O.S. and 22-20548S to M.K.); European Union’s Horizon 2020 research and innovation program under grant agreement No 802878 (ERC Starting Grant FunDiT); core funding of the Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic (IMG ASCR) (RVO 68378050); the National Institute of Virology and Bacteriology (Programme EXCELES, LX22NPO5103) and the National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102), both funded by the European Union – Next Generation EU; and institutional research concept of the Institute of Microbiology of the Czech Academy of Sciences (RVO 6138897). Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic. V.N. is a student of the Faculty of Science, Charles University, Prague. Figures were created with BioRender (<ext-link xlink:href=\"https://www.biorender.com/\" ext-link-type=\"uri\">BioRender.com</ext-link>).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><title>Interleukin (IL)-2 and IL-2 modifications used in immunotherapy approaches.</title><p>(A) Wild-type (WT) IL-2 can bind to dimeric intermediate-affinity IL-2 receptor (IL-2Rβγ) or trimeric high-affinity IL-2 receptor (IL-2Rαβγ) [##REF##20732639##1##]. (B) Multiple IL-2 variants (IL-2v) were designed with a mutated IL-2Rα-binding site. These variants can bind only to IL-2Rβγ receptors, regardless of the availability of IL-2Rα [##REF##23677467##48##–##REF##35817480##51##,##REF##34373459##59##]. (C,D) The apparent affinity of binding of IL-2 variants (IL2v) to IL-2Rβγ is increased in <italic toggle=\"yes\">cis</italic> by fusion to (C) monovalent [##REF##35104810##7##] or (D) bivalent [##REF##36171284##8##,##REF##36171288##11##] anti-PD-1 antibodies, regardless of the availability of IL-2Rα. These fusion proteins can impede PD-1 inhibitory signaling to some extent [##REF##36171284##8##]. When PD-1 is not expressed, PD1-IL2v can still bind to IL-2Rβγ, but with lower affinity. ‘+’ indicates the binding affinity of IL-2v to IL-2R.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><title>Generation of ‘better effector’ CD8<sup>+</sup> T cells upon PD-1–interleukin (IL)-2v treatment in mice.</title><p>(A,B) Uniform manifold approximation and projection (UMAP) plots based on single cell RNA sequencing (scRNAseq), constructed for illustration purposes only, showing (A) CD8<sup>+</sup> T cells isolated from pancreatic carcinoma in RIP1-Tag5 mice [##REF##36630914##9##], (B) CD8<sup>+</sup> T cells isolated from subcutaneous Panc02-H7-Fluc pancreatic carcinoma in C57BL/6 mice [##REF##36171284##8##], (C) antigen-specific GP33<sup>+</sup>CD8<sup>+</sup> T cells isolated from spleens of C57BL/6 mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 [##REF##36171288##11##]. UMAP plots on the left show the localization of different subpopulations in the dimensional reduction space. UMAP plots on the right show the density of cell populations upon different treatments. ScRNAseq data were obtained from the following studies: Study 1 [##REF##36630914##9##] (GSE197854), Study 2 [##REF##36171284##8##] (E-MTAB-11773), and Study 3 [##REF##36171288##11##] (GSE206739).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><title>Gene expression signatures of CD8<sup>+</sup> T cells in response to interleukin (IL)-2 based therapies.</title><p>(A,B) Heatmaps showing the relative gene expression of selected genes in splenic or intratumoral mouse CD8<sup>+</sup> T cells, constructed for illustration purposes only: (A) upon treatment with IL-2-based compounds [wild-type (WT) IL-2 [##REF##36171288##11##], PD-1-laIL-2 (intermediate-affinity IL-2 conjugated to anti-PD-1 monoclonal antibodies; mAbs; monovalent) [##REF##35104810##7##], Erb-laIL-2 (intermediate-affinity IL-2 conjugated to anti-EGFR mAb that serves as a control conjugate to PD-1-laIL-2 [##REF##35104810##7##]), FAP-IL2v (intermediate-affinity IL-2 variant fused to a mAb against fibroblast-activating protein [##REF##36171284##8##]), PD1-IL2v (intermediate-affinity IL-2 variant fused to an anti-PD-1 mAb; bivalent) [##REF##36171284##8##,##REF##36630914##9##,##REF##36171288##11##]), and/or checkpoint inhibition therapy (anti-PD-1 or anti-PD-L1 antibody)], or (B) upon regulatory T cell (Treg) depletion [##REF##36705564##3##]. Selected genes represent the signature genes of better effector cells [##REF##36171284##8##,##REF##36171288##11##]. (C) Bar plots, constructed for illustration purposes only, showing the enrichment of KLRK1<sup>+</sup> IL-7Rα<sup>+</sup> (KILR) T cell signature genes (genes induced in activated CD8<sup>+</sup> T cells upon Treg depletion) in splenic CD8<sup>+</sup> T cells upon treatment with IL-2-based compounds and/or checkpoint inhibitors [##REF##36705564##3##]. (A–C) Single cell RNA sequencing data were obtained from the following studies: Study 1 [##REF##36171284##8##] (E-MTAB-11773), Study 2 [##REF##36630914##9##] (GSE197854), Study 3 [##REF##36171288##11##] (GSE206739), Study 4 [##REF##35104810##7##], and Study 5 [##REF##36705564##3##] (GSE183940). Abbreviations: NK, natural killer; LMCV, lymphocytic choriomeningitis virus; OVA, ovalbumin.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><title>Key figure. Proposed mechanism of interleukin (IL)-2-based tumor clearance mediated by CD8<sup>+</sup> T cells</title><p>In this model, we propose that, under physiological conditions, antigen-activated CD8<sup>+</sup> T cells produce the cytokine IL-2, which is largely sequestered by regulatory T cells (Tregs), expressing the high-affinity IL-2 receptor (IL-2R). High amounts of exogenous IL-2 may not only activate both Tregs and CD8<sup>+</sup> T cells, but also lead to life-threatening adverse effects caused by damage to endothelial cells with subsequent vascular leak syndrome. Intermediate-affinity IL-2 variants (IL-2v) may bind preferentially to an intermediate-affinity IL-2R expressed by CD8<sup>+</sup> T cells, but are not potent enough to trigger full activation <italic toggle=\"yes\">per se</italic>. Novel molecules, represented by IL-2v fused to an antibody against inhibitory receptor PD-1 (PD1-IL2v), may act specifically on PD-1<sup>+</sup> CD8<sup>+</sup> T cells (enriched in tumor-specific cells), which proliferate and differentiate into better effector cells with superior anticancer cytotoxic activity. A similar effect might be achieved by Treg depletion, increasing IL-2 availability for CD8<sup>+</sup> T cells. Abbreviation: KILR, KLRK1<sup>+</sup>IL-7Rα<sup>+</sup>CD8<sup>+</sup> T cells.</p></caption></fig>" ]
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[ "<boxed-text id=\"BX1\" position=\"float\"><caption><title>Highlights</title></caption><p>The therapeutic potential of interleukin (IL)-2 in cancer and chronic infections has triggered the development of novel IL-2-based biologicals and combinatorial treatment strategies to achieve high efficacy and low off-target adverse effects in mouse models, before being tested in clinical trials.</p><p>Suboptimal IL-2 variants fused to an anti-PD-1 antibody specifically promote robust therapeutic effects in various preclinical cancer and infection mouse models (e.g., implanted lymphoma, pancreatic adenocarcinoma, glioma, colon carcinoma, spontaneous pancreatic cancer, and chronic viral infection). The effect occurs by targeting IL-2 to PD-1<sup>+</sup> T cells and enhancing IL-2 binding to an ‘intermediate-affinity IL-2 receptor’ in <italic toggle=\"yes\">cis</italic>.</p><p>Strong IL-2 signals are required to induce the formation of a unique differentiation state of antigen-stimulated CD8<sup>+</sup> T cells, termed ‘better effectors’, with superior antitumor and antiviral properties in mice.</p><p>Based on preclinical mouse models, we propose that regulatory T cells suppress CD8<sup>+</sup> T cells and the formation of ‘better effectors’ via IL-2 sequestration, which is disabled upon administration of exogenous IL-2-based biologicals.</p></boxed-text>", "<boxed-text id=\"BX2\" position=\"float\"><caption><title>Significance</title></caption><p>Strong IL-2 receptor stimulation of antigen-activated CD8<sup>+</sup> T cells induces a unique gene expression program in which the cells harbor a superior cytotoxicity and anticancer/antiviral potential. This effect can be achieved by exogenous IL-2-based therapy or depletion of Tregs. Recent progress in the design of targeted IL-2-derived biologicals and single cell transcriptomics provide a novel framework for the development of putative therapies for treating cancer and chronic infections in humans.</p></boxed-text>", "<boxed-text id=\"BX3\" position=\"float\"><label>Box 1</label><caption><title>The history of IL-2 therapies</title></caption><p>The therapeutic potential of IL-2 was revealed ~40 years ago, when it was observed that <italic toggle=\"yes\">in vitro</italic> IL-2-stimulated CD8<sup>+</sup> T cells kill tumor cells [##REF##6975652##52##,##REF##6176669##53##]. A recombinant IL-2 therapy (aldesleukin) demonstrated clinical efficacy in metastatic renal cell carcinoma and melanoma, leading to its US Food and Drug Administration approval during the 1990s [##REF##2993418##54##–##REF##10561265##56##]. However, this therapy also caused severe adverse effects, including <xref rid=\"G24\" ref-type=\"other\">vascular leak syndrome</xref>, resulting from an off-target effect on endothelial cells [##REF##27601468##12##,##REF##20547866##13##,##REF##31412088##57##].</p><p>The second generation of IL-2-based antitumor therapies featured drugs with modified binding to IL-2R aiming to stimulate cytotoxic CD8<sup>+</sup> T cells and NK cells, but not endothelial cells or Tregs in mouse models and, subsequently, in clinical trials (NCT05267626<sup><xref rid=\"FN1\" ref-type=\"fn\">i</xref></sup>, NCT02983045<sup><xref rid=\"FN2\" ref-type=\"fn\">ii</xref></sup>, and NCT04855929<sup><xref rid=\"FN3\" ref-type=\"fn\">iii</xref></sup>) [##REF##35058325##18##–##REF##34255535##20##,##REF##23677467##48##–##REF##35817480##51##,##REF##36459543##58##–##REF##20133862##69##]. The respective strategies included modifications of the IL-2Rα binding site [##REF##23677467##48##–##REF##35817480##51##,##REF##36459543##58##,##REF##34373459##59##], selecting IL-2 variants with increased affinity to IL-2Rβ [##REF##35058325##18##,##REF##22446627##60##], complexes and fusion of IL-2 with anti-IL-2-antibodies [##REF##33353953##61##–##REF##19801515##64##] or IL-2Rα [##REF##32317293##65##,##REF##34362793##66##], or by designing novel IL-2-like drugs [##REF##30626941##67##,##REF##36316485##68##]. These innovative strategies resolved the adverse effects, but have not yet shown promising results in clinical trials [##REF##37004361##16##], potentially due to their limited antitumor efficacy.</p><p>The third generation of IL-2-based antitumor drugs combined immune checkpoint blockade and specific IL-2 delivery to CD8<sup>+</sup> PD-1<sup>+</sup> T cells, which are found particularly in tumors, using a chimeric molecule of anti-PD-1 antibody fused to IL-2 variants binding specifically to intermediate-affinity receptor (IL-2Rβγ) (henceforth termed ‘intermediate-affinity IL-2 variants’). The promising effects of this approach in preclinical models of cancer and chronic infection [##REF##35104810##7##–##REF##36630914##9##] led to a current Phase 1 clinical trial (NCT04303858<sup><xref rid=\"FN4\" ref-type=\"fn\">iv</xref></sup>) (reviewed in [##REF##37004361##16##]).</p><p>In contrast to antitumor therapy, <xref rid=\"G15\" ref-type=\"other\">low-dose IL-2 therapy</xref> can promote the expansion and differentiation of Tregs in humans [##REF##30282826##70##,##REF##36399073##71##] and, therefore, has been tested for safety and effectivity in Phase 2 clinical trials in type 1 diabetes mellitus (NCT01862120<sup><xref rid=\"FN5\" ref-type=\"fn\">v</xref></sup>) [##REF##32607749##72##] and other autoimmune diseases (NCT01988506<sup><xref rid=\"FN6\" ref-type=\"fn\">vi</xref></sup>) [##REF##30472651##73##].</p><p>Besides the rapid development of IL-2-based drugs for <italic toggle=\"yes\">in vivo</italic> administration, IL-2 is routinely used ex <italic toggle=\"yes\">vivo</italic> to stimulate cells for adoptive therapies, such as <xref rid=\"G3\" ref-type=\"other\">chimeric antigen receptor (CAR) T cells</xref> [##REF##29641319##74##]. Moreover, T cells for adoptive transfer can be engineered <italic toggle=\"yes\">in vitro</italic> to enhance their stimulation by IL-2 <italic toggle=\"yes\">in vivo</italic>. One example is based on an engineered IL-2:IL-2Rβ pair, which enables using modified IL-2 to stimulate adoptively transferred T cells expressing the engineered IL-2Rβ, but not endogenous T cells [##REF##29496879##75##]. A corresponding treatment using CAR-T cells reduced tumor burden, prolonged mouse survival, and exhibited lower toxicity compared with the systemic administration of WT IL-2 in two xenograft tumor models using immunodeficient <xref rid=\"G16\" ref-type=\"other\">NOD scid gamma (NSG)</xref> mice implanted with human Nalm6 leukemia cells [##REF##34936380##76##] or human Raji lymphoma cells [##REF##34936383##77##].</p><p>Overall, multiple IL-2-based approaches have been developed aiming to treat cancer, chronic infection, or autoimmunity, and over 100 clinical trials have been registered worldwide, suggesting that IL-2 is among the most promising candidates for immunotherapy [##REF##37004361##16##].</p></boxed-text>", "<boxed-text id=\"BX4\" position=\"float\"><caption><title>Glossary</title></caption></boxed-text>", "<boxed-text id=\"BX5\" position=\"float\"><caption><title>Outstanding questions</title></caption><p>How does strong IL-2 signaling induce a unique gene expression program leading to the formation of better effector CD8<sup>+</sup> T cells? Although the proximal IL-2 signaling pathway is relatively well understood, it is unclear how strong IL-2 signals synergize with antigenic signaling to induce this transcriptional program leading to superior anti-cancer activity. Uncovering the respective signaling pathways leading to the triggering of the causative transcription factors and epigenetic regulators might reveal molecular mechanisms required for better effector formation that can be therapeutically induced or enhanced.</p><p>What is the role of Tregs in IL-2-based cancer immunotherapy? We posit that they might not be as crucial as once believed, especially in terms of their ability to suppress CD8<sup>+</sup> T cells, but this should be thoroughly tested.</p><p>What is the optimal molecular structure of the PD1-IL2v chimera to provide strong targeted IL-2R signals and block PD-1:PD-L1 interactions? Different types of PD1-IL2v molecules appear to have different efficacies and potential to induce a better effector CD8<sup>+</sup> T cell signature. Apparently, features such as anti-PD-1 antibody valency and topological orientation of fused proteins might have a role, but this has not been extensively investigated. Emerging PD1-IL2v drugs do not block PD-1 signaling completely and their efficacy can be improved in a combination with anti-PD-L1 antibodies. Thus, it might be possible to develop even more potent PD1-IL2 chimeric biologicals.</p><p>Is the effect of PD1-IL2v similar in humans? Since most of the emerging findings in IL-2-based immunotherapy are from preclinical mouse and humanized mouse models, the <italic toggle=\"yes\">in vivo</italic> effects of IL-2-based therapy need to be addressed in humans, including the eventual formation of better effector human T cells.</p></boxed-text>" ]
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[ "<fn-group><title>Resources</title><fn id=\"FN1\"><label>i</label><p id=\"P26\">\n<ext-link xlink:href=\"https://clinicaltrials.gov/study/NCT05267626\" ext-link-type=\"uri\">https://clinicaltrials.gov/study/NCT05267626</ext-link>\n</p></fn><fn id=\"FN2\"><label>ii</label><p id=\"P27\">\n<ext-link xlink:href=\"https://clinicaltrials.gov/study/NCT02983045\" ext-link-type=\"uri\">https://clinicaltrials.gov/study/NCT02983045</ext-link>\n</p></fn><fn id=\"FN3\"><label>iii</label><p id=\"P28\">\n<ext-link xlink:href=\"https://clinicaltrials.gov/study/NCT04855929\" ext-link-type=\"uri\">https://clinicaltrials.gov/study/NCT04855929</ext-link>\n</p></fn><fn id=\"FN4\"><label>iv</label><p id=\"P29\">\n<ext-link xlink:href=\"https://clinicaltrials.gov/study/NCT04303858\" ext-link-type=\"uri\">https://clinicaltrials.gov/study/NCT04303858</ext-link>\n</p></fn><fn id=\"FN5\"><label>v</label><p id=\"P30\">\n<ext-link xlink:href=\"https://clinicaltrials.gov/study/NCT01862120\" ext-link-type=\"uri\">https://clinicaltrials.gov/study/NCT01862120</ext-link>\n</p></fn><fn id=\"FN6\"><label>vi</label><p id=\"P31\">\n<ext-link xlink:href=\"https://clinicaltrials.gov/study/NCT01988506\" ext-link-type=\"uri\">https://clinicaltrials.gov/study/NCT01988506</ext-link>\n</p></fn></fn-group>", "<fn-group><fn id=\"FN7\" fn-type=\"COI-statement\"><p id=\"P32\">\n<bold>Declaration of interests</bold>\n</p><p id=\"P33\">M.K. is listed as a co-inventor on patent entitled ‘Methods and materials for targeted expansion of immune effector cells’, which was filed on June 26, 2020, and now bears International Application Number PCT/US2020/039857. The other authors have no conflict of interest to declare.</p></fn><fn id=\"FN8\"><p id=\"P34\">\n<bold>Declaration of Generative AI and AI-assisted technologies in the writing process</bold>\n</p><p id=\"P35\">During the preparation of this work, the authors used ChatGPT-3.5 (OpenAI) to improve language use and readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"EMS193164-f001\" position=\"float\"/>", "<graphic xlink:href=\"EMS193164-f002\" position=\"float\"/>", "<graphic xlink:href=\"EMS193164-f003\" position=\"float\"/>", "<graphic xlink:href=\"EMS193164-f004\" position=\"float\"/>" ]
[]
[{"label": ["62"], "person-group": ["\n"], "surname": ["Arenas-Ramirez"], "given-names": ["N"], "article-title": ["Improved cancer immunotherapy by a CD25-mimobody conferring selectivity to human interleukin-2"], "source": ["Sci Transl Med"], "year": ["2016"], "volume": ["8"], "elocation-id": ["367ra166"]}]
{ "acronym": [ "Better effector", "Bystander-activated CD8+ T cells", "Chimeric antigen receptor (CAR) T cells", "Cis (interaction)", "FTY720 (fingolimod)", "IL-2-based immunotherapies", "IL-2 immunocomplexes (IL2ICx)", "IL-2 immunocytokines", "IL-2 mutein", "IL-2 receptor (IL-2R)", "IL-2 variant (IL-2v)", "Immune checkpoint inhibitors", "KILR T cell", "Killer cell lectin like receptor K1 (KLRK1/NKG2D)", "Low-dose IL-2 therapy", "NOD scid gamma (NSG) mice", "OT-I", "PD1-IL15m", "PD1-IL2v", "Single cell RNA sequencing (scRNAseq)", "Stem-like T cells", "Synthetic effectors", "TCF7", "Vascular leak syndrome" ], "definition": [ "differentiation state of CD8+ T cells with superior antitumor and antiviral cytotoxic capacity; these develop in vivo after PD-1 blockade and IL-2 treatment.", "CD8+ T cells, usually with memory phenotype, that become activated in an antigen-independent manner due to inflammatory signals during an immune response.", "T cells engineered to express a synthetic receptor called a CAR. The CAR combines the antigen-binding domain of an antibody with TCR signaling components, enabling T cells to recognize specific antigens on target cells and initiate signaling and ensuing immune responses. CAR-T cells are used to treat various malignancies, mostly of hematopoietic origin.", "in the context of receptor–ligand interactions, cis binding occurs when two molecules on the same cell interact with the ligand. By contrast, trans binding occurs when two molecules on different cells interact with the ligand.", "sphingosine-1-phosphate receptor modulator, which induces the internalization of the receptor and, thus, prevents T cells from egressing the lymph nodes.", "therapeutic interventions that utilize IL-2 and its engineered variants to modify immune response to diseases, such as cancer and autoimmunity.", "complexes of IL-2 and anti-IL-2 antibodies. The immunocomplexes have a longer in vivo half-life than IL-2 alone and thus, are potent immunomodulators of the IL-2 response. Based on the clone of the anti-IL-2 antibody, different IL-2 immunocomplexes have different in vivo effects (i.e., if the antibody blocks the IL-2Rα binding site, IL-2Rα-expressing cells are not preferentially stimulated).", "engineered therapeutic molecules that combine IL-2 with specific monoclonal antibodies or antibody fragments to target immune cells expressing a particular cell surface receptor.", "mutated variant of the natural IL-2 cytokine. Through alterations in its amino acid sequence, it is designed to have modified functional properties, such as enhanced affinity for specific IL-2 receptors or reduced interactions with regulatory immune cells, while retaining the core immune-stimulating functions of IL-2.", "cell-surface protein complex that binds IL-2 and triggers the intracellular IL-2 signaling pathways. IL-2R has three subunits: IL-2Rα (alias CD25, intermediate-affinity IL-2 receptor, KD ~10 nM), IL-2Rβ (alias CD122 or IL-15Rβ), and IL-2Rγ (alias CD132, common γ-chain). Naïve and memory T cells and NK cells express a dimeric IL-2Rβγ receptor with intermediate affinity to IL-2 (KD ~1 nM). Tregs and recently activated T cells express a high-affinity trimeric IL-2Rαβγ receptor (KD ~10 pM). One mechanism of Treg-mediated suppression of conventional T cells is using high-affinity receptors for sequestering IL-2 from the environment. IL-2R signaling induces the expression of IL-2Rα, which provides a positive feed-back loop.", "modified version of IL-2 that has been engineered to alter its properties, especially the receptor binding. Here, we use IL-2v as a synonym for mutant IL-2, the binding of which to IL-2Rα is reduced or eliminated.", "antibodies or other molecules that block inhibitory receptors, such as PD-1 or CTLA-4, on T cells, enhancing their proliferative capacity and/or effector function, mostly in the context of tumors. These inhibitors are widely used in cancer immunotherapy.", "differentiation state of CD8+ T cells that develops in vivo after antigenic stimulation in combination with supraphysiological IL-2R signals, occurring upon depletion of Tregs or administration of exogenous IL-2R agonists. It is characterized by expression of KLRK1, IL-7R, and other specific markers. KILR effectors and better effectors share striking gene expression similarities.", "activating cell surface receptor expressed by NK cells and some CD8+ T cells. Its expression is characteristic of KILR+ effector and better effector cells.", "administration of low doses of IL-2 in multiple dosing schedules aiming to suppress autoimmune responses in conditions such as type 1 diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, and graft-versus-host disease, by expanding Treg cells.", "mouse strain with a set of genetic mutations (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ); develop severe combined immunodeficiency presenting with one of the most severe phenotypes without any mature T, B, or NK cells. Notably, NSG mice carry a complete null mutation in the gene encoding IL-2Rγ.", "transgenic OT-I mice produce monoclonal CD8+ T cells expressing OT-I TCRs specific to mouse H-2Kb MHC-I loaded with a chicken ovalbumin peptide (amino acid sequence is SIINFEKL). OT-I mice are commonly used for monitoring antigen-specific responses in mouse studies.", "fusion protein comprising an engineered variant of IL-15 that does not interact with IL-15Rα and that has a reduced affinity to IL2Rβγ, fused to the anti-PD-1 antibody.", "chimeric protein comprising an engineered variant of IL-2 not interacting with IL-2Rα fused to the anti-PD-1 antibody. It has superior antitumor effects compared with the IL-2v itself due to the increased in vivo half-life and cis-interaction with target T cells.", "technology used to quantify mRNA transcripts on a per cell basis, providing a high-resolution view of cell-to-cell variation in a biological sample.", "have the properties of stem cells (i.e., self-renewal potential and ability to differentiate into terminal effector T-cell stages: TCF7+). Stem-like properties are generally associated with memory T cells.", "genetically engineered CD8+ T cells with transgenic production of particular cytokines, which could be used in an adoptive transfer therapy to treat cancer. Synthetic effector CD8+ T cells producing IL-2v and IL-33 alarmin exhibited potent antitumor activity in a preclinical model.", "transcriptional factor that is a marker of T cell stemness; expressed in central memory T cells and stem-like T cells in chronic infection or in tumors. Also known as TCF-1.", "increase in vascular permeability causing the escape of blood plasma through capillary vessels into the tissue. This results in edema and subsequent tissue damage." ] }
77
CC BY
no
2024-01-13 00:12:45
Trends Immunol. 2023 Nov 1; 44(11):890-901
oa_package/f3/af/PMC7615502.tar.gz
PMC7615507
38213311
[ "<title>Introduction</title>", "<p id=\"P2\">Transition to adulthood has been steadily delayed in Western societies since the 1970s (##REF##21652599##Mills et al., 2011##). This process contributes to later fertility, with first births to women aged 40 and older accounting for between 2% and 7% of the total first birth rate in European countries in 2018, a figure that is growing rapidly (##UREF##1##Beaujouan &amp; Sobotka, 2022##). The prevalence of later fertility (above the age of 35 or 40) varies widely across national and subnational contexts (##UREF##29##Leibert, 2020##). The importance of national settings, in particular, countries’ economic situation, attitudes towards the family or family policies, is often emphasised to explain crosscountry differences in fertility in later reproductive life (##UREF##2##Beaujouan &amp; Toulemon, 2021##; ##UREF##48##Sobotka et al., 2011##). A more refined picture of later fertility can be obtained by understanding the aspects that account for its wide subnational variation.</p>", "<p id=\"P3\">Childbearing is often delayed in urban environments, and late fertility more prevalent (e.g., ##UREF##4##Buelens, 2021##; ##REF##17979002##Kulu et al., 2007##; ##REF##31736514##Riederer &amp; Buber-Ennser, 2019##. Urban and rural areas differ in many ways that may be relevant to the timing of births, such as the educational, recreational and occupational opportunities they offer, their economic structures (prevalence of knowledge work, high-tech sectors and international companies), their wealth, their lifestyle, their family cultures, their internationality and the age structure and mobility of their populations (##UREF##5##Burtenshaw et al., 2021##; ##UREF##18##Hamnett, 2021##; ##UREF##32##Lichter et al., 2020##; ##UREF##41##Riederer et al., 2021##). Factors associated to urbanisation level may thus also be related to the prevalence of later fertility in subnational areas and its variation by degree of urbanisation. In this paper, we examine the relationship between population density and prevalence of later fertility at the subnational level, and whether this relationship is explained by differences in contextual characteristics.</p>", "<p id=\"P4\">After theoretically assessing why people living in urban environments may delay fertility more, we detail how a range of socioeconomic variables can be related to both later fertility and degree of urbanisation at the aggregate level. In the method part, we present the database and model used to assess this interrelationship. In the results part, we first highlight the strong relationship between cities and later fertility by using several examples of cities all across Europe. We then check whether differences between urbanisation levels persist across Europe once contextual characteristics are accounted for. The final discussion emphasises the importance of educational and professional opportunities for fertility delay across residential contexts.</p>" ]
[ "<title>Data And Methods</title>", "<title>Construction of indicators</title>", "<p id=\"P18\">Our dependent variable is an indicator of later fertility at the subnational level across European countries. As the relevance of later fertility obviously depends on the number (and share) of women of the respective age group in a region, we use age-specific fertility rates (ASFR) instead of number of births to calculate late fertility prevalence. Hence, our indicator corresponds to the contribution of women aged 35+ (or 40+) at birth to the TFR per calendar year.<sup><xref rid=\"FN1\" ref-type=\"fn\">1</xref></sup></p>", "<p id=\"P19\">Variation of the late fertility indicator is observed along the urban–rural continuum. Going beyond the urban–rural dichotomy, the urban–rural continuum (i.e., the degree of urbanisation) is indicated by population density (persons per square kilometre). At the nomenclature des unités territoriales statistiques (NUTS) 3 level, the population density variable represents urbanisation along the gradient from very dense cities to rural areas through less densely populated urban areas (e.g., suburbs and towns). Using population density at NUTS 3 level makes it possible to clearly distinguish between metropolitan, urban and rural areas (##UREF##6##Campisi et al., 2020##), and to identify specific areas in many metropolitan regions (e.g., Athens, Greater London, Île-de-France). Our analyses are, nonetheless, also conducted at NUTS 2 level. First, the geographic detail of NUTS 3 regions differs across European countries, whereas region sizes do not vary that strongly at NUTS 2 level. Second, some of the other covariates (see below) are only available at NUTS 2 level. In short, NUTS 3 level analyses allow for a more accurate and fine-grained picture and NUTS 2 level analyses may be preferable in terms of comparability across indicators and countries.</p>", "<p id=\"P20\">Other covariates indicate aspects of education, the economic environment, family and gender norms and population composition. <italic toggle=\"yes\">Education</italic> is measured by the share of women with tertiary education among all women aged 25–64. The <italic toggle=\"yes\">economic and educational environment</italic> in each area is captured by gross domestic product per capita (in purchasing power standards and as percentage of the European Union average) and the share of the high-tech sector in the economy (i.e., employment in high-technology manufacturing and knowledge-intensive high-technology services). Indicators of dominant <italic toggle=\"yes\">family and gender norms</italic> comprise the share of divorced persons (including persons whose registered partnership was legally dissolved) in the population and the ratio of male-to-female employment (age 25–54). Finally, the <italic toggle=\"yes\">composition of the population</italic> is described by the share of the population aged 60 and over among the total population and the share of foreign-born women among female population (age 25–54). Most measures refer to the year 2018.<sup><xref rid=\"FN2\" ref-type=\"fn\">2</xref></sup> Exceptions are figures for Germany that refer to 2017 and figures for the share of divorced persons that have been calculated on the basis of the population censuses in 2011.<sup><xref rid=\"FN3\" ref-type=\"fn\">3</xref></sup> The aggregated data has been retrieved from various EUROSTAT databases (for details, see ##SUPPL##0##Supporting Information: Table A.1##).</p>", "<title>Analytical strategy</title>", "<p id=\"P21\">Our analytical strategy comprises several steps. First, we compare the share of ASFR 35+ and 40+ in total fertility in major cities to the respective country average in a descriptive analysis. Exemplary presentations of the results for Germany and the United Kingdom provide a more detailed picture of regional differences. Second, we employ multilevel models with varying model specifications. For multilevel analyses, we use aggregated data for 1328 NUTS 3 and 270 NUTS 2 regions from 28 European countries.<sup><xref rid=\"FN4\" ref-type=\"fn\">4</xref></sup></p>", "<p id=\"P22\">Although ##UREF##6##Campisi et al. (2020)## argue in favour of spatial models to analyse regional variations in fertility, we decided to use multilevel mixed-effects models (i.e., models with both random intercepts and random slopes). First, we want to avoid overlooking cross-country heterogeneity in urban–rural differences (i.e., the magnitude of coefficients). Differences between regions are larger in some countries than in others for manifold reasons (e.g., the process of defining NUTS regions, the degree of urbanisation of a country, geographical structure) and this affects estimated associations. Erroneously assuming invariant coefficients across countries reduces the estimation precision (##UREF##21##Heisig et al., 2017##) and can lead to serious biases even if the fixed effects are specified correctly (##UREF##44##Schmidt-Catran &amp; Fairbrother, 2016##). The estimation of random slopes avoids such problems. Second, we focus on the role of regional characteristics for fertility differences in the urban and rural contexts, explicitly assuming differences by degree of urbanisation due to characteristics of cities and the urban context that go beyond geographical proximity. Given our research interest, the correct specification and interpretation of spatial effects are not straightforward (see ##UREF##17##Golgher &amp; Voss, 2016##).<sup><xref rid=\"FN5\" ref-type=\"fn\">5</xref></sup> As covariates and the correct estimation of random slopes, rather than spatial effects per se, are central to our main research question, we prefer multilevel mixed-effects models to spatial models in our specific case. Nevertheless, we present sensitivity analyses in the Supporting Information: Appendix to check for spatial autocorrelation in multilevel models (computing Moran’s I with residuals) and whether results in standard spatial models are similar to those in multilevel mixed-effects models.</p>", "<p id=\"P23\">A series of three-level mixed-effect models with random intercepts and slopes serves to analyse the association between population density (indicating the degree of urbanisation) and later fertility on NUTS 3 level. In the first model, we include only a fixed effect of population density. In the second model, we additionally introduce a random effect on the country level to explore how the coefficient varies over countries (random slope). In the third model, we additionally include context variables available at NUTS 3 level (fixed and random effects). The fourth model also contains context variables at NUTS 2 level (only fixed effects due to low case numbers). This final model can be expressed as: \n where <italic toggle=\"yes\">y</italic><sub>ijc</sub> is the dependent variable ‘later fertility’, <italic toggle=\"yes\">α, ζ</italic><sub>jc</sub> and <italic toggle=\"yes\">ζ</italic><sub>c</sub> are intercept(s), with <italic toggle=\"yes\">ζ</italic><sub>jc</sub> varying at NUTS 2 level and <italic toggle=\"yes\">ζ</italic><sub>c</sub> varying at country level (random intercepts); <italic toggle=\"yes\">x</italic><sub>ijc</sub> and <italic toggle=\"yes\">x</italic><sub>jc</sub> represent explanatory variables at NUTS 3 and NUTS 2 level, respectively; vector <italic toggle=\"yes\">β</italic><sub>1</sub> includes the regression slope coefficients of NUTS 3 level variables and vector <italic toggle=\"yes\">β</italic><sub>2</sub> the regression slope coefficients of NUTS 2 level variables; <italic toggle=\"yes\">ζ</italic><sub>1c</sub> allows the regression coefficients of NUTS 3 level variables to vary across countries (random slope). Summarising (<italic toggle=\"yes\">α</italic> + <italic toggle=\"yes\">β</italic><sub>1</sub>\n<italic toggle=\"yes\">x</italic><sub>ijc</sub> + <italic toggle=\"yes\">β</italic><sub>2</sub>\n<italic toggle=\"yes\">x</italic><sub>jc</sub>) is the fixed part and (<italic toggle=\"yes\">ζ</italic><sub>jc</sub> + <italic toggle=\"yes\">ζ</italic><sub>c</sub> + <italic toggle=\"yes\">ζ</italic><sub>1c</sub>\n<italic toggle=\"yes\">x</italic><sub>ijc</sub> + <italic toggle=\"yes\">ε</italic><sub>ijc</sub>) is the random part of the model. Most importantly, random intercepts account for different levels of later fertility by country and NUTS 2 region, while random slopes reflect cross-country heterogeneity regarding all NUTS 3 level covariates, including urban–rural differences in later fertility. The estimated relationship between later fertility (<italic toggle=\"yes\">y</italic>) and population density (<italic toggle=\"yes\">d</italic>) can thus be described as (<italic toggle=\"yes\">β</italic><sub>pd</sub> + <italic toggle=\"yes\">ζ</italic><sub>pdc</sub>) <italic toggle=\"yes\">d</italic><sub>ijc</sub> in: where (<italic toggle=\"yes\">β</italic><sub>d</sub> + ζ<sub>dc</sub>) describes the regression slope coefficients of population density (<italic toggle=\"yes\">d</italic><sub>ijc</sub>) that vary across countries (<italic toggle=\"yes\">ζ</italic><sub>dc</sub>), whereas <italic toggle=\"yes\">β</italic><sub>1</sub>, <italic toggle=\"yes\">ζ</italic><sub>1c</sub> and <italic toggle=\"yes\">x</italic><sub>ijc</sub> refer to other NUTS 3 level covariates.</p>", "<p id=\"P24\">Afterwards, we use a series of two-level models to refine which groups of context variables explain the association between population density and later fertility. We conduct this analysis on NUTS 2 level because of better comparability of NUTS 2 regions across European countries and because not all necessary context information is available on NUTS 3 level. As the number of observations is much smaller at NUTS 2 level, these models include a random slope only for population density. We estimate models separately considering indicators of female education, economic environment, family and gender norms and population composition, before including all of them in a final model. In sensitivity analyses (shown in the Supporting Information: Appendix), we follow the same procedure with different model specifications (including spatial models) to demonstrate the robustness of our findings and to test changes in the coefficients of population density across models.</p>", "<title>Choice of standardisation method</title>", "<p id=\"P25\">All described models are run for both the share of ASFR contributed by women aged 35+ and by women aged 40+ to TFR. In addition, standardisation is meaningful to make coefficient sizes comparable. We test two different methods of standardisation to the sample mean, using the country-specific standard deviation (SD) in one case and the total sample’s SD in the other.<sup><xref rid=\"FN6\" ref-type=\"fn\">6</xref></sup> Within-country standardisation is valuable in multilevel settings (e.g., ##UREF##22##Horn, 2007##): it accounts for differing degrees of variation within countries and avoids standardised coefficients above 1. Regions are thus compared to other regions within their country and coefficients indicate an increase in one SD within a country instead of one SD across all regions.</p>", "<p id=\"P26\">We identify the most accurate method of standardisation here and then focus on models employing the preferred method throughout the rest of the article. ##TAB##0##Table 1## gives the results of our multilevel models on NUTS 3 level applying both ways of standardisation, using the grand mean and within-country SD (M1–M4, left columns)<sup><xref rid=\"FN7\" ref-type=\"fn\">7</xref></sup> or the grand mean and the total sample’s SD (N1–N4, right columns). Overall, all models show a positive association between population density and later fertility: the higher the population density, the larger the share of ASFR 35+ (or 40+) in TFR in a region on average. As expected, coefficients of population density are larger in the models without covariates (M1–M2, N1–N2) and become smaller when context characteristics are included (M3–M4, N3–N4). Akaike information criterion and Bayesian information criterion improve with every step, justifying the decision to account for between-country variation and to add further covariates (##TAB##1##Tables 2## and ##TAB##2##3##).</p>", "<p id=\"P27\">Models M1 and N1 only include country-fixed effects. In models M2 and N2, the coefficient of population density is allowed to vary across countries (country random effect). Although SDs of 0.18 (panel A) and 0.21 (panel B) indicate considerable variation between countries, the coefficient of population density in model M2 is barely different from that in model M1 (0.53). However, coefficients for population density in models N1 and N2 dramatically increase from 0.40 to 0.71 (panel A) and from 0.46 to 0.70 (panel B). In addition, SDs in model N2 are larger than 0.40, which indicates that standardised coefficients for some countries are larger than 1 and that we overestimate the strength of the association if using the total sample’s standardisation. The same conclusions can be drawn from models at NUTS 2 level where coefficients in model Y2 exceed values of 1 (1.13 in panel A and 1.18 in panel B of ##SUPPL##0##Supporting Information: Table A.3##).</p>" ]
[ "<title>Descriptive Results: Major Cities And Later Fertility</title>", "<p id=\"P28\">In all the European countries considered, later fertility appears to be more prominent in the capital cities and other large cities than in the national average (##FIG##0##Figure 1##). The share of fertility by women 35+ in total fertility is, for instance, larger in Paris than in France, larger in Berlin or Munich than in Germany, larger in Praha than in Czechia, larger in Athens (especially in the North) than in Greece, or larger in (particularly Inner) London than in the United Kingdom on average. The shares of later fertility differ considerably by countries and cities—Inner London West is characterised by the highest share of births at ages 35+ (45%) and births at ages 40+ (13%), while Romania is the country with the lowest share of ASFR 35+ in total fertility (slightly above 17%)—but the general citycountry contrast is very consistent.</p>", "<p id=\"P29\">The examples of Germany and the United Kingdom provide a more complete picture of the regional variation in later fertility. The darker patches in ##FIG##1##Figure 2## show regions with a higher share of later fertility (ASFR 35+) in total fertility, compared to the respective national average. Later fertility tends to be more common in metropolitan areas and large cities. Prominent examples are Cologne, Hamburg or Munich in Germany and London or Manchester in the United Kingdom. In addition, even cities with lower levels of later fertility often have higher levels than surrounding areas. This is the case for eastern German cities such as Leipzig or Dresden, and for Liverpool or Glasgow in the United Kingdom. The high level of geographical detail of NUTS 3 divisions allows for additional insights: In Germany, many small commercial or university towns such as Bamberg, Heidelberg, Karlsruhe or Passau are also characterised by high prevalence of later fertility. The representation of Greater London reveals differences within metropolitan regions: inner London areas like Wandsworth (48%), Lambeth (45%) or Camden and the City of London (44%) have much higher shares of late fertility than outer London areas like Croydon (26%), Harrow and Hillingdon (25%) or Barking and Dagenham and Havering (23%).</p>", "<p id=\"P30\">Although the general pattern of higher late fertility in cities holds across Europe and can be observed at both NUTS 3 and NUTS 2 levels (see Supporting Information: ##SUPPL##0##Figures A.1## and ##SUPPL##0##A.2##), there are also exceptions. These exceptions are usually workingclass cities with a mining and manufacturing heritage, like Gelsenkirchen in the German Ruhr Area or Katowice in Poland, or harbour cities, like Middlesbrough in the United Kingdom or Naples in Italy. Nevertheless, these exceptions do not contradict our general assumption. Rather, they point to the relevance of the economic context for urban–rural differences in later fertility.</p>" ]
[ "<title>Summary And Discussion</title>", "<p id=\"P39\">Previous research consistently shows that European cities and urban regions are characterised by lower fertility than rural regions (e.g., ##UREF##19##Hank, 2001##; ##REF##26047550##Kulu &amp; Washbrook, 2014##). Some studies explicitly address postponement of childbearing in cities (e.g., ##REF##17979002##Kulu et al., 2007##; ##REF##31736514##Riederer &amp; Buber-Ennser, 2019##), but differences in later fertility across the urban–rural continuum and its factors have not been examined. Addressing this research gap, we analysed differences in later fertility in Europe by degree of urbanisation, using aggregated Eurostat data of 1328 NUTS 3 and 270 NUTS 2 regions from 28 countries.</p>", "<p id=\"P40\">First, our findings confirmed that later fertility is much more common in cities than on average. Rare exceptions are some working-class cities. Second, results from multilevel random coefficient models indicated a remarkable link between population density and later fertility with some variation in effect size across countries. Third, stepwise model building suggested that the association between population density and later fertility can be largely explained by the share of highly educated women and the economic environment.</p>", "<p id=\"P41\">Methodologically, our multilevel mixed-effect models demonstrated that it is important to consider not only random intercepts but also random slopes. The strength of the association between population density and the share of later fertility in total fertility varied significantly across countries (as did the associations with other contextual characteristics). We also showed that caution is needed in assessing the effect sizes of contextual variables, as only standardisation using within-country SDs allowed to compare countries.</p>", "<p id=\"P42\">The particular relevance of the educational and economic environment for later fertility in general, and for the association between population density and later fertility in particular, supports the claim that occupational opportunities in urban environments are highly relevant for urban–rural differentials in childbearing postponement (e.g., ##REF##31736514##Riederer &amp; Buber-Ennser, 2019##). Although gender and family norms as well as population composition also matter for later fertility, they seem to be less relevant for differentials in later fertility across the urban–rural continuum. In line with the demographic literature, the tertiarisation of urban economies seems to be most important for urban-rural differences in (later) fertility (e.g., ##UREF##35##Michielin, 2004##; ##UREF##53##Vobecká &amp; Piguet, 2012##).</p>", "<p id=\"P43\">The high relevance of the economic context for urban-rural differences in later fertility reinforces also the picture of a divide between the city and less densely populated, ageing areas, where economic differences may also exacerbate other behaviours. Together with findings on education, it furthermore points to issues of work-family compatibility. In the wake of women’s empowerment, the tertiarisation of urban economies and skill-biased technological change, women’s educational and economic aspirations and opportunities often compete with bearing and raising children. Particularly in cities, where later fertility is likely to be more prevalent, policies that promote work-family reconciliation through increased gender equality and the availability of quality childcare are crucial (##REF##27795600##Matysiak &amp; Węziak-Białowolska, 2016##).</p>", "<p id=\"P44\">Our indicators of family and gender norms did not affect the association between population density and later fertility. Either family and gender norms are little active in explaining the urban–rural gradient or better measures of family and gender norms are needed. The huge variation of associations between the share of divorcees and later fertility across countries suggest that effects of divorce on fertility may depend on the country-specific institutional context. Furthermore, our results were not consistent with usual expectations: the more conservative family and gender norms were (indicated by lower divorce rate, higher male-to-female employment ratio), the higher later fertility was. This makes more sense, when we think about large families and higher-order births than about ‘postponement’ of the first birth.</p>", "<p id=\"P45\">The primary limitation of our study is that we could not distinguish between later births indicating ‘postponement’ of the first birth and those indicating family enlargement by mothers. Despite the current development of a larger variety of fertility indicators at NUTS 2 and NUTS 3 levels (##REF##33597840##Nisén et al., 2021##), the coverage remains limited to countries where large data sets and notably registers are made available for such calculation. Future studies should draw a more detailed picture that draws on other indicators such as the share of women remaining childless. Indeed, though this has not (yet) been found at the cross-country level in the past generations, one can expect that with the increase in age at first birth, the share of women having children later and of women remaining childless will become increasingly correlated, and this would be better observed at a more refined geographical scale.</p>", "<p id=\"P46\">Unfortunately, information on some context characteristics were only available at NUTS 2 level. The NUTS 2 scale may not be refined enough to seize the subtility of local variations, which is consistent with the fact that public service infrastructure is constructed at a very local scale. For other characteristics, information was not even available at NUTS 2 level. For instance, we did not find indicators referring to environmental quality (e.g., air quality, availability of green spaces), housing (e.g., crowded housing, housing costs) and other aspects of quality of life (e.g., childcare, crime rates, living costs, poverty, public transport) for enough regions to include them in our analyses. They are all parts of the context in which childbearing decisions are made, realised, postponed or abandoned. Information at a more refined geographical scale would also allow to dig into the complexity of metropolitan areas, and future studies of fertility quantum and timing could focus on the heterogeneity of the populations living in urban areas. Finally, modelling strategies that reflect our understanding that urban contextual factors affect the surrounding areas (but not vice versa) and that simultaneously integrate multilevel mixed-effects models and spatial models to avoid that random slopes at the country level are affected by spatial autocorrelation, would be promising.</p>" ]
[ "<title>Conclusion</title>", "<p id=\"P47\">Our findings primarily point to the importance of the educational and economic context in shaping alternative life goals and opportunity costs of childbearing. The relationship of elements such as lifestyle, quality of life and wealth to the timing of childbearing can be further explored, as they still tend to vary across urban–rural contexts and countries (##UREF##45##Shucksmith et al., 2009##; ##UREF##49##Sørensen, 2014##). The literature indicates that early parenthood may have negative consequences for parents’ subjective well-being, while the evidence on later fertility remains inconclusive (##UREF##24##Kohler et al., 2005##; ##REF##11989963##Mirowsky &amp; Ross, 2002##; ##UREF##40##Riederer, 2018##). Our main message that context matters is very much in line with the research on later fertility. As ##UREF##2##Beaujouan and Toulemon (2021##, p. 13) recently found at the country level, contextual effects can dominate individual constraints (including biological ones) and are often decisive for the occurrence of later births. Cities as economic centres of knowledge societies with important high-tech sectors, characterised by international competition, digitalisation and a high dependence on high education, will probably remain distinct from rural areas in the future. Their characteristics appear to be particularly relevant to later childbearing and are likely to shape future demographic trends and behaviours.</p>" ]
[ "<p id=\"P1\">Demographic research shows that, in Europe, fertility takes place later and is lower in cities than in rural areas. One might expect fertility to be delayed in urban areas because of longer periods in education and enhanced career opportunities. We, therefore, examine how prevalent later fertility (35+ and 40+) is along the urban–rural axis, and whether differences can be explained by economic, cultural and compositional factors. We estimate multilevel random coefficient models, employing aggregated Eurostat data of 1328 Nomenclature des unités territoriales statistiques (NUTS) 3 and 270 NUTS 2 regions from 28 European countries. The urban–rural gradient in later fertility considerably diminishes once factors describing the economic environment, family and gender norms as well as population composition are accounted for. The higher prevalence of later fertility in cities is particularly associated with higher female education, greater wealth and a higher share of employment in high-technology sectors.</p>" ]
[ "<title>Contextual Factors Relevant To Fertility Delay And Its Variation By Degree Of Urbanisation</title>", "<p id=\"P5\">Cities usually have lower fertility rates than suburban and rural regions in Europe (##UREF##3##de Beer &amp; Deerenberg, 2007##; ##UREF##6##Campisi et al., 2020##; ##UREF##19##Hank, 2001##, ##UREF##20##2002##; ##UREF##27##Kulu, 2013##; ##REF##26047550##Kulu &amp; Washbrook, 2014##). According to ##UREF##51##Trovato and Grindstaff (1980)##, many scholars in the 1960s and 1970s thought that educational, income or employment differences between urban and rural populations were most decisive to explain the fertility contrast (compositional hypothesis). At the same time, a stronger orientation towards large families and other attitudinal and lifestyle differences pointed to the potential influence of traditional norms in rural areas (subcultural hypothesis). In the framework of the second demographic transition (SDT), ##REF##20734551##Lesthaeghe (2010##, p. 232) explains the fertility delay with a set of reasons that also refer to structural versus ideational factors. On the side of the former, he places mechanical factors, such as prolonged formal education and career preparation, as well as economic and social structure. On the other are cultural factors related to higher-order needs and individual autonomy, such as a greater need for self-actualisation and increased economic aspirations. As a result of these changes, men and women have different priorities in their youth before starting to think about having a family, which contributes to fewer marriages, more separations, declining fertility rates at younger reproductive ages and delayed fertility (##UREF##47##Sobotka, 2008##).</p>", "<p id=\"P6\">Theories emphasising the importance of gender-egalitarian norms for family formation in high-income countries complement this approach. On the societal level, fertility is lower where dominant family norms are in conflict with egalitarian gender roles and female employment (##UREF##10##Esping-Andersen &amp; Billari, 2015##; ##UREF##16##Goldscheider et al., 2015##; ##UREF##34##McDonald, 2000##; ##REF##33321056##Raybould &amp; Sear, 2021##). In general, female economic empowerment corresponds to both later and reduced childbearing, as it promotes women’s independence from the family (##REF##21652599##Mills et al., 2011##; ##UREF##37##Osiewalska, 2018##). Also, women are more likely to wait to have their first child in unsupportive societal contexts, where work and family life are difficult to reconcile and opportunity costs of childbearing are higher (##UREF##36##Neyer, 2006##). Important conditions notably entail family policies (especially affordable, high-quality childcare), flexible working arrangements and father’s involvement in the family (##REF##27795600##Matysiak &amp; Węziak-Białowolska, 2016##).</p>", "<title>Later Fertility According To The Degree Of Urbanisation</title>", "<p id=\"P7\">An interesting dimension of studying later fertility across residential contexts is that several of the aspects that have contributed to its development are also attributes of city life. As developed above, childbearing delay is partly driven by increase in educational attainment, later entry into the labour force and women labour force participation in more qualified jobs. Such structural features first developed in cities and remain much more common in the urban context (##UREF##5##Burtenshaw et al., 2021##). In addition, cities are often assumed to be more progressive and less traditional than rural places (##UREF##7##Carter &amp; Borch, 2005##; ##UREF##15##Glenn &amp; Hill, 1977##), people there embracing a lifestyle more oriented towards culture or work (##UREF##38##Pisman et al., 2011##; ##UREF##42##Riederer &amp; Buber-Ennser, 2018##). Thus, metropolitan areas are often emphasised as the forerunners of the SDT, where attitudes and norms towards the family are more flexible and alternative couple and fertility patterns develop quickly, whereas traditional family views and behaviours remain longer predominant in rural areas (##REF##20734551##Lesthaeghe, 2010##; ##UREF##52##Valkonen et al., 2008##; ##UREF##54##Walford &amp; Kurek, 2016##). Alternative family forms that characterise the SDT, such as unmarried cohabitation, short childless unions and separation, are highly relevant for later fertility as they contribute to (re)starting childbearing at a later age (see, e.g., ##REF##22259032##Thomson et al., 2012##). During the last decades, urban-rural gradients in SDT-related attitudes and behaviours may have been attenuated due to ongoing secularisation, globalisation, technological change and the spread of (new) mass media, resulting in mixed empirical evidence (##UREF##31##Lesthaeghe &amp; Neels, 2002##; ##UREF##52##Valkonen et al., 2008##).</p>", "<p id=\"P8\">Although research often refers to urban–rural contrasts, there is rather an urban–rural continuum, consisting of a broad spectrum of intermediate levels of urbanisation, with corresponding differences in residential environment and in fertility behaviour. Within a country, fertility is particularly late in the metropolitan regions that include the country’s capital; other types of less densely populated urban areas, such as smaller cities and local industrial centres, generally postpone fertility less; and rural areas have the earliest birth schedules (##UREF##4##Buelens, 2021##). Metropolitan areas themselves are diverse: fertility rates are higher in suburban areas (##REF##26047550##Kulu &amp; Washbrook, 2014##). First, they offer a better environment for a family, such as larger housing with garden and green areas (##UREF##53##Vobecká &amp; Piguet, 2012##). Often, people move there to start a family in an environment that is better suited to their children’s needs and activities, while still being close to the opportunities the city has to offer (##REF##26047550##Kulu &amp; Washbrook, 2014##). Their values do not seem to differ much from those of the city dwellers (##UREF##38##Pisman et al., 2011##). Second, people in low-paid jobs for whom the city is unaffordable tend to live in (very different) suburban areas, marking strong suburban inequalities (##UREF##0##Bailey &amp; Minton, 2018##; ##UREF##55##Weck et al., 2023##). This group, often comprised of migrants from non-European origin and people from modest background, generally has more children than the average so that their last children are often born at older maternal ages (##UREF##28##Kulu et al., 2017##; ##UREF##50##Toulemon, 2004##). Overall, we expect a fairly linear variation in later fertility across the urbanisation gradient, with large shares of late fertility in very high-density areas.</p>", "<p id=\"P9\">We also note different levels of urbanisation between urban and rural areas within and between European countries (##UREF##11##European Environment Agency, 2009##), as well as very different prevalence of late fertility (##UREF##4##Buelens, 2021##). Population density is particularly low in the Nordic and Baltic countries while it is the highest along the diagonal from the United Kingdom to Italy. The number of urban areas also varies considerably between countries. In this paper, we use population density to represent the range of urbanisation levels, with the drawback that density is not exactly the same in urban and rural areas of different countries. Later fertility, represented as the contribution of women aged 35+ (or 40+) to the total fertility rate (TFR), is also subject to national as well as international variations. It is much more prevalent in the South of Europe, and much less so in countries of Central and Eastern Europe (##REF##32733116##Beaujouan, 2020##). These limitations are partly dealt with in our models, as detailed later.</p>", "<title>Factors Of Later Fertility Differentials Along The Urban–Rural Continuum</title>", "<p id=\"P10\">In this section, we explore which contextual factors may underlie the variation in later fertility along the urban–rural continuum, highlighting the importance of socioeconomic, cultural and compositional factors. Such elements are hardly independent of each other (##REF##31736514##Riederer &amp; Buber-Ennser, 2019##). For instance, highly educated women are more likely to live in cities where they pursue professional careers, and tend to have less traditional attitudes and lifestyles. The urban environment, including the educational and economic opportunities and the corresponding lifestyles, also leads to a different composition of the population than in small towns and rural areas. Despite difficulties in disentangling their effects, the analytical distinction between these elements has been fruitful in identifying reasons for differences in fertility levels by degree of urbanisation (see ##UREF##6##Campisi et al., 2020##; ##UREF##20##Hank, 2002##; ##REF##26047550##Kulu &amp; Washbrook, 2014##; ##REF##31736514##Riederer &amp; Buber-Ennser, 2019##). In this section, we discuss the potential role of the socioeconomic environment, family and gender norms and population composition for differences in fertility timing along the urban–rural continuum.</p>", "<title>Level of education</title>", "<p id=\"P11\">The role of education for delay and later recovery of childbearing is emphasised in research on regional fertility variations. Indeed, highly educated women are more likely to have children at later ages, when less educated women have already completed their family (##REF##22833187##Neels et al., 2012##). Findings of ##REF##17979002##Kulu et al. (2007)## for Sweden indicate that ‘the higher fertility of older women in the large cities is mostly the result of the larger proportion of highly educated women‘ (##REF##17979002##Kulu et al., 2007##, p. 277). This is in line with findings of ##REF##31736514##Riederer and Buber-Ennser (2019)## who report that postponing fertility intentions is more common in urban than in rural regions in Europe, likely owing to the larger proportion of highly educated women. ##UREF##35##Michielin (2004)## even supposed that urban fertility is mainly driven by female education as the highly educated may be ‘more oriented to urban ways of life’ (##UREF##35##Michielin, 2004##, p. 343). ##REF##17979002##Kulu et al. (2007##, p. 279) also refer to a ‘preferred lifestyle’ in urban settlements, but discuss in addition other mechanisms possibly leading to later fertility, such as high competition on urban labour markets.</p>", "<title>Economic environment</title>", "<p id=\"P12\">Several studies suggest that economic factors are at least partly responsible for urban-rural fertility differentials (##UREF##6##Campisi et al., 2020##). ##REF##26047550##Kulu and Washbrook (2014##, p. 169f.), for instance, state that living costs are higher in cities (e.g., housing), that children are more expensive and time-consuming for parents (e.g., after-school activities, extracurricular activities, availability of various shops and attractions), that opportunity costs of childbearing are higher (more opportunities for work and leisure), and that life is more competitive in large cities than small towns and rural regions. In this context, higher living costs and economic competitiveness alone may contribute to later births if young people postpone starting a family until they are financially secure (##UREF##27##Kulu, 2013##; ##REF##17979002##Kulu et al., 2007##). In addition, European cities are characterised by professionalisation trends (##UREF##18##Hamnett, 2021##; ##UREF##41##Riederer et al., 2021##), offering more opportunities and jobs in tertiary education, business, science and technology, sectors that require a high initial career and time investment and may contribute to lower fertility at younger ages (##UREF##25##Kravdal, 1994##). These trends often go hand in hand with better childcare infrastructure, making it easier to combine a career with motherhood later in life (##UREF##42##Riederer &amp; Buber-Ennser, 2018##). Although economic conditions in metropolitan areas vary, the urban economic environment seems to encourage later fertility.</p>", "<title>Family and gender norms</title>", "<p id=\"P13\">More liberal norms towards family and acceptance of women’s work may lead to more favourable conditions for fertility at a later age. Specifically, broad acceptance of maternal employment and respective policy support (particularly, affordable and good quality childcare) are understood as a prerequisite for combining a career and motherhood in modern societies (##UREF##9##Esping-Andersen, 2009##; ##UREF##16##Goldscheider et al., 2015##; ##REF##27795600##Matysiak &amp; Węziak-Białowolska, 2016##). Women’s work is more accepted in cities, and the necessary childcare facilities have developed more quickly across urban than rural areas in the last decades (##REF##31832030##Wood &amp; Neels, 2019##). Hence, in urban regions with a higher prevalence of female employment, circumstances may be more favourable to fertility recovery at later ages.</p>", "<p id=\"P14\">Flexible family norms, by allowing childbearing in diverse family circumstances, may also facilitate fertility at later ages. In particular, research on the United States (##UREF##13##Fuguitt et al., 1989##; ##UREF##46##Snyder et al., 2004##) describes urban–rural fertility differentials as a consequence of differences in marital status and of the lower age at first marriage in rural areas (cf. ##UREF##53##Vobecká and Piguet, 2012##, p. 226). Higher divorce rates indicate more liberal family norms, and divorce rates tend to be higher in European cities (e.g., ##UREF##14##Gautier et al., 2009##; ##UREF##33##Lyngstad &amp; Jalovaara, 2010##). The repartnering of divorcees may then contribute to later fertility in urban areas (##REF##22259032##Thomson et al., 2012##).</p>", "<title>Population composition</title>", "<p id=\"P15\">The composition of the population varies systematically along the urban–rural continuum and has likely an impact on later fertility. The age structure also affects the prevalence of births by women age 35+, and we explain below how we account for this in the construction of our dependent variable. Rural regions in the intertwined processes of depopulation and population ageing are often characterised by out-migration, lower economic development and problems in the provision of social services; including childcare services and schools (##UREF##30##Leibert &amp; Golinski, 2017##; ##UREF##39##Reynaud &amp; Miccoli, 2018##). Given out-migration due to economic uncertainty, a lack of future prospects and missing possibilities to combine work and family, later fertility is probably less relevant in such settings.</p>", "<p id=\"P16\">Migration particularly matters for regional fertility differentials (e.g., ##UREF##26##Kulu, 2006##; ##UREF##35##Michielin, 2004##; ##UREF##53##Vobecká &amp; Piguet, 2012##). Selective migration to cities may contribute to later fertility, as metropolitan regions attract highly qualified natives and foreigners who are more likely to postpone childbearing (e.g., ##UREF##41##Riederer, 2021##). In addition, international immigrants are generally more often found in urban than in rural areas. Metropolitan areas and cities have larger share of migrants (##UREF##32##Lichter et al., 2020##), who have larger family size (##UREF##28##Kulu et al., 2017##), which may contribute to later fertility. Indeed, while they may begin to postpone childbearing as part of an adjustment process, their fertility may remain temporarily high at older ages, thereby reinforcing the differentials in later fertility along the urban–rural continuum.</p>", "<p id=\"P17\">In summary, urban areas—and particularly international cities and metropolitan regions—in Europe are characterised by higher levels of later fertility than rural areas. We hypothesise that characteristics of the economic environment, family and gender norms and population composition account for differences along the urban–rural continuum. Finally, we expect our hypotheses to hold (broadly) across different national contexts.</p>", "<title>Results From Multilevel Models: Degree Of Urbanisation And Later Fertility</title>", "<p id=\"P31\">To explore how much the association between population density and later fertility can be explained by education, economic context, family and gender norms and population composition, we conducted a series of models with all variables measured at NUTS 2 level (##TAB##1##Table 2##). The results for the share of ASFR 35+ (panel A) and 40+ (panel B) are not substantially different and lead to the same conclusions. The bivariate association between population density and later fertility (models X1 and X2) is remarkable. The values of the coefficients <italic toggle=\"yes\">b</italic> range between 0.55 and 0.65 and are of a comparable magnitude to those obtained with measures at NUTS 3 level (compare ##TAB##0##Table 1##, M1 and M2). Further, in line with the results at NUTS 3 level, SD<sub>c</sub> of 0.28 and 0.29 (model X2) indicate considerable variation in the association between countries.</p>", "<p id=\"P32\">Our findings indicate that regional variations in education are important for differences in later fertility along the urban–rural continuum. The coefficients indicating the association between population density and later fertility (b) are substantially smaller when education (share of women with tertiary education) is included into the model (model X3 in ##TAB##1##Table 2##). The coefficients shrink from 0.55 to 0.30 (ASFR 35+) and from 0.62 to 0.39 (ASFR 40+), respectively. The reduction in coefficients is even larger when indicators of economic environment are included instead of education (0.16 and 0.29 in model X4). As expected, a higher share of highly educated women, a higher average gross domestic product (GDP) and a higher share of the high-tech sector are all associated with a higher share of late fertility. Comparing the coefficient of population density between models in which each of the three variables is entered separately (##TAB##2##Table 3##), the magnitude of the reduction in the coefficients seems comparable (coefficients between 0.26 and 0.30). When indicators of family and gender norms are included (model X5), there is no change in the coefficient of population density (##TAB##1##Table 2##). Some changes, albeit smaller than those in models considering the educational or economic environment, are found when indicators of population composition are included (coefficients of 0.45 and 0.54 in model X6, respectively). These findings are also consistent across the other model specifications, and tests of differences between coefficients support our interpretations (##SUPPL##0##Supporting Information: Table A.4##).</p>", "<p id=\"P33\">Finally, both the coefficient of population density b and its variation between countries SDc are reduced most when all variables are entered simultaneously in model X7 (##TAB##1##Table 2##). The coefficient now amounts to 0.12 (compared to 0.53 in model X2) and 0.23 (compared to 0.62 in model X2); the SDc to 0.15 (compared to 0.28 in model X2) and 0.18 (compared to 0.29 in model X2). Altogether, our analyses suggest that regional characteristics, and in particular the educational and economic environment, explain a very substantial part of the relationship between the degree of urbanisation and later fertility on NUTS 2 level and its variation across countries.</p>", "<title>Further Findings And Sensitivity Analyses</title>", "<p id=\"P34\">Other findings stand out as well. First, we observe significant associations of the indicators of gender and family norms and population composition with indicators of later fertility (##TAB##1##Table 2##): The lower the share of divorcees, the higher the share of ASFR 35+ in total fertility (models X5 and X7), and the higher the male-to-female employment ratio, the higher the share of ASFR 35+ and 40+ in total fertility (model X7). The higher the share of population age 60+, the lower the share of ASFR 35+ and 40+ (model X6). This association disappears, when all covariates are introduced (model X7), indicating that the association is mostly driven by the difference in the socioeconomic context of ageing areas. The higher the share of the foreign-born female population, the higher the share of ASFR 35+ and 40+, but the magnitude of the coefficient is small (models X6 and X7).</p>", "<p id=\"P35\">Second, results of multilevel models with population density and later fertility measured at NUTS 3 level are very consistent with results obtained at NUTS 2 level. The coefficient for population density and its decrease when contextual covariates are added are of comparable magnitude, and associations between later fertility and contextual covariates hardly differ from those obtained in models at NUTS 2 level (##SUPPL##0##Supporting Information: Tables A.5## and ##SUPPL##0##A.6##). Nevertheless, two deviations have also to be noted. In NUTS 3 level models, only the contextual characteristics measured at NUTS 3 level explain a substantial part of the positive association between population density and later fertility. The coefficient of population density hardly changes when contextual characteristics only available at NUTS 2 level are additionally entered (compare M3 and M4 in ##TAB##0##Table 1##). This suggests a high relevance of measuring all covariates at the same level to avoid inaccuracies.</p>", "<p id=\"P36\">In addition, the higher the share of divorcees at NUTS 3 level, the higher the share of ASFR 40+ in total fertility at NUTS 3 level (##SUPPL##0##Supporting Information: Table A.5##). Seemingly, this is in contrast to the NUTS 2 level analysis (negative effect on the share of ASFR 35+). The variation in the coefficient across countries SD<sub>c</sub>, however, is larger than the estimated coefficient <italic toggle=\"yes\">b</italic>. On NUTS 3 level, estimated associations are positive for most countries (e.g., France, Belgium), close to zero for some (e.g., Poland, Czechia), and even negative for others (in particular for Italy and the United Kingdom). When we add a random slope for the share of divorces on NUTS 2 level, the variation in the coefficient across countries is also larger than the estimated coefficient. Estimated associations are again negative for Italy and the United Kingdom and positive for France. Both, negative effects due to separations as well as positive effects due to repartnering may be at work (##REF##22259032##Thomson et al., 2012##).</p>", "<p id=\"P37\">Finally, in models both at NUTS 2 and NUTS 3 levels, female education is by far the most important predictor of later fertility. In models at NUTS 3 level, NUTS 3 level measures of population density and GDP show coefficients of similar magnitude as female education, but female education is the only NUTS 2 level measure with a strong association with later fertility at NUTS 3 level. Female education is thus confirmed to be the most important predictor for later fertility (e.g., ##UREF##8##Compans, 2021##; ##REF##29398739##Neels et al., 2017##).</p>", "<p id=\"P38\">We conduct analyses to check whether spatial autocorrelation could seriously bias our results. Computations of Moran’s <italic toggle=\"yes\">I</italic> show that there is huge spatial dependence in later fertility (values ranging from 0.75 to 0.81). However, most spatial dependence is accounted for in our multilevel models: In models at NUTS 3 level, remaining spatial dependence indicated by Moran’s <italic toggle=\"yes\">I</italic> on residuals is close to zero (between 0.01 and 0.03). In models at NUTS 2 level, Moran’s <italic toggle=\"yes\">I</italic> ranges from 0.11 to 0.30 (##SUPPL##0##Supporting Information: Table A.7##). In addition, conclusions obtained with spatial autoregressive models are the same as with other model specifications. When all the covariates are introduced, the coefficients for population density are almost identical (##SUPPL##0##Supporting Information: Table A.8##).</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>Bernhard Riederer’s work was supported by the Austrian Science Fund (FWF), under grant agreement no. P31171-G29 (‘Later Fertility in Europe’; see <ext-link xlink:href=\"https://fertilitychange.wordpress.com/\" ext-link-type=\"uri\">https://fertilitychange.wordpress.com/</ext-link>). Eva Beaujouan’s work was supported by the European Union’s Horizon 2020 research and innovation programme, grant Agreement No 101001410 (BIC.LATE; see <ext-link xlink:href=\"https://biclate.univie.ac.at/\" ext-link-type=\"uri\">https://biclate.univie.ac.at/</ext-link>).</p>", "<title>Funding information</title>", "<p>Austrian Science Fund, Grant/Award Number: P31171-G29; European Union Horizon 2020, Grant/Award Number: 101001410</p>", "<title>Data Availability Statement</title>", "<p id=\"P48\">The article combines Eurostat data from different sources that is publicly available. Detailed descriptions and links are presented to the reader in a table in the Supporting Information: Appendix.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><title>Share of age-specific fertility rates 35–39 and 40+ in total fertility rate in Europe (in %). <italic toggle=\"yes\">Source</italic>: ##UREF##12##Eurostat (2021)##; figures refer to 2018, except for Germany (2017); own figure.</title></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><title>Share of age-specific fertility rates 35+ in total fertility rate by NUTS 3 region for (a) Germany and (b) the United Kingdom, deviation from the respective national average. <italic toggle=\"yes\">Source:</italic>\n##UREF##12##Eurostat (2021)##; figures refer to 2017 (Germany) and 2018 (United Kingdom); own figure.</title></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Associations between population density and later fertility on <bold>NUTS</bold> 3 level according to standardisation method (multilevel mixed regression models)</title></caption><table frame=\"below\" rules=\"none\"><thead><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"bottom\" rowspan=\"2\" colspan=\"1\">Model</th><th align=\"left\" valign=\"top\" colspan=\"4\" style=\"border-bottom: 1px solid\" rowspan=\"1\">Standardisation with grand mean and within-country standard deviation</th><th align=\"left\" valign=\"top\" colspan=\"4\" style=\"border-bottom: 1px solid\" rowspan=\"1\">Standardisation with grand mean and standard deviation across all NUTS 3 or NUTS 2 regions</th></tr><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M1</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M2</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M3</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">M4</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N1</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N2</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N3</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N4</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" colspan=\"9\" rowspan=\"1\">A. Share of ASFR 35+ in TFR</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Population density (b)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.52<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.53<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.26<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.24<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.40<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.71<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.26<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0 21<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SD<sub>C</sub> population density</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">AIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3171</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3142</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2840</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2808</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2211</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2112</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1776</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1726</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3197</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3173</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2902</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2891</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2237</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2143</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1839</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1809</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">LR <italic toggle=\"yes\">χ</italic><sup>2</sup> test against previous model</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30.7<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">344.9<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40.2<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">101.3<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">347.1<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">58.4<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"9\" rowspan=\"1\">B. Share of ASFR 40+ in TFR</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Population density (<italic toggle=\"yes\">b</italic>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.53<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.53<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.30<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.46<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.70<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.32<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SD<sub>C</sub> population density</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.42</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">AIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3189</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3141</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2949</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2914</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2150</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2060</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1853</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1802</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3215</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3172</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3011</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2997</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2176</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2091</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1915</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1885</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">LR <italic toggle=\"yes\">χ</italic><sup>2</sup> test against previous model</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50.5<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">203.5<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43.4<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">91.9<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">219.0<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">58.7<xref rid=\"TFN2\" ref-type=\"table-fn\">***</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"9\" rowspan=\"1\">Included in model</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NUTS 3 covariates</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Incl.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Incl.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Incl.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Incl.</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NUTS 2 covariates</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Incl.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Incl.</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Associations between population density and later fertility on NUTS 2 level (multilevel mixed regression models), standardisation with grand mean and within-country standard deviation.</title></caption><table frame=\"below\" rules=\"none\"><thead><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"bottom\" rowspan=\"2\" colspan=\"1\">Model</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X1</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X2</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X3</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X4</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X5</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X6</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">X7</th></tr><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">A. Share of ASFR 35+ in TFR</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Population density</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.55<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.55<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.30<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.16<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.58<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.45<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.12<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      SD<sub>c</sub> Population density</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.26</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Education</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Share of women with tertiary education</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.49<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.41<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Economic environment</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      GDP/capita (%)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.27<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.20<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of high-tech sector</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.37<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.17<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Family and gender norms</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of divorced persons (2011)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.09<xref rid=\"TFN7\" ref-type=\"table-fn\">****</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.14<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Ratio of male-to-female employment (age 25–54)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.03</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.16<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Population composition</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of population age 60+</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.15<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of foreign-born female population (age 25–54)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.04<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.03<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Intercept</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.48<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.80<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.35</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.83<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.89<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    SD<sub>c</sub> intercept</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.87</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.68</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.87</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.54</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.44</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    SD residual</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.76</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.72</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.61</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.61</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.72</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.71</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.55</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    AIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">750</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">729</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">644</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">640</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">729</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">715</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">590</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    BIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">764</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">747</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">666</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">665</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">755</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">741</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">633</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    LR χ<sup>2</sup> test against X1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23.1<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    LR χ<sup>2</sup> test against X2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">86.6<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">92.4<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17.3<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">152.6<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"8\" style=\"background-color:#e7e7e9\" rowspan=\"1\">B. Share of ASFR 40+ in TFR</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Population density</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.66<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.62<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.39<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.61<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.54<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.23<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    SD<sub>c</sub> Population density</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.18</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Education</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of women with tertiary education</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.43<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.37<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Economic environment</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      GDP/capita (%)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.22<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.13<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of high-tech sector</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.31<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.12<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Family and gender norms</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of divorced persons (2011)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.01</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Ratio of male-to-female employment (age 25-54)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.03</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.12<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"8\" rowspan=\"1\">Population composition</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of population age 60+</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.10<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.03</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      Share of foreign-born female population (age 25-54)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.04<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.03<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Intercept</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.75<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.79<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.40</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.27</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.74<xref rid=\"TFN4\" ref-type=\"table-fn\">*</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.89<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">      SD<sub>c</sub> intercept</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.77</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.62</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.57</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.75</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.52</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.46</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    SD residual</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.56</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.57</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.53</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    AIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">704</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">675</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">596</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">604</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">678</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">667</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">573</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    BIC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">719</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">693</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">617</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">629</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">703</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">692</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">617</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    LR χ<sup>2</sup> test against X1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31.1<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    LR χ<sup>2</sup> test against X2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81.4<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75.1<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.98</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12.2<xref rid=\"TFN5\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">115.8<xref rid=\"TFN6\" ref-type=\"table-fn\">***</xref></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><title>Associations between covariates and later fertility on NUTS 2 level in models with population density and only one other covariate (multilevel mixed regression models), standardisation with grand mean and within-country standard.</title></caption><table frame=\"below\" rules=\"none\"><thead><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">Models incl. population density and one additional covariate Other covariate</th><th align=\"left\" valign=\"top\" colspan=\"3\" rowspan=\"1\">Share of ASFR 35+ in TFR (NUTS 2)</th><th align=\"left\" valign=\"top\" colspan=\"3\" rowspan=\"1\">Share of ASFR 40+ in TFR (NUTS 2)</th></tr><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Population density</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Other covariate</th><th align=\"left\" valign=\"top\" colspan=\"2\" rowspan=\"1\">Population density</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Other covariate</th></tr><tr style=\"background-color:#d1d2d4\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SD<sub>c</sub></th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SD<sub>c</sub></th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.55<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.62<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"7\" style=\"background-color:#e7e7e9\" rowspan=\"1\">Education</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Share of women with tertiary education</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.30<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.49<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.39<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.43<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"7\" rowspan=\"1\">Economic environment</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    GDP/capita (%)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.27<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.22</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.44<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.38<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.36<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Share of high-tech sector</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.26<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.49<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.37<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.22</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.41<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" colspan=\"7\" rowspan=\"1\">Family and gender norms</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Share of divorced persons (2011)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.57<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.08<xref rid=\"TFN13\" ref-type=\"table-fn\">****</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.61<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.03</td></tr><tr style=\"background-color:#e7e7e9\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Ratio of male-to-female employment</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.55<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.01</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.61<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.04</td></tr><tr><td align=\"left\" valign=\"top\" colspan=\"7\" rowspan=\"1\">Population composition</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Share of population age 60+</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.49<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.15<xref rid=\"TFN11\" ref-type=\"table-fn\">**</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.58<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.27</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">-0.10<xref rid=\"TFN10\" ref-type=\"table-fn\">*</xref></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">    Share of foreign-born female population (age 25–54)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.51<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.04<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.58<xref rid=\"TFN12\" ref-type=\"table-fn\">***</xref></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.04<xref rid=\"TFN11\" ref-type=\"table-fn\">**</xref></td></tr></tbody></table></table-wrap>" ]
[ "<disp-formula id=\"FD1\"><mml:math id=\"M1\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>y</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>α</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mi>c</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mn>1</mml:mn><mml:mi>c</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ε</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo></mml:math></disp-formula>", "<disp-formula id=\"FD2\"><mml:math id=\"M2\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>y</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>α</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mi>c</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mn>1</mml:mn><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ε</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi><mml:mi>c</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>,</mml:mo></mml:math></disp-formula>", "<disp-formula id=\"FD3\"><mml:math id=\"M3\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>y</mml:mi><mml:mrow><mml:mtext>ijc</mml:mtext></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>α</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mtext>jc</mml:mtext></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mtext>c</mml:mtext></mml:msub><mml:mo>+</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mtext>d</mml:mtext></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mtext>dc</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msub><mml:mi>d</mml:mi><mml:mrow><mml:mtext>ijc</mml:mtext></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ζ</mml:mi><mml:mrow><mml:mn>1</mml:mn><mml:mtext>c</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msub><mml:mtext>x</mml:mtext><mml:mrow><mml:mtext>ijc</mml:mtext></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:msub><mml:mtext>x</mml:mtext><mml:mrow><mml:mtext>jc</mml:mtext></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ε</mml:mi><mml:mrow><mml:mtext>ijc</mml:mtext></mml:mrow></mml:msub><mml:mo>,</mml:mo></mml:math></disp-formula>" ]
[]
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[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supplementary material</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\"><label>1</label><p id=\"P49\">In most countries, figures are available for 5-year age groups (10−14,15−19, 20−24, 25−29, 30−34. 35−39, 40−44, 45−49, 50+). For the United Kingdom and Germany, figures begin with the age group ‘below age 20’ and end with the age group ‘age 45+’. Computing agespecific fertility, we assumed 10-year intervals for ‘below age 20’ and ‘age 45+’ and a 5-year interval for ‘age 50+’.</p></fn><fn id=\"FN2\"><label>2</label><p id=\"P50\">If data for 2018 was not available for single regions, we used the chronologically closest available information to fill these gaps (usually, 2017).</p></fn><fn id=\"FN3\"><label>3</label><p id=\"P51\">As categories have been changed in several countries between NUTS 2010 and NUTS 2013 (France, Germany, Greece, Poland, Portugal, Slovenia, United Kingdom), NUTS 2013 and NUTS 2016 (Finland, Germany, Ireland, Netherlands, Poland, United Kingdom) and NUTS 2016 and NUTS 2021 (Croatia, Norway), and given the restricted availability of more detailed subregional data, we imputed the values for NUTS 2016 regions using the NUTS converter of the Joint Research Centre (2023) and based on population size in 2010 (for 2011 census data) or 2018 (share of high-tech sector 2018), respectively. Changes in and of NUTS regions are listed in the ##SUPPL##0##Supporting Information: Table A.2##.</p></fn><fn id=\"FN4\"><label>4</label><p id=\"P52\">Some countries that have been included in the descriptive analyses had to be excluded due to missing information on context characteristics. The remaining 28 countries are Austria, Belgium, Bulgaria, Croatia, Cyprus, Czechia, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Lithuania, Luxembourg, Malta, the Netherlands, Norway, Poland, Portugal, Romania, Sweden, Slovakia, Slovenia, Spain and the United Kingdom. Within those countries, some NUTS 2 and NUTS 3 regions had to be omitted from the analyses because of missing context information as well (Finland: 1 NUTS 3 region; France: 5; Greece: 10; Italy: 1; Poland: 2; Portugal: 8; Spain: 2). Most of them are autonomous regions, islands or oversea departments (a limitation for prior research as well; e.g., ##UREF##6##Campisi et al., 2020##).</p></fn><fn id=\"FN5\"><label>5</label><p id=\"P53\">Spatial models with standard weight matrices account for proximity effects from bidirectional spillover. If similarity due to proximity results from effects of urban centres on surrounding areas but similarity of surrounding areas to urban areas is used to explain effects of urban regions in a cross-sectional setting, findings could be misleading. Using information from population density to generate a weight matrix that guarantees larger influences of regions with higher population density, for instance, would further complicate the interpretation of direct and indirect effects of population density. In addition, the correct specification of multilevel models that allow for spatial random effects and spatial random slopes (e.g., by following Bayesian approaches) and its estimation are not straightforward.</p></fn><fn id=\"FN6\"><label>6</label><p id=\"P54\">Unfortunately, the number of NUTS 2 regions has not been sufficient to compute meaningful standard deviations for Croatia, Latvia, Lithuania, Luxembourg and Malta; for NUTS 3 regions for Luxembourg and Malta. In these exceptional cases, the standard deviation of the total sample of regions had to be used.</p></fn><fn id=\"FN7\"><label>7</label><p id=\"P55\">See Footnote 6.</p></fn><fn id=\"FN8\" fn-type=\"COI-statement\"><p id=\"P56\">\n<bold>Conflict Of Interest Statement</bold>\n</p><p id=\"P57\">The authors declare no conflicts of interest.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><p id=\"P58\"><italic toggle=\"yes\">Note</italic>: <italic toggle=\"yes\">N</italic><sub>country</sub> = 28; <italic toggle=\"yes\">N</italic><sub>NUTS 2</sub> = 270; <italic toggle=\"yes\">N</italic><sub>NUTS 3</sub> = 1328. Population density at NUTS 3 level. NUTS 3 covariates: GDP/capita (%), share of divorced persons (%; in 2011), share of couples with four or more children (%; in 2011), share of population age 60+. NUTS 2 covariates: share of high-tech sector (%), share of women with tertiary education (%), share of foreign-born female population (%; age 25–54), ratio of male-to-female employment (ratio of %, age 25–54). b, regression coefficients. SD<sub>C</sub>, random-effect on country level (i.e., variation of coefficient b across countries).</p><p id=\"P59\">Abbreviations: AIC, Akaike information criterion; ASFR, age-specific fertility rates; BIC, Bayesian information criterion; GDP, gross domestic product; LR, likelihood ratio; TFR, total fertility rate.</p></fn><fn id=\"TFN2\"><label>***</label><p id=\"P60\">p≤ 0.001</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN3\"><p id=\"P61\"><italic toggle=\"yes\">Note</italic>: <italic toggle=\"yes\">N</italic><sub>country</sub> = 28; <italic toggle=\"yes\">N</italic><sub>NUTS</sub>\n<sub>2</sub> = 270. b, regression coefficients. SD<sub>c</sub>, random-effect on country level (i.e., variation of coefficient <italic toggle=\"yes\">b</italic> across countries). Abbreviations: AIC, Akaike information criterion; ASFR, age-specific fertility rates; BIC, Bayesian information criterion; GDP, gross domestic product; LR, likelihood ratio; TFR, total fertility rate.</p></fn><fn id=\"TFN4\"><label>*</label><p id=\"P62\">p ≤ 0.05</p></fn><fn id=\"TFN5\"><label>**</label><p id=\"P63\">p ≤ 0.01</p></fn><fn id=\"TFN6\"><label>***</label><p id=\"P64\">p ≤ 0.001</p></fn><fn id=\"TFN7\"><label>****</label><p id=\"P65\">p ≤ 0.10</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN8\"><p id=\"P66\"><italic toggle=\"yes\">Note</italic>: <italic toggle=\"yes\">N</italic><sub>country</sub> = 28; <italic toggle=\"yes\">N</italic><sub>NUTS 2</sub> = 270; <italic toggle=\"yes\">N</italic><sub>NUTS 3</sub> = 1328. b, regression coefficients. SD<sub>c</sub>, random-effect on country level (i.e., variation of coefficient <italic toggle=\"yes\">b</italic> across countries).</p></fn><fn id=\"TFN9\"><p id=\"P67\">Abbreviation: GDP, gross domestic product.</p></fn><fn id=\"TFN10\"><label>*</label><p id=\"P68\">p ≤ 0.05</p></fn><fn id=\"TFN11\"><label>**</label><p id=\"P69\">p ≤ 0.10;</p></fn><fn id=\"TFN12\"><label>***</label><p id=\"P70\">p ≤ 0.001;</p></fn><fn id=\"TFN13\"><label>****</label><p id=\"P71\">p ≤ 0.10</p></fn></table-wrap-foot>" ]
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[{"person-group": ["\n"], "surname": ["Bailey", "Minton"], "given-names": ["N", "J"], "article-title": ["The suburbanisation of poverty in British cities, 2004-16: Extent, processes and nature"], "source": ["Urban Geography"], "year": ["2018"], "volume": ["39"], "issue": ["6"], "fpage": ["892"], "lpage": ["915"], "pub-id": ["10.1080/02723638.2017.1405689"]}, {"person-group": ["\n", "\n"], "surname": ["Beaujouan", "Sobotka", "Nikolaou", "Seifer"], "given-names": ["\u00c9", "T", "D", "D"], "part-title": ["Is 40 the new 30? Increasing reproductive intentions and fertility rates beyond age 40"], "source": ["Optimizing the management of fertility in women over 40"], "publisher-name": ["Cambridge University Press"], "year": ["2022"], "fpage": ["3"], "lpage": ["18"], "pub-id": ["10.1017/9781009025270.002"]}, {"person-group": ["\n"], "surname": ["Beaujouan", "Toulemon"], "given-names": ["\u00c9", "L"], "article-title": ["European countries with delayed childbearing are not those with lower fertility"], "source": ["Genus"], "year": ["2021"], "volume": ["77"], "issue": ["2"], "fpage": ["2"], "pub-id": ["10.1186/s41118-020-00108-0"]}, {"person-group": ["\n"], "surname": ["de Beer", "Deerenberg"], "given-names": ["J", "I"], "article-title": ["An explanatory model for projecting regional fertility differences in the Netherlands"], "source": ["Population Research and Policy Review"], "year": ["2007"], "volume": ["26"], "fpage": ["511"], "lpage": ["528"], "pub-id": ["10.1007/s11113-007-9040-y"]}, {"person-group": ["\n"], "surname": ["Buelens"], "given-names": ["M"], "article-title": ["Subnational spatial variations of fertility timing in Europe since 1990"], "source": ["Cybergeo: European Journal of Geography"], "year": ["2021"], "pub-id": ["10.4000/cybergeo.37887"]}, {"person-group": ["\n"], "surname": ["Burtenshaw", "Bateman", "Ashworth"], "given-names": ["D", "M", "GJ"], "source": ["European city: A western perspective"], "publisher-name": ["Routledge"], "year": ["2021"], "comment": ["\n"], "ext-link": ["https://books.google.at/books?hl=en&lr=&id=m1EwEAAAQBAJ&oi=fnd&pg=PT6&dq=urban+system+in+europe&ots=zF1wRaT_zT&sig=FERkd6aSyaICz-uxZI9q0EmYL9E#v=onepage&q&f=false"]}, {"person-group": ["\n"], "surname": ["Campisi", "Kulu", "Mikolai", "Kl\u00fcsener", "Myrskyl\u00e4"], "given-names": ["N", "H", "J", "S", "M"], "article-title": ["Spatial variation in fertility across Europe: Patterns and determinants"], "source": ["Population, Space and Place"], "year": ["2020"], "volume": ["26"], "issue": ["4"], "pub-id": ["10.1002/psp.2308"]}, {"person-group": ["\n"], "surname": ["Carter", "Borch"], "given-names": ["JS", "CA"], "article-title": ["Assessing the effects of urbanism and regionalism on gender-role attitudes, 1974-1998"], "source": ["Sociological Inquiry"], "year": ["2005"], "volume": ["75"], "issue": ["4"], "fpage": ["548"], "lpage": ["563"], "pub-id": ["10.1111/j.1475-682X.2005.00136.x"]}, {"person-group": ["\n"], "surname": ["Compans"], "given-names": ["MC"], "article-title": ["Late motherhood, late fatherhood, and permanent childlessness: Trends by educational level and cohorts (1950-1970) in France"], "source": ["Demographic Research"], "year": ["2021"], "volume": ["45"], "month": ["July"], "fpage": ["329"], "lpage": ["344"], "pub-id": ["10.4054/DEMRES.2021.45.10"]}, {"person-group": ["\n"], "surname": ["Esping-Andersen"], "given-names": ["G"], "source": ["The incomplete revolution: Adapting welfare states to women\u2019s new roles"], "publisher-name": ["Polity Press"], "year": ["2009"]}, {"person-group": ["\n"], "surname": ["Esping-Andersen", "Billari"], "given-names": ["G", "FC"], "article-title": ["Re-theorizing family demographics"], "source": ["Population and Development Review"], "year": ["2015"], "volume": ["41"], "issue": ["1"], "fpage": ["1"], "lpage": ["31"], "pub-id": ["10.1111/j.1728-4457.2015.00024.x"]}, {"collab": ["European Environment Agency"], "source": ["Population density"], "year": ["2009"], "comment": ["\n"], "ext-link": ["https://www.eea.europa.eu/data-and-maps/figures/population-density-2"]}, {"collab": ["Eurostat"], "source": ["Live births by age group of the mothers and NUTS 3 region"], "year": ["2021"], "comment": ["Retrieved from: "], "ext-link": ["https://ec.europa.eu/eurostat/web/rural-development/data"]}, {"person-group": ["\n"], "surname": ["Fuguitt", "Brown", "Beale"], "given-names": ["GV", "DL", "CL"], "source": ["Rural and small town America"], "publisher-name": ["Russel Sage Foundation"], "year": ["1989"]}, {"person-group": ["\n"], "surname": ["Gautier", "Svarer", "Teulings"], "given-names": ["PA", "M", "CN"], "article-title": ["Sin City? Why is the divorce rate higher in urban areas?"], "source": ["The Scandinavian Journal of Economics"], "year": ["2009"], "volume": ["111"], "issue": ["3"], "fpage": ["439"], "lpage": ["456"], "pub-id": ["10.1111/j.1467-9442.2009.01571.x"]}, {"person-group": ["\n"], "surname": ["Glenn", "Hill"], "given-names": ["ND", "L"], "article-title": ["Rural-Urban differences in attitudes and behavior in the United States"], "source": ["The Annals of the American Academy of Political and Social Science"], "year": ["1977"], "volume": ["429"], "issue": ["1"], "fpage": ["36"], "lpage": ["50"], "pub-id": ["10.1177/000271627742900105"]}, {"person-group": ["\n"], "surname": ["Goldscheider", "Bernhardt", "Lappeg\u00e5rd"], "given-names": ["F", "E", "T"], "article-title": ["The gender revolution: A framework for understanding changing family and demographic behavior"], "source": ["Population and Development Review"], "year": ["2015"], "volume": ["41"], "issue": ["2"], "fpage": ["207"], "lpage": ["239"], "pub-id": ["10.1111/j.1728-4457.2015.00045.x"]}, {"person-group": ["\n"], "surname": ["Golgher", "Voss"], "given-names": ["AB", "PR"], "article-title": ["How to interpret the coefficients of spatial models: Spillovers, direct and indirect effects"], "source": ["Spatial Demography"], "year": ["2016"], "volume": ["4"], "fpage": ["175"], "lpage": ["205"], "pub-id": ["10.1007/s40980-015-0016-y"]}, {"person-group": ["\n"], "surname": ["Hamnett"], "given-names": ["C"], "article-title": ["The changing social structure of global cities: Professionalisation, proletarianisation or polarisation"], "source": ["Urban Studies"], "year": ["2021"], "volume": ["58"], "issue": ["5"], "fpage": ["1050"], "lpage": ["1066"], "pub-id": ["10.1177/0042098020940556"]}, {"person-group": ["\n"], "surname": ["Hank"], "given-names": ["K"], "article-title": ["Regional fertility differences in western Germany: An overview of the literature and recent descriptive findings"], "source": ["International Journal of Population Geography"], "year": ["2001"], "volume": ["7"], "issue": ["4"], "fpage": ["243"], "lpage": ["257"], "pub-id": ["10.1002/ijpg.228"]}, {"person-group": ["\n"], "surname": ["Hank"], "given-names": ["K"], "article-title": ["Regional social contexts and individual fertility decisions: A multilevel analysis of first and second births in western Germany"], "source": ["European Journal of Population/Revue Europenne de Dmographie"], "year": ["2002"], "volume": ["18"], "fpage": ["281"], "lpage": ["299"], "pub-id": ["10.1023/A:1019765026537"]}, {"person-group": ["\n"], "surname": ["Heisig", "Schaeffer", "Giesecke"], "given-names": ["JP", "M", "J"], "article-title": ["The costs of simplicity: Why multilevel models may benefit from accounting for crosscluster differences in the effects of controls"], "source": ["American Sociological Review"], "year": ["2017"], "volume": ["82"], "issue": ["4"], "fpage": ["796"], "lpage": ["827"], "pub-id": ["10.1177/0003122417717901"]}, {"person-group": ["\n"], "surname": ["Horn"], "given-names": ["D"], "source": ["A note on the importance of within country standardization when conducting multilevel analysis (MZESWorking Paper 104)"], "publisher-name": ["Mannheim Centre for European Social Research"], "year": ["2007"], "comment": ["\n"], "ext-link": ["http://www.mzes.uni-mannheim.de/publications/wp/wp-104.pdf"]}, {"collab": ["Joint Research Centre"], "source": ["NUTS converter [Computer software]"], "year": ["2023"], "comment": ["\n"], "ext-link": ["https://urban.jrc.ec.europa.eu/nutsconverter"]}, {"person-group": ["\n"], "surname": ["Kohler", "Behrman", "Skytthe"], "given-names": ["H-P", "JR", "A"], "article-title": ["Partner + children = happiness? the effects of partnerships and fertility on well-being"], "source": ["Population and Development Review"], "year": ["2005"], "volume": ["31"], "month": ["September"], "fpage": ["407"], "lpage": ["445"], "pub-id": ["10.1111/j.1728-4457.2005.00078.x"]}, {"person-group": ["\n"], "surname": ["Kravdal"], "given-names": ["\u00d8"], "article-title": ["The importance of economic activity, economic potential and economic resources for the timing of first births in Norway"], "source": ["Population Studies"], "year": ["1994"], "volume": ["48"], "issue": ["2"], "fpage": ["249"], "lpage": ["267"], "pub-id": ["10.1080/0032472031000147786"]}, {"person-group": ["\n"], "surname": ["Kulu"], "given-names": ["H"], "article-title": ["Fertility of internal migrants: Comparison between Austria and Poland"], "source": ["Population, Space and Place"], "year": ["2006"], "volume": ["12"], "issue": ["3"], "fpage": ["147"], "lpage": ["170"], "pub-id": ["10.1002/psp"]}, {"person-group": ["\n"], "surname": ["Kulu"], "given-names": ["H"], "article-title": ["Why do fertility levels vary between urban and rural areas"], "source": ["Regional Studies"], "year": ["2013"], "volume": ["47"], "issue": ["6"], "fpage": ["895"], "lpage": ["912"], "pub-id": ["10.1080/00343404.2011.581276"]}, {"person-group": ["\n"], "surname": ["Kulu", "Hannemann", "Pailh\u00e9", "Neels", "Krapf", "Gonz\u00e1lez-Ferrer", "Andersson"], "given-names": ["H", "T", "A", "K", "S", "A", "G"], "article-title": ["Fertility by birth order among the descendants of immigrants in selected european countries"], "source": ["Population and Development Review"], "year": ["2017"], "volume": ["43"], "issue": ["1"], "fpage": ["31"], "lpage": ["60"], "pub-id": ["10.1111/padr.12037"]}, {"person-group": ["\n"], "surname": ["Leibert"], "given-names": ["T"], "article-title": ["Geburten in Europa: Junge M\u00fctter, alte M\u00fctter. N aktuell, Leibniz-Institut f\u00fcr L\u00e4nderkunde"], "year": ["2020"], "comment": ["\n"], "ext-link": ["https://aktuell.nationalatlas.de/wp-content/uploads/20_06_Geburten_Europa.pdf"]}, {"person-group": ["\n"], "surname": ["Leibert", "Golinski"], "given-names": ["T", "S"], "article-title": ["Peripheralisation: The missing link in dealing with demographic change"], "source": ["Comparative Population Studies"], "year": ["2017"], "volume": ["41"], "issue": ["3-4"], "pub-id": ["10.12765/CPoS-2017-02"]}, {"person-group": ["\n"], "surname": ["Lesthaeghe", "Neels"], "given-names": ["R", "K"], "article-title": ["From the first to the second demographic transition: An interpretation of the spatial continuity of demographic innovation in France, Belgium and Switzerland"], "source": ["European Journal of Population/Revue Europenne de Dmographie"], "year": ["2002"], "volume": ["18"], "issue": ["4"], "fpage": ["325"], "lpage": ["360"], "pub-id": ["10.1023/A:1021125800070"]}, {"person-group": ["\n"], "surname": ["Lichter", "Parisi", "Ambinakudige"], "given-names": ["DT", "D", "S"], "article-title": ["The spatial integration of immigrants in Europe: A cross-national study"], "source": ["Population Research and Policy Review"], "year": ["2020"], "volume": ["39"], "issue": ["3"], "fpage": ["465"], "lpage": ["491"], "pub-id": ["10.1007/s11113-019-09540-3"]}, {"person-group": ["\n"], "surname": ["Lyngstad", "Jalovaara"], "given-names": ["TH", "M"], "article-title": ["A review of the antecedents of union dissolution"], "source": ["Demographic Research"], "year": ["2010"], "volume": ["23"], "fpage": ["257"], "lpage": ["291"], "pub-id": ["10.4054/DemRes.2010.23.10"]}, {"person-group": ["\n"], "surname": ["McDonald"], "given-names": ["P"], "article-title": ["Gender equity in theories of fertility transition"], "source": ["Population and Development Review"], "year": ["2000"], "volume": ["26"], "issue": ["3"], "fpage": ["427"], "lpage": ["439"], "comment": ["\n"], "ext-link": ["http://www.jstor.org/stable/172314"]}, {"person-group": ["\n"], "surname": ["Michielin"], "given-names": ["F"], "article-title": ["Lowest low fertility in an urban context: The role of migration in Turin, Italy"], "source": ["Population, Space and Place"], "year": ["2004"], "volume": ["10"], "issue": ["4"], "fpage": ["331"], "lpage": ["347"], "pub-id": ["10.1002/psp.337"]}, {"person-group": ["\n"], "surname": ["Neyer"], "given-names": ["G"], "source": ["Family policies and fertility in Europe: Fertility policies at the intersection of gender policies, employment policies and care policies, MPIDR Working Paper, WP 2006-010"], "publisher-name": ["Max Planck Institute for Demographic Research"], "year": ["2006"], "comment": ["\n"], "ext-link": ["https://www.demogr.mpg.de/papers/working/wp-2006-010.pdf"]}, {"person-group": ["\n"], "surname": ["Osiewalska"], "given-names": ["B"], "article-title": ["Partners\u2019 empowerment and fertility in ten European countries"], "source": ["Demographic Research"], "year": ["2018"], "volume": ["38"], "fpage": ["1495"], "lpage": ["1534"], "pub-id": ["10.4054/DemRes.2018.38.49"]}, {"person-group": ["\n"], "surname": ["Pisman", "Allaert", "Lombaerde"], "given-names": ["A", "G", "P"], "article-title": ["Urban and suburban lifestyles and residential preferences in a highly urbanized society: Experiences from a case study in Ghent (Flanders, Belgium)"], "source": ["Belgeo"], "year": ["2011"], "volume": ["1\u20132"], "fpage": ["89"], "lpage": ["104"], "pub-id": ["10.4000/belgeo.6394"]}, {"person-group": ["\n"], "surname": ["Reynaud", "Miccoli"], "given-names": ["C", "S"], "article-title": ["Depopulation and the aging population: The relationship in Italian municipalities"], "source": ["Sustainability"], "year": ["2018"], "volume": ["10"], "issue": ["4"], "fpage": ["1004"], "pub-id": ["10.3390/su10041004"]}, {"person-group": ["\n"], "surname": ["Riederer"], "given-names": ["B"], "source": ["Elternschaft und Wohlbefinden: Kinder im individuellen, partnerschaftlichen und gesellschaftlichen Kontext"], "publisher-name": ["Springer VS"], "year": ["2018"], "pub-id": ["10.1007/978-3-658-22561-2"]}, {"person-group": ["\n"], "surname": ["Riederer"], "given-names": ["B"], "source": ["Urban-rural differences in fertility: A longitudinal analysis using Austrian register data from the female 1984 cohort"], "publisher-name": ["Annual Meeting of the EAPS Working Group on Register Based Fertility Research"], "year": ["2021"], "month": ["December"], "day": ["13"], "comment": ["\n"], "ext-link": ["https://www.eaps.nl/page/register-based-fertility-research"]}, {"person-group": ["\n"], "surname": ["Riederer", "Buber-Ennser"], "given-names": ["B", "I"], "article-title": ["Regional context and realization of fertility intentions: Are capitals different? The examples of Austria and Hungary"], "source": ["Demogr\u00e1fia"], "year": ["2018"], "volume": ["61"], "issue": ["5"], "fpage": ["29"], "lpage": ["58"], "pub-id": ["10.21543/DEE.2018.2"]}, {"person-group": ["\n", "\n"], "surname": ["Riederer", "Verwiebe", "Ahn", "Kazepov", "Verwiebe"], "given-names": ["B", "R", "B", "Y", "R"], "part-title": ["Professionalisation, polarisation or both? Economic restructuring and new divisions of labour"], "source": ["Vienna: Still a just city?"], "publisher-name": ["Routledge"], "year": ["2021"], "edition": ["1st"], "fpage": ["99"], "lpage": ["114"], "pub-id": ["10.4324/9781003133827-10"]}, {"person-group": ["\n"], "surname": ["Schmidt-Catran", "Fairbrother"], "given-names": ["AW", "M"], "article-title": ["The random effects in multilevel models: Getting them wrong and getting them right"], "source": ["European Sociological Review"], "year": ["2016"], "volume": ["32"], "issue": ["1"], "fpage": ["23"], "lpage": ["38"], "pub-id": ["10.1093/esr/jcv090"]}, {"person-group": ["\n"], "surname": ["Shucksmith", "Cameron", "Merridew", "Pichler"], "given-names": ["M", "S", "T", "F"], "article-title": ["Urban-rural differences in quality of life across the European Union"], "source": ["Regional Studies"], "year": ["2009"], "volume": ["43"], "issue": ["10"], "fpage": ["1275"], "lpage": ["1289"], "pub-id": ["10.1080/00343400802378750"]}, {"person-group": ["\n"], "surname": ["Snyder", "Brown", "Condo"], "given-names": ["AR", "SL", "EP"], "article-title": ["Residential differences in family formation: The significance of cohabitation"], "source": ["Rural Sociology"], "year": ["2004"], "volume": ["69"], "issue": ["2"], "fpage": ["235"], "lpage": ["260"], "pub-id": ["10.1526/003601104323087598"]}, {"person-group": ["\n"], "surname": ["Sobotka"], "given-names": ["T"], "article-title": ["Overview Chapter 6: The diverse faces of the second demographic transition in Europe"], "source": ["Demographic Research"], "year": ["2008"], "volume": ["19"], "issue": ["8"], "fpage": ["171"], "lpage": ["224"], "pub-id": ["10.4054/DemRes.2008.19.8"]}, {"person-group": ["\n"], "surname": ["Sobotka", "Zeman", "Lesthaeghe", "Frejka"], "given-names": ["T", "K", "RJ", "T"], "article-title": ["Postponement and Recuperation in Cohort Fertility: Austria, Germany and Switzerland in a European Context"], "source": ["Comparative Population Studies"], "year": ["2011"], "volume": ["36"], "issue": ["2\u20133"], "pub-id": ["10.12765/CPoS-2011-10"]}, {"person-group": ["\n"], "surname": ["S\u00f8rensen"], "given-names": ["JFL"], "article-title": ["Rural-urban differences in life satisfaction: Evidence from the European Union"], "source": ["Regional Studies"], "year": ["2014"], "volume": ["48"], "issue": ["9"], "fpage": ["1451"], "lpage": ["1466"], "pub-id": ["10.1080/00343404.2012.753142"]}, {"person-group": ["\n"], "surname": ["Toulemon"], "given-names": ["L"], "article-title": ["Fertility among immigrant women: New data, a new approach"], "source": ["Population and Societies"], "year": ["2004"], "volume": ["400"], "fpage": ["4"], "comment": ["\n"], "ext-link": ["https://www.ined.fr/fichier/s_rubrique/18833/publi_pdf2_pop_and_soc_english_400.en.pdf"]}, {"person-group": ["\n"], "surname": ["Trovato", "Grindstaff"], "given-names": ["F", "CF"], "article-title": ["Decomposing the urban-rural fertility differential: Canada 1971"], "source": ["Rural Sociology"], "year": ["1980"], "volume": ["45"], "issue": ["3"], "fpage": ["448"], "lpage": ["468"], "comment": ["\n"], "ext-link": ["https://www.proquest.com/openview/a74c5678035731f9468974e3719375d9/"]}, {"person-group": ["\n"], "surname": ["Valkonen", "Blomgren", "Kauppinen", "Martikainen", "M\u00e4enp\u00e4\u00e4"], "given-names": ["T", "J", "TM", "P", "E"], "article-title": ["The effects of socioeconomic and cultural characteristics of regions on the spatial patterns of the Second Demographic Transition in Finland"], "source": ["Demographic Research"], "year": ["2008"], "volume": ["19"], "fpage": ["2043"], "lpage": ["2056"], "pub-id": ["10.4054/DemRes.2008.19.61"]}, {"person-group": ["\n"], "surname": ["Vobeck\u00e1", "Piguet"], "given-names": ["J", "V"], "article-title": ["Fertility, natural growth, and migration in the Czech Republic: An urban-suburban-rural gradient analysis of long-term trends and recent reversals"], "source": ["Population, Space and Place"], "year": ["2012"], "volume": ["18"], "issue": ["3"], "fpage": ["225"], "lpage": ["240"], "pub-id": ["10.1002/psp.698"]}, {"person-group": ["\n"], "surname": ["Walford", "Kurek"], "given-names": ["N", "S"], "article-title": ["Outworking of the second demographic transition: National trends and regional patterns of fertility change in Poland, and England and Wales, 2002-2012: outworking of the second demographic transition"], "source": ["Population, Space and Place"], "year": ["2016"], "volume": ["22"], "issue": ["6"], "fpage": ["508"], "lpage": ["525"], "pub-id": ["10.1002/psp.1936"]}, {"person-group": ["\n"], "surname": ["Weck", "Dobusch", "Pfaffenbach", "Fina", "Schmitz"], "given-names": ["S", "D", "C", "S", "J"], "article-title": ["New peripheries in the making? Analysing German cities from the lens of a suburbanisation of poverty"], "source": ["Journal of Housing and the Built Environment"], "year": ["2023"], "pub-id": ["10.1007/s10901-023-10036-y"]}]
{ "acronym": [], "definition": [] }
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Popul Space Place. 2023 Oct 31; 30(1):psp.2720
oa_package/1f/85/PMC7615507.tar.gz
PMC7615508
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[ "<title>Introduction</title>", "<p id=\"P2\">Career plans and funding calls in biostatistical methodology often revolve around “novelty” and “innovation.” This stimulates the development of new methods, and leads to the publication of results that show a new method working well. For example, asymptotic properties of a method are established and the finite-sample case investigated using simulation studies. Recently, it was demonstrated that how “new” methods can easily be proven to be optimal using simulation studies (##UREF##11##Pawel et al., 2022##). A paper introducing a new method and demonstrating its superiority over existing methods with simulation studies should therefore be treated with caution; we must be aware that these simulations may be prone to inventor bias (##REF##23637855##Boulesteix et al., 2013##). Narrow asymptotic results and simulation studies may not create a sufficiently broad base of evidence to ensure the trustworthiness of that method. While new methods are essential to solve existing and new problems, users of methods need to understand which methods work well when. A trustworthy method keeps its essential operational characteristics in a wide variety of settings where it might be applied, or is sufficiently well understood such that a user of the method would know when to use the method and when to avoid it. More and more new methods are proposed without ever being fully investigated and adequately compared in a wider variety of situations. This creates the problem that, even though there is a plethora of methods available to the analyst, many of them are not trustworthy enough to be used in practical analyses. In order to improve this unfortunate situation, we propose a framework to think about how a piece of methodological research contributes to the evidence base for a specific method. A much needed side-effect is that such a concept gives more gravitas to carefully planned method comparison studies and to studies that explore the empirical properties of existing methods in a wider range of problems (##REF##29193206##Boulesteix et al., 2018##). All authors of this paper are members of the international stratos Initiative (STRengthening Analytical Thinking for Observational Studies) and support the initiative’s overarching aim to provide guidance for relevant methodological topics in the design and analysis of observational studies for specialist and non-specialist audiences (##REF##25074480##Sauerbrei et al., 2014##). The proposed framework aims at refining the notion of evidence in methodological research that is central to STRATOS’ efforts.</p>" ]
[ "<title>Introducing A Framework Of Phases Of Methodological Research</title>", "<p id=\"P6\">Methodological phase I may introduce a new idea and try to prove that the proposed method is valid from a theoretical point of view and has the potential to improve on existing methods, or may constitute the first solution to a particular problem. It often includes logical reasoning, proofs and investigation of asymptotic properties such as consistency or normality, etc. This does not mean that further proofs and investigations of asymptotic properties in a wider variety of situations will not be needed in later phases. In practice, phase I studies’ results may often reveal no or only a small benefit of a new method and researchers do not try to disseminate them or, if they do, some journals may be reluctant to publish them. We claim that such “negative” studies, if they can explain why a method does not work in a specific situation, are sometimes needed to increase the community’s understanding, to stimulate further research and to stop other researchers from investing time and resources in the same dead-end idea (##UREF##2##Boulesteix et al., 2015##).</p>", "<p id=\"P7\">Methodological phase II may have the aim to prove that a method can be used with caution in an applied setting which is not completely identical to the developer’s target setting. A phase II study may provide empirical evidence to demonstrate validity in finite samples using simulation studies with a limited set of scenarios, or by illustrative data analyses. When browsing the table-of-contents of typical biostatistical journals, one gets the impression that phase II study reports are abundant in the biostatistical literature (see also below). Often, a given paper includes both phases I and II contributions. By the end of phase II, an openly available software implementation of the method may facilitate the uptake of the method for “early adopters” and ease further investigation in later phases.</p>", "<p id=\"P8\">Methodological phase III may investigate how the method performs in a wide range of settings and, if applicable, for different types of outcomes. This may include empirical comparisons with any existing methods. From such studies, researchers may learn in which situations and under which assumptions the method can be safely used and performs better than or at least as well as alternative methods. This includes understanding which of the method’s assumptions are critical and which are not: for example, in linear regression with large samples, the assumption of normally distributed residuals is not critical in terms of consistency of point and variance estimators. Hence, a phase III study must provide substantial evidence to demonstrate a method’s validity and relative performance. It should be replicable (##UREF##7##Lohmann et al., 2022##) and, if possible, avoid “inventor bias” by making efforts to ensure neutral comparisons (##REF##23637855##Boulesteix et al., 2013##) or at least disclose possible biases. Furthermore, it typically includes “broad” simulations in different, practically relevant settings. Phase III may detect previously unknown implicit assumptions of a method. Methods that were not properly validated may show undesirable or unintended properties when applied in a situation, where such an assumption is not met. Several examples involving real data would have to demonstrate how to properly apply the method in question and how to interpret its results. By the end of phase III, a software implementation of the method should be reasonably fast and user-friendly to be applicable for a wider audience.</p>", "<p id=\"P9\">Methodological phase IV should establish that a method is now suitably well-understood, that is, we know when it is the preferred method and when it is not. A phase IV study is based on extensive experience with the method. For example, a phase IV study may systematically review applications of a method, or may deal with applying the method in new settings that were not considered initially. In this phase, pitfalls of a method may be discovered and highlighted, that is, things that are likely to go wrong if the method is applied carelessly by a user. Likewise, a phase IV study may propose essential, practically useful diagnostics that help a data analyst to assess if any critical assumptions of a method were violated for the data in hand. Using simulations, new mathematical results (##REF##31009064##Wang et al., 2019##) and example analyses of interesting case studies beyond previous applications of the method, it may identify “sweet spots” and “breakdown scenarios” for a method (by analogy to optimal use and long-term or rare adverse effects of a drug). Breakdown scenarios in which the method gives suboptimal results may not have been obvious when the method was introduced and may only be discovered through extensive experience, for example, also by evaluating its behavior when competing methods are known to break. Such breakdown scenarios may give rise to modifications and further developments. A modification may make the method applicable in further settings, for which it may undergo another phase II and possibly phase III. After that phase III evaluation, it may turn out that the modification is suitable only for very special target settings. In some areas, such as machine learning, “adversarial examples,” that is, analysis situations or datasets where a method fails, are frequently published, and often stimulate research toward robustifying existing methods (##UREF##1##Biggio &amp; Roli, 2018##). In biostatistics, this is very rarely the case, or such examples are hidden in phase II studies intended to motivate the need for another method. Nevertheless, empirical studies on the breakdown of a method, particularly if they contain explanations of why a breakdown happens, will increase our understanding, prevent others from wasting their time on it, and are therefore worth publishing. By phase IV, a robust implementation of the method should be easily accessible to practitioners and the understanding of the method so advanced that in principle alternative implementations could be developed in several software packages.</p>", "<p id=\"P10\">The different phases of research may be summarized by different scopes, elements, and outcomes of a study (##TAB##0##Table 1##). For example, studies in phase I will often focus on the introduction of a new method, while only later comparisons become more important and lastly, in phase IV the focus is more on investigating where a method works and where it fails in a broad spectrum of applications.</p>" ]
[]
[]
[ "<title>Conclusion</title>", "<p id=\"P30\">We believe that a framework such as the one outlined in this paper may make method development more trustworthy, provide an efficient tool to communicate about methods’ applicability, and increase visibility of research concerned with making the applications of methods safe and successful. Rather than the vague, cliched “more research is needed,” our framework provides a constructive way of thinking about <italic toggle=\"yes\">what</italic> research would move the development of a method forward.</p>" ]
[ "<p id=\"P1\">Although new biostatistical methods are published at a very high rate, many of these developments are not trustworthy enough to be adopted by the scientific community. We propose a framework to think about how a piece of methodological work contributes to the evidence base for a method. Similar to the well-known phases of clinical research in drug development, we propose to define four phases of methodological research. These four phases cover (I) proposing a new methodological idea while providing, for example, logical reasoning or proofs, (II) providing empirical evidence, first in a narrow target setting, then (III) in an extended range of settings and for various outcomes, accompanied by appropriate application examples, and (IV) investigations that establish a method as sufficiently well-understood to know when it is preferred over others and when it is not; that is, its pitfalls. We suggest basic definitions of the four phases to provoke thought and discussion rather than devising an unambiguous classification of studies into phases. Too many methodological developments finish before phase III/IV, but we give two examples with references. Our concept rebalances the emphasis to studies in phases III and IV, that is, carefully planned method comparison studies and studies that explore the empirical properties of existing methods in a wider range of problems.</p>" ]
[ "<title>Learning From Drug Development</title>", "<p id=\"P3\">In drug development, the concept of <italic toggle=\"yes\">phases of research</italic> was defined decades ago (##REF##24906716##Sedgwick, 2014##). As research progresses from one phase to the next, many candidate treatments are dismissed because of intolerability (phase I), lack of safety or of efficacy (phase II), or ineffectiveness when compared to a placebo or standard of care (phase III), while promising treatments advance to the next phase. After licensing of a drug, a phase IV trial investigates long-term effects and effectiveness in the real world; this may allow identification of, for example, an expanded safety profile, expansion of indication or treatment effect heterogeneity. Previous work has also defined <italic toggle=\"yes\">phases of prognostic factor research</italic> to identify underlying methodological issues and provide guidelines for the conduct of prognostic factor studies (##REF##10066088##Altman &amp; Lyman, 1998##; ##UREF##4##Hayden et al., 2008##; ##REF##23393429##Riley et al., 2013##). Here, also four phases were defined, where in phase I exploratory, hypothesis generating studies would propose a new prognostic marker to have prognostic importance, and in phase II exploratory studies would attempt to use the marker to discriminate between patients at high and low risk of disease progression (prognostic ability) or to identify which patients are likely to benefit from therapy (predictive ability). Phase III would be confirmative studies to proof a priori hypotheses about the prognostic or predictive abilities of the marker. Finally, further studies may combine several prognostic markers into a prognostic or predictive model.</p>", "<p id=\"P4\">We argue that a similar concept of “phases with well-defined aims” helps to build the evidence base for methodological research.</p>", "<p id=\"P5\">The aim of methodological research is to give applied researchers methods to obtain accurate answers to relevant questions (and to identify methods that fail to do this), along with the necessary understanding to use the methods properly. Similarly, the aim of drug development is to precisely estimate a drug’s beneficial and adverse causal effects in various potential application areas. In drug development, regulatory involvement ensures that the development phases achieve these aims, being efficient with early pulling out for unpromising drugs. In methodological research, just as in drug development, new methods can be worse than existing ones or have unexpected properties in some situations. For promising methods, it is not just a matter of introducing the method and getting it used; similarly to a drug, it needs to be carefully evaluated broadly in a way that onlookers can trust. Further, just as in drug development, existing methods may be repurposed. While we do not envision a single governing body that regulates this, methodological research may benefit from considering how evidence is created through the phases in drug development.</p>", "<title>Examples</title>", "<p id=\"P11\">For a given method, it is still unusual to have all four phases of methodological research represented in publications. As positive exceptions, we describe two developments representing some of the authors’ interests.</p>", "<title>Example 1: Firth’s correction</title>", "<p id=\"P12\">Firth’s correction is a bias correction method for maximum likelihood estimators. As a side effect, the correction gives finite estimates of regression coefficients in generalized linear models even with data constellations, where maximum likelihood estimates do not exist.</p>", "<p id=\"P13\">Phase I: In his 1993 paper in Biometrika, David Firth presented the correction for the first time, derived it algebraically, and gave some simple examples (##UREF##3##Firth, 1993##).</p>", "<p id=\"P14\">Phase II: In 2002, Heinze and Schemper took up the idea and provided, for the first time, evidence from a simulation study with logistic regression with binary covariates, demonstrating that the method improved on previously available methods to deal with non-existing maximum likelihood estimates (##REF##12210625##Heinze &amp; Schemper, 2002##).</p>", "<p id=\"P15\">Phase III: A comprehensive simulation study on logistic regression was performed by van Smeden and colleagues in 2016 using 465 scenarios (##REF##27881078##van Smeden et al., 2016##). The study confirmed the earlier results that regression coefficients are less biased and more precise when estimated using Firth’s correction. The study was intended as neutral in that none of the authors had published work on Firth’s correction. Similar results were obtained by Puhr et al., who also suggested two modifications of Firth’s correction, both of which make probabilities predicted from the model more precise compared to using the original correction, while retaining the favorable properties of the estimators of the regression coefficients (##REF##28295456##Puhr et al., 2017##). These modifications were compared to some Bayesian approaches using weakly informative priors which—in the meantime—had been suggested as alternative solutions to solve the problem.</p>", "<p id=\"P16\">Phase IV: In 2018, Mansournia and colleagues summarized the evidence on the topic and explained failure of maximum likelihood estimation and solutions by means of two data examples (##REF##29020135##Mansournia et al., 2018##). They also explained why different methods to deal with separation lead to different results and gave general advice on how to detect and to deal with separation in practice.</p>", "<title>Example 2: Predictive mean matching</title>", "<p id=\"P17\">The literature on multiple imputation is vast and includes several strands that have effectively undergone phases of development. Here, we review predictive mean matching, a type of “hot deck” procedure that multiply imputes each missing value with a “borrowed” observed value.</p>", "<p id=\"P18\">Phase I: The idea of predictive mean matching was introduced by ##UREF##6##Little (1988)## as a way of imputing only observable values by replacing missing values with observed values of “donors,” based on a model. There was no proof of its validity but the idea was linked to multiple imputation and Rubin’s theoretical work on hot deck multiple imputation procedures, published a year earlier (##UREF##12##Rubin, 1987##). A multivariate imputation extension was outlined.</p>", "<p id=\"P19\">Phase II: ##UREF##5##Heitjan and Little (1991)## used predictive mean matching to multiply impute seatbelt use and blood alcohol content in the Fatal Accident Reporting System (<sc>fars</sc>) database. This was followed by a limited simulation study, with data generation involving the fars data, to evaluate the performance of predictive mean matching. The simulation results showed promising, though not ideal, performance.</p>", "<p id=\"P20\">Phase III: ##UREF##13##Schenker and Taylor (1996)## conducted a simulation study which, in particular, compared methods for identifying “donors” when using predictive mean matching. They conducted a reasonably broad simulation study with incomplete continuous outcomes. This explored the performance of three predictive mean matching variants, among other methods, and found that they performed reasonably well across a range of scenarios.</p>", "<p id=\"P21\">Phase IV: ##UREF##8##Morris et al. (2014)## reviewed the existing literature on predictive mean matching and considered how to “tune” its implementation. Some of their simulation studies used scenarios where predictive mean matching might be expected to break: for example, with small-sample size and data strongly missing at random (i.e., with missingness depending strongly on observed variables). This showed when the method performed poorly, and how poorly, and clarified how and when it would be expected to outperform alternative methods.</p>", "<title>Classifying articles into phases: A pilot study</title>", "<p id=\"P22\">In a pilot evaluation of the phases that are published, we analyzed a volume of each of four biostatistical journals. The evaluation revealed that most articles of an issue of <italic toggle=\"yes\">Biometrika</italic> dealt with phase I studies, while phase II dominated in <italic toggle=\"yes\">Biometrical Journal, Statistics in Medicine</italic> and <italic toggle=\"yes\">Statistical Methods in Medical Research</italic>. Overall, only a few papers were found that could be classified as phase IV. The protocol and detailed results of the pilot study can be found in the ##SUPPL##0##Supporting information##. This pilot study had several limitations. First, it was based on the judgment of the main phase contribution and many papers will span more than one phase. Second, papers were assessed by a single evaluator only and the judgment may vary between different evaluators. Third, for simplicity the pilot study was conducted with a one-rater-one-journal design, so a journal’s assessment was probably confounded with personal judgment.</p>", "<title>Further Steps</title>", "<p id=\"P23\">Our proposal aims to provide a framework to communicate about the development stage of a method, that is, to understand the limitations of current research, and what further work would be necessary for wide understanding and application of a method. So far we have sketched how the phases of methodological research could be defined, but one may also think of phases when developing software packages to implement methods. In order to define phases such that they are useful and practical for the scientific community, a larger systematic assessment of methodological papers in different biostatistical journals building on our pilot study, and a Delphi process aiming at reaching an agreement on the definitions, are probably needed. After such work, a tool could be developed that enables a methodologist to assess and specify the phase of their research. The technology readiness level calculator of NASA may be template for such a tool (##UREF##0##Altunok &amp; Cakmak, 2010##). In addition, signaling questions may help to rule in or rule out certain phases. Given a broad consensus and existence of guidance for phase identification, the wide adoption of such a framework may facilitate efficient communication and “peer-regulation” of the current state of knowledge about a method. Thereby, the framework would increase trustworthiness in the methodological development process, which is to the benefit of scientific communication, that is, it helps authors, journal editors, and readers and reviewers of manuscripts and grants. The ultimate goal is to ensure that users of methods are equipped with a solid evidence base that allows them to choose the appropriate method for a given challenge. Transparently labeled methodological studies of whatever phase may also stimulate other biostatistical researchers to get interested in a method or a methodological problem and may encourage them to conduct a study in the next phase. For example, good phase IV studies which show shortcomings of existing methods can help focus thought on solutions, and so may lead to “inventing” better methods.</p>", "<p id=\"P24\">Research in all four phases is important for scientific advancement, but currently there are many obstacles to achieving an appropriate balance. First, many funding agencies are inclined to fund only early phase methods research while unrealistically expecting “phase-IV-like results” within a too short time frame, and many biostatistical journals favor papers on new methods over articles comparing existing methods. Similarly, early career methodologists are often pushed to publish “original” research in order to get tenure. However, the classical definition of <italic toggle=\"yes\">originality</italic> is a very narrow one. If this same standard were applied to funding for randomized trials, we would not have studies like Recovery (##UREF##10##Nuffield Department of Publication Health, 2022##) or Stampede (##UREF##9##MRC Clinical Trials Unit at UCL, 2022##) which re-examine existing treatments in new and highly relevant settings. We claim that phases III and IV methodological studies are undervalued in the statistical community and often downplayed as “yet another” simulation study or application, yet planning and conducting properly designed studies covering a broad variety of clearly defined settings is not trivial. Successful phase III or phase IV research involves careful consideration of the practical impact of the methods: selection and implementation of reasonable simulation setups, identification of relevant example datasets, finding out how the methods might be used in practice and extracting relevant evidence from the numerical results, as well as a good working understanding of the methods themselves. Such studies provide novelty by increasing the scientific evidence base for methods and extending the scope of their safe applicability and hence are as important to scientific advancement as “inventing” a new statistical method. The acceptance of phase III and IV studies could be increased by introducing new ideas related to the design and conduct of simulation and comparison studies toward the same rigor as clinical trials. This includes, for example, clarifying the assumptions and range of problems that the study seeks to address, publishing a protocol before conducting the study (##REF##36191290##Kipruto &amp; Sauerbrei, 2022##), or distributing the roles of data generator, data analyst, and performance evaluator between different parties. Furthermore, inventor bias should be identified and avoided or at least disclosed (##REF##30016950##Couronné et al., 2018##; ##REF##32823283##Herrmann et al., 2021##).</p>", "<p id=\"P25\">Concerning the reporting of such studies, the main task is to transparently clarify the level of trustworthiness of methods, both in absolute and comparative terms. The biostatistical community would benefit from phase IV studies, especially when these studies clarify when a method can—rather than cannot—be recommended for the task at hand. Experienced applied statisticians may to some extent develop a good gut feeling for this difficult task, but phase IV studies would provide the objective evidence for this intuition and aid decision making for less experienced researchers.</p>", "<p id=\"P26\">While some of our ideas may seem ambitious and not easy to reach within short term, we are confident that a discussion on phases of methodological research, inspired by this paper, will reach short-term goals such as: <list list-type=\"simple\" id=\"L1\"><list-item><label>(i)</label><p id=\"P27\">Giving frustrated researchers doing early-phase work a framework to understand why their method has not been universally adopted, and suggestions on how to achieve wider adoption: for example, by conducting broad comparison studies while acknowledging possible biases, by supporting neutral comparisons and by making methods easily accessible to other researchers.</p></list-item><list-item><label>(ii)</label><p id=\"P28\">Giving applied researchers a way to articulate their scepticism about new methods that have not undergone several phases of testing.</p></list-item><list-item><label>(iii)</label><p id=\"P29\">Giving more legitimacy to phase III and IV studies and encouraging researchers to conduct and journal editors to publish them.</p></list-item></list></p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>We are grateful to two anonymous reviewers, the handling guest editor, Willi Sauerbrei, Pamela Shaw and another anonymous reviewer of the STRATOS publication panel for suggestions that helped improving the first version of the manuscript. Members of the STRATOS simulation panel at time of first submission were: Michal Abrahamowicz, Anne-Laure Boulesteix, Harald Binder, Rolf Groenwold, Victor Kipnis, Jessica Myers Franklin, Tim Morris, Willi Sauerbrei, Pamela Shaw, Ewout Steyerberg, Ingeborg Waernbaum, Max Westphal.</p>", "<title>Funding information</title>", "<p>Medical Research Council, Grant/Award Number: MC_UU_00004/07; Deutsche Forschungsgemeinschaft, Grant/Award Number: BO3139/4-3</p>", "<title>Data Availability Statement</title>", "<p id=\"P31\">The full data of the pilot study can be found in the ##SUPPL##0##Supporting information##.</p>" ]
[]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>A brief description of the proposed scheme of phases of methodological research</title></caption><table frame=\"void\" rules=\"none\"><thead><tr style=\"background-color:#cccccc\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Phase</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Scope: A study in that phase will typically aim at…</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Elements: Typically, a study in that phase will consist of…</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Outcome: after that phase, we know…</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">I</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… introducing a new idea, demonstrating its validity by investigation of (asymptotic or finite-sample) properties, showing potential to improve on existing methods or to be the only solution.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… mathematical derivations and proofs, very simple example data analyses.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… whether a method is valid or invalid from a theoretical point of view.</td></tr><tr style=\"background-color:#e5e5e5\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">II</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… demonstrating the use of the method with real data, probably introducing refinements and extensions; it will consider only a limited range of possible applications.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… simulations including limited comparisons with other methods, simple example data analyses.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… whether a method can be used with caution or should not be used in certain applied settings.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">III</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… comparing a relatively new method with competitors and demonstrating its use in practice; it will consider a wide range of applications.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… simulations with wide range of scenarios and different outcome types (ideally set up as neutral comparison studies), realistic comparative example data analyses.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… in which settings (among many) a method can be safely used and in which it outperforms competing methods.</td></tr><tr style=\"background-color:#e5e5e5\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">IV</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… summarizing the evidence about a method, also in comparison with competing methods; uncovering previously unknown behavior of the method in complex data analyses; considering an extended range of possible and actual applications.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… a review of the existing evidence about a method, simulations with extended range of scenarios, complex comparative example data analyses.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">… when a method is and when it is not the preferred method; what diagnostics are available and which pitfalls may occur with its application.</td></tr></tbody></table></table-wrap>" ]
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[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supporting Information</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P32\">\n<bold>Conflict Of Interest Statement</bold>\n</p><p id=\"P33\">The authors declare no conflicts of interest.</p></fn></fn-group>" ]
[]
[ "<media xlink:href=\"EMS177446-supplement-Supporting_Information.docx\" id=\"d64e347\" position=\"anchor\"/>" ]
[{"person-group": ["\n"], "surname": ["Altunok", "Cakmak"], "given-names": ["T", "T"], "article-title": ["A technology readiness levels (TRLs) calculator software for systems engineering and technology management tool"], "source": ["Advances in Engineering Software"], "year": ["2010"], "volume": ["41"], "issue": ["5"], "fpage": ["769"], "lpage": ["778"], "pub-id": ["10.1016/j.advengsoft.2009.12.018"]}, {"person-group": ["\n"], "surname": ["Biggio", "Roli"], "given-names": ["B", "F"], "article-title": ["Wild patterns: Ten years after the rise of adversarial machine learning"], "source": ["Pattern Recognition"], "year": ["2018"], "volume": ["84"], "fpage": ["317"], "lpage": ["331"], "pub-id": ["10.1016/j.patcog.2018.07.023"]}, {"person-group": ["\n"], "surname": ["Boulesteix", "Stierle", "Hapfelmeier"], "given-names": ["AL", "V", "A"], "article-title": ["Publication bias in methodological computational research"], "source": ["Cancer Informatics"], "year": ["2015"], "volume": ["14"], "issue": ["Suppl 5"], "fpage": ["11"], "lpage": ["19"], "pub-id": ["10.4137/CIN.S30747"]}, {"person-group": ["\n"], "surname": ["Firth"], "given-names": ["D"], "article-title": ["Bias reduction of maximum likelihood estimates"], "source": ["Biometrika"], "year": ["1993"], "volume": ["80"], "issue": ["1"], "fpage": ["27"], "lpage": ["38"]}, {"person-group": ["\n"], "surname": ["Hayden", "C\u00f4t\u00e9", "Steenstra", "Bombardier"], "given-names": ["JA", "P", "IA", "C"], "collab": ["QUIPS-LBP Working Group"], "article-title": ["Identifying phases of investigation helps planning, appraising, and applying the results of explanatory prognosis studies"], "source": ["Journal ofClinical Epidemiology"], "year": ["2008"], "volume": ["61"], "issue": ["6"], "fpage": ["552"], "lpage": ["560"], "pub-id": ["10.1016/j.jclinepi.2007.08.005"]}, {"person-group": ["\n"], "surname": ["Heitjan", "Little"], "given-names": ["DF", "RJA"], "article-title": ["Multiple imputation for the fatal accident reporting system"], "source": ["Journal of the Royal Statistical Society, Series C (Applied Statistics)"], "year": ["1991"], "volume": ["40"], "issue": ["1"], "fpage": ["13"], "lpage": ["29"], "pub-id": ["10.2307/2347902"]}, {"person-group": ["\n"], "surname": ["Little"], "given-names": ["RJA"], "article-title": ["Missing-data adjustments in large surveys"], "source": ["Journal of Business Economic Statistics"], "year": ["1988"], "volume": ["6"], "fpage": ["287"], "lpage": ["296"]}, {"person-group": ["\n"], "surname": ["Lohmann", "Astivia", "Morris", "Groenwold"], "given-names": ["A", "OLO", "TP", "RHH"], "article-title": ["It\u2019s time! 10 + 1 reasons we should start replicating simulation studies"], "source": ["Frontiers in Epidemiology"], "year": ["2022"], "volume": ["2"], "elocation-id": ["973470"], "pub-id": ["10.3389/fepid.2022.973470"]}, {"person-group": ["\n"], "surname": ["Morris", "White", "Royston"], "given-names": ["TP", "IR", "P"], "article-title": ["Tuning multiple imputation by predictive mean matching and local residual draws"], "source": ["BMCMedical Research Methodology"], "year": ["2014"], "volume": ["14"], "fpage": ["75"], "pub-id": ["10.1186/1471-2288-14-75"]}, {"collab": ["MRC Clinical Trials Unit at UCL"], "source": ["STAMPEDE\u2014Systemic therapy in advancing or metastatic prostate cancer: Evaluation of drug efficacy"], "year": ["2022"], "date-in-citation": ["Accessed 11 August 2022"], "comment": ["\n"], "ext-link": ["http://www.stampedetrial.org/"]}, {"collab": ["Nuffield Department of Publication Health"], "source": ["RECOVERY\u2014Randomised evaluation of COVID-19 therapy"], "year": ["2022"], "date-in-citation": ["Accessed 11 August 2022"], "comment": ["\n"], "ext-link": ["https://www.recoverytrial.net/"]}, {"person-group": ["\n"], "surname": ["Pawel", "Kook", "Reeve"], "given-names": ["S", "L", "K"], "source": ["Pitfalls and potentials in simulation studies"], "year": ["2022"], "pub-id": ["10.48550/arXiv.2203.13076"]}, {"person-group": ["\n"], "surname": ["Rubin"], "given-names": ["D"], "source": ["Multiple imputation for nonresponse in surveys"], "publisher-name": ["Wiley"], "year": ["1987"]}, {"person-group": ["\n"], "surname": ["Schenker", "Taylor"], "given-names": ["N", "JMG"], "article-title": ["Partially parametric techniques for multiple imputation"], "source": ["Computational Statistics Data Analysis"], "year": ["1996"], "volume": ["22"], "issue": ["4"], "fpage": ["425"], "lpage": ["446"], "pub-id": ["10.1016/0167-9473(95)00057-7"]}]
{ "acronym": [], "definition": [] }
28
CC BY
no
2024-01-13 00:12:45
Biom J. 2023 Feb 3;:e2200222
oa_package/9b/9a/PMC7615508.tar.gz
PMC7615510
38113417
[ "<title>Introduction</title>", "<p id=\"P6\">Studies have shown that hormonal changes along the hypothalamus-pituitary-ovarian axis during the menopause transition (MT) may be associated with adverse changes in cardiometabolic health in midlife women.<sup>##REF##18663170##1##,##REF##20082925##2##</sup> One report from the Study of Women Across the Nation (SWAN), highlighted that despite the levels of total testosterone (T) remaining constant during the MT, the more rapid decline of estradiol (E2) creates a more androgenic sex hormone profile termed the relative androgen excess, which contributes to increased risk of the metabolic syndrome.<sup>##REF##11549553##3##</sup> Reports have also shown that declining E2 and increasing follicle stimulating hormone (FSH) levels during the MT are associated with drastic changes in body fat composition and distribution.<sup>##UREF##0##4##,##REF##23460719##5##</sup> These changes have been associated with central obesity and increased secretion of pro-inflammatory adipokines and free fatty acids which in turn increase the risk of insulin resistance, and hypertension.<sup>##UREF##1##6##,##REF##33414495##7##</sup></p>", "<p id=\"P7\">Studies have shown a higher prevalence of obesity among women compared with men from\nLMICs, and these differences are reported to be more apparent in midlife than in\nchildhood years.<sup>##UREF##2##8##</sup> As a result, an\nin-depth analysis of the contribution of menopause to obesity and associated CMD\nrisk factors in women in LMICs is warranted. Furthermore, a meta-analysis showed\nthat women from LMICs reach menopause at an earlier age than those from high-income\ncountries (HICs).<sup>##REF##24771324##9##</sup> In this\nmeta-analysis involving thirty-six studies across the six continents, the mean (with\n95% CIs) age at menopause was lower in Africa (48.4 (48.1–48.7)), Latin\nAmerica (47.2 (45.9–48.6)), Asia (48.8 (48.1– 49.4)), and the Middle\nEast (47.4 (46.9–47.8)), compared to Australia (51.3 (49.8–52.8)),\nEurope (50.5 (50.0–51.1)) and the United States (49.1\n(48.8–49.4)).<sup>##REF##24771324##9##</sup> Early\nage at menopause has been linked with increased CMD risk factors,<sup>##REF##32252968##10##</sup> therefore suggesting heightened\nrisk in LMICs.</p>", "<p id=\"P8\">At present, there are no data quantifying the differences between the levels of CMD risk factors in pre- and postmenopausal women in studies from LMICs despite an increasing prevalence of obesity and associated CMD in these countries. The objective of this systematic review and meta-analysis was therefore to evaluate evidence from the literature on the links between menopause and CMD risk factors in midlife women living in LMICs.</p>" ]
[ "<title>Methods</title>", "<title>Protocol</title>", "<p id=\"P9\">This systematic review and meta-analysis were performed using the Preferred Reporting Items for Systematic Review (PRISMA) 2020 guidelines and was registered with the International Prospective Register of Systematic Reviews (PROSPERO) with the number CRD42021295401.<sup>##UREF##3##11##</sup></p>", "<title>Search strategy and data sources</title>", "<p id=\"P10\">We searched the databases; PubMed, PubMed Central, Scopus, and ISI Web of Science, for original articles of all study designs from inception until April 24, 2023. The query terms consisted of the key words related to “premenopause”, “postmenopause”, “cardiometabolic disease risk factors” and “LMICs”. The search strategy is fully detailed in the ##SUPPL##0##Supplemental Table 1##.</p>", "<title>Eligibility criteria</title>", "<p id=\"P11\">We only included studies conducted in LMICs as defined by the World Bank list of economies (June 2020).<sup>##UREF##4##12##</sup> These studies assessed differences in CMD risk factors between pre- and postmenopausal women. The inclusion criteria were: 1) studies that enrolled both pre- and postmenopausal women, 2) studies evaluating differences in CMD risk factors according to the menopausal stage, and 3) studies published in English. Articles were excluded if they were reviews, editorials, or preliminary reports.</p>", "<title>Data extraction</title>", "<p id=\"P12\">One researcher (RPC) independently screened all initially identified articles and abstracts using the Rayyan software.<sup>##UREF##5##13##</sup> The number of included and excluded records is mapped in ##FIG##0##Figure 1##. Studies deemed to potentially meet inclusion criteria underwent a full-text assessment by two independent reviewers (RPC and NGM). The consensus between two authors satisfied the inclusion criteria. Disagreements were resolved by a third reviewer, NJC.</p>", "<title>Quality Assessment</title>", "<p id=\"P13\">Two reviewers, RPC and NGM, independently used the modified Newcastle-Ottawa Scale (NOS)<sup>##UREF##6##14##</sup> to assess the methodological quality of selected articles. Two separate NOS tools developed for cross-sectional and longitudinal studies were used in the quality assessment. Based on the total score, the risk of bias was assigned into two categories: low risk (7–9) and high risk (0–6). Only studies with a low risk of bias were included in this study. Any disagreements were referred to a third reviewer, NJC.</p>", "<title>Statistical Analyses</title>", "<p id=\"P14\">To quantitatively assess the association between menopause stage and CMD risk factors i.e. metabolic syndrome (MetS), blood pressure, triglycerides, HDL-C, blood glucose, and carotid intima thickness (cIMT) levels, obesity, waist circumference (WC), waist-to-hip ratio (WHR) and type 2 diabetes mellitus, we calculated the pooled estimates of odds ratios and associated 95% confidence intervals using the inverse variance fixed-effect model. In the analyses, studies were grouped based on the defined outcome of interest (CMD risk factor).</p>", "<p id=\"P15\">Heterogeneity between studies was assessed using Cochran’s Q statistic (p&lt;0.01 indicative of heterogeneity) and the I<sup>2</sup> index (values 25%, 50% and 75% suggestive of low, moderate, and high heterogeneity, respectively). All statistical analyses were performed using Stata 16.1 (StataCorp LLC, College Station, TX).</p>" ]
[ "<title>Results</title>", "<title>Search results</title>", "<p id=\"P16\">##FIG##0##Figure 1## shows the PRISMA flow chart on the screening and selection of the research articles. Briefly, the initial search identified 7,124 abstracts. After removing duplicates, 4,849 titles and abstracts were screened. Of these, 4,767 irrelevant articles were excluded, leaving 82 articles for full-text review. Thirty-eight of the 82 articles were excluded in the quality assessment. As a result, 44 articles constituted the systematic review. Of these 44 articles, 16 were eligible for the quantitative analysis and 28 were excluded due to the following reasons: reporting of a CMD risk factor that was uncommon to other articles (n=3), no combined comparison of pre- and postmenopausal stages on CMD risk factors (n=1), different definition criterion for MetS (n=1), and studies that did not report odds ratios as measures of association (n=23).</p>", "<title>Study characteristics and populations</title>", "<p id=\"P17\">##TAB##0##Tables 1## and ##TAB##1##2## show the characteristics of the 44 studies included in the systematic review. The studies were from the following countries: China<sup>##REF##28980832##15##–##UREF##11##27##</sup> (n=14), Brazil<sup>##REF##20658092##28##–##UREF##14##34##</sup> (n=7), Iran<sup>##REF##30929733##35##–##REF##24575135##43##</sup> (n=9), India<sup>##REF##22923976##44##–##UREF##16##46##</sup> (n=3), Tunisia<sup>##REF##27149156##47##–##REF##22883486##49##</sup> (n=3), Thailand<sup>##UREF##18##50##,##REF##29975281##51##</sup> (n=2), Mexico <sup>##REF##21971210##52##</sup> (n=1), the Democratic Republic of Congo<sup>##REF##24156784##53##</sup> (n=1), Ghana<sup>##REF##32420328##54##</sup> (n=1), South Africa<sup>##REF##26031506##55##</sup> (n=1), Bangladesh<sup>##REF##23281703##56##</sup> (n=1), and Chile<sup>##REF##11449080##57##</sup> (n=1). In total, the studies comprised of 353,589 participants, with sample sizes ranging from 122 to 281,319. Staging of natural menopause in all the studies was performed by asking the study participants about their menstrual history, with slight variations in 3 articles<sup>##UREF##12##31##,##REF##29975281##51##,##REF##21971210##52##</sup> where additional confirmation was done by measuring the levels of the sex hormones oestradiol and follicle stimulating hormone. In 31 articles, the differences between CMD risk factors were compared between two menopausal stages namely, the pre- and postmenopause. In these studies, premenopause was defined as regular menses while postmenopause was amenorrhea for 12 consecutive months. In the remaining 13 articles, a third menopausal group, the perimenopause group was included. Perimenopause was defined as irregular menses within the past 12 months. Women who had a history of surgical menopause were excluded from most of the reviewed articles. Only 4 articles<sup>##REF##28980832##15##,##REF##30130291##16##,##UREF##9##24##,##REF##23281703##56##</sup> in this review included participants with a known history of surgical menopause. Furthermore, the use of hormone therapy (HT) was confirmed in only 4 articles<sup>##REF##16645541##30##,##UREF##12##31##,##REF##23157491##40##,##REF##24575135##43##</sup>. Articles were later grouped according to each CMD risk factor as shown in ##TAB##0##Table 1##.</p>", "<p id=\"P18\">##TAB##1##Table 2## presents the 44 articles included in the systematic review. In the 17 articles describing the MetS, 11 showed higher MetS in post- than premenopause<sup>##REF##28980832##15##,##UREF##8##19##,##REF##33625107##20##,##UREF##11##27##,##UREF##14##34##,##REF##23157491##40##,##REF##17768019##41##,##REF##18776958##45##,##REF##22883486##49##,##REF##23281703##56##</sup>, and 6 showed no differences<sup>##UREF##8##19##,##REF##20658092##28##,##UREF##12##31##,##UREF##13##32##,##UREF##15##39##,##UREF##18##50##</sup>. In the 14 articles focused on obesity, 3 showed higher obesity risk in post- than premenopause,<sup>##REF##22549168##29##,##REF##30929733##35##,##UREF##16##46##</sup> and 11 showed no differences<sup>##UREF##7##17##,##REF##17675039##22##,##UREF##9##24##,##REF##22549168##29##,##REF##16645541##30##,##REF##23895384##37##,##UREF##17##48##,##REF##26031506##55##,##REF##11449080##57##,##UREF##19##58##</sup>. One of these studies<sup>##REF##26031506##55##</sup> also measured total body fat mass which was higher in post- than premenopausal women. In the 11 articles on WC and WHR, 5 showed that postmenopausal women had higher WC<sup>##REF##30130291##16##,##UREF##9##24##,##REF##16645541##30##,##REF##27149156##47##,##UREF##19##58##</sup> and 4 articles showed higher WHR<sup>##REF##22445219##21##,##REF##17675039##22##,##REF##16645541##30##,##UREF##19##58##</sup>, but no menopausal differences were reported on WC in 4 studies<sup>##REF##22549168##29##,##REF##23895384##37##,##REF##23432168##42##,##REF##26031506##55##</sup>. One of these studies showed no difference in abdominal subcutaneous and visceral fat between the menopause groups<sup>##REF##26031506##55##</sup>. In the 12 articles on blood lipids, HDL-C was lower in post- than in premenopause in one study<sup>##REF##26228673##36##</sup>, but no differences were reported in triglycerides in a separate study<sup>##REF##23432168##42##</sup>. Elevated total cholesterol, LDL-C, lipoperoxides, and triglyceride-rich lipoprotein-cholesterol (TLR-C) levels were reported in 9 articles<sup>##REF##30130291##16##,##REF##22445219##21##–##REF##2376439##23##,##REF##31706433##33##,##REF##25267788##38##,##REF##24575135##43##,##REF##22923976##44##,##REF##21971210##52##</sup> in post- compared to premenopausal women. In the 10 articles on blood glucose and insulin levels, 3 showed higher glucose levels in post- than premenopausal women<sup>##REF##22923976##44##,##REF##27149156##47##,##UREF##17##48##</sup> but 5 showed no difference,<sup>##REF##30130291##16##,##REF##22445219##21##,##REF##17675039##22##,##UREF##9##24##,##REF##23895384##37##</sup> 2 showed higher insulin in post- than premenopausal women<sup>##REF##27149156##47##,##UREF##17##48##</sup> and in 2 studies diabetes was more prevalent in postmenopausal women<sup>##REF##22445219##21##,##UREF##10##25##</sup>. In the 9 articles on blood pressure, 6 showed higher blood pressure levels in post- than premenopause<sup>16,##REF##2376439##23##,##UREF##9##24##,##REF##22923976##44##,##UREF##16##46##,##REF##27149156##47##</sup> and 3 showed no differences.<sup>##REF##22445219##21##,##REF##17675039##22##,##REF##23895384##37##</sup> In the 2 articles describing cIMT, postmenopausal women had higher cIMT levels than their premenopausal counterparts<sup>##REF##25627797##18##,##REF##29975281##51##</sup> and in 1 study 10-year risk of cardiovascular disease was higher postmenopausally.<sup>##REF##31601203##26##</sup></p>", "<p id=\"P19\">In the meta-analysis, 16 studies from the following countries, China<sup>##REF##28980832##15##–##REF##25627797##18##,##REF##22445219##21##,##UREF##11##27##</sup> (n=7), Brazil<sup>##REF##20658092##28##,##REF##16645541##30##,##UREF##13##32##</sup> (n=3), Tunisia<sup>##REF##27149156##47##–##REF##22883486##49##</sup> (n=3), Thailand<sup>##UREF##18##50##,##REF##29975281##51##</sup> (n=2), Bangladesh<sup>##REF##23281703##56##</sup> (n=1), and Iran (n=1)<sup>##REF##23895384##37##</sup> constituted a total of 29,361 women. Studies were further categorised according to standard definitions of the CMD risk factors as follows: 1) MetS defined by the National Cholesterol Education Program Expert Panel on the Detection, Evaluation, and Treatment of High Blood Cholesterol in Adult Treatment Panel III (NCEP-ATP III criteria)<sup>##REF##20658092##28##,##UREF##13##32##,##UREF##17##48##–##UREF##18##50##,##REF##23281703##56##</sup> (n=6), 2) elevated serum triglycerides (≥1.69 mmol/L)<sup>##REF##28980832##15##,##REF##30130291##16##,##REF##22883486##49##,##REF##23281703##56##</sup> (n=4), 3) elevated fasting glucose (≥6.1 mmol/L)<sup>##REF##28980832##15##,##REF##22883486##49##,##REF##23281703##56##</sup>, 4) low HDL-C (&lt;1.29 mmol/L)<sup>##REF##28980832##15##,##REF##23281703##56##</sup> (n=2), 5) hypertension (SBP ≥140 mmHg, DBP ≥90 mmHg and use of antihypertensives)<sup>##REF##30130291##16##,##REF##22445219##21##,##UREF##11##27##,##REF##23895384##37##,##REF##27149156##47##</sup> (n=5), 6) hypertension (SBP ≥135 mmHg, DBP ≥85 mmHg and/or use of antihypertensives)<sup>##REF##28980832##15##,##REF##22883486##49##,##REF##23281703##56##</sup> (n=3), 7) high WC (≥80 cm)<sup>##REF##28980832##15##,##REF##30130291##16##</sup> (n=2) and 8) high WC (≥88 cm)<sup>##REF##16645541##30##,##REF##22883486##49##,##REF##23281703##56##</sup> (n=3), 9) high WHR (≥0.86)<sup>##UREF##7##17##,##REF##16645541##30##</sup> (n=2), and 10) obesity (BMI ≥28kg/m<sup>2</sup>)<sup>##REF##30130291##16##,##UREF##7##17##</sup> (n=2).</p>", "<title>Primary outcomes</title>", "<p id=\"P20\">##FIG##1##Figure 2## shows the combined effect size estimates in studies that evaluated differences in CMD risk factors according to menopausal stage. Overall, postmenopausal stage was associated with greater CMD risk as supported by significant odds ratios for MetS, hypertension and high triglyceride, fasting blood glucose, waist circumference, and WHR levels. However, odds ratios were not significant for BMI, HDL-C and cIMT levels in post- relative to premenopausal stage (##FIG##1##Figure 2##). The individual forest plots for each CMD risk factor are shown in the ##SUPPL##0##Supplemental Figures 1–11##.</p>", "<title>Metabolic Syndrome</title>", "<p id=\"P21\">Six studies involving 5,177 women from Brazil<sup>##REF##20658092##28##,##UREF##13##32##</sup> (n=2), Thailand<sup>##UREF##18##50##</sup> (n=1), Tunisia<sup>##UREF##17##48##,##REF##22883486##49##</sup> (n=2), and Bangladesh<sup>##REF##23281703##56##</sup> (n=1) were included in the meta-analysis for MetS. Pooled analysis of these studies showed that the risk of MetS was higher in post- than premenopausal women (OR=1.18; 95% CI, 1.11–1.27, P=0.19, and I<sup>2</sup>=33.4% (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 1##; with a moderate heterogeneity between the studies).</p>", "<title>Blood Pressure</title>", "<p id=\"P22\">Five studies involving 16,602 women from China<sup>##REF##30130291##16##,##REF##22445219##21##,##UREF##11##27##</sup> (n=3), Tunisia<sup>##REF##27149156##47##</sup> (n=1), and Iran<sup>##REF##23895384##37##</sup> (n=1) showed that when using a definition of hypertension of SBP ≥140 mmHg and/or DBP ≥90 mmHg, postmenopausal women had a higher risk of hypertension compared to their premenopausal peers (OR=1.10; 95% CI 1.04–1.16, P=0.22, and I<sup>2</sup> =29.9%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 2##, with a moderate heterogeneity between the studies). A similar trend was shown in 3 studies <sup>##REF##28980832##15##,##REF##22883486##49##,##REF##23281703##56##</sup> that defined hypertension as SBP ≥130 mmHg and DBP ≥85 mmHg (OR=1.32; 95% CI 1.26–1.38, P&lt;0.001, and I<sup>2</sup> =97.9%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 3##), however these studies showed a highly significant level of heterogeneity.</p>", "<title>Triglycerides and HDL-C</title>", "<p id=\"P23\">Four studies involving 13,465 women from China<sup>##REF##28980832##15##,##REF##30130291##16##</sup> (n=2), Bangladesh<sup>##REF##23281703##56##</sup> (n=1), and Tunisia<sup>##REF##22883486##49##</sup> (n=1) showed that the risk of elevated triglyceride levels (≥1.69 mmol/L) was higher in post-than in premenopausal women (OR=1.16; 95% CI 1.11–1.21, P&lt;0.001, and I<sup>2</sup> =87.7%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 4##). When low HDL-C levels (&lt;1.29 mmol/L) were compared in two studies <sup>##REF##28980832##15##,##REF##23281703##56##</sup> with a combined sample size of 4,313 women, no differences were present between pre- and postmenopausal women (OR=0.95; 95% CI 0.89–1.01, P=0.001, and I<sup>2</sup> =91.6%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 5##). All these analyses showed a high level of heterogeneity.</p>", "<title>Glucose</title>", "<p id=\"P24\">Three studies involving 5,274 women from China<sup>##REF##28980832##15##</sup>, Bangladesh<sup>##REF##23281703##56##</sup> and Tunisia<sup>##REF##22883486##49##</sup> showed that the odds ratio of impaired blood glucose levels (≥6.1 mmol/L) was higher in post- than premenopausal women (OR=1.21; 95% CI 1.15–1.28, P=0.001, and I<sup>2</sup>=91.1%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 6##), but with a high level of heterogeneity.</p>", "<title>Obesity</title>", "<p id=\"P25\">Pooled results of two studies from China<sup>##REF##30130291##16##,##UREF##7##17##</sup> involving 13,654 women showed that the risk of obesity (BMI ≥28 kg/m<sup>2</sup>) was similar in pre- and postmenopausal women (OR=1.05; 95% CI 0.96–1.14, P=0.13, and I<sup>2</sup> =56.8%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 7##), with a moderate level of heterogeneity between the studies.</p>", "<title>Waist circumference and waist-to-hip ratio</title>", "<p id=\"P26\">In two studies from China<sup>##REF##28980832##15##,##REF##30130291##16##</sup> involving 10,702 women, postmenopausal women had an increased WC (≥80 cm) than their premenopausal peers (OR=1.16; 95% CI 1.08–1.25, P=0.02,and I<sup>2</sup>=81.9%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 8##). A similar trend was observed when three studies from Brazil<sup>##REF##16645541##30##</sup>, Bangladesh<sup>##REF##23281703##56##</sup>, and Tunisia<sup>##REF##22883486##49##</sup>, that defined high WC as ≥88 cm were meta-analysed (OR=1.09; 95% CI 1.02–1.17, P=0.01, and I<sup>2</sup>=77.3%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 9##).Furthermore, pooled analyses from two studies from China<sup>##UREF##7##17##</sup> and Brazil<sup>##REF##16645541##30##</sup> showed that postmenopausal women had higher WHR (≥0.85) than premenopausal women (OR=1.22; 95% CI 1.12–1.32, P=0.14, and I<sup>2</sup> =54.5%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 10##). The level of heterogeneity between all these studies was moderate to high.</p>", "<title>cIMT</title>", "<p id=\"P27\">In two studies from China<sup>##REF##25627797##18##</sup> and Thailand<sup>##REF##29975281##51##</sup> involving 2,253 women, there were no differences in the risk of high cIMT levels (≥0.70mm) between post- and premenopausal women (OR=1.09; 95% CI 0.87–1.36 <italic toggle=\"yes\">P</italic>=0.09, and I<sup>2</sup>=64.4%) (##FIG##1##Figure 2## and ##SUPPL##0##Supplemental Figure 11##). There was a moderate level of heterogeneity across these studies.</p>" ]
[ "<title>Discussion</title>", "<p id=\"P28\">This systematic review and meta-analysis on midlife women from LMICs show that the postmenopausal stage is associated with higher risk of MetS, elevated triglycerides, elevated blood glucose, high blood pressure, and high waist circumference but no differences when obesity, HDL-C and cIMT levels were compared between the two menopausal groups. These observations highlight a disproportionate burden of CMD risk factors in post- compared to premenopausal women in LMICs.</p>", "<p id=\"P29\">Our study broadens the understanding of the association of menopause with CMD risk factors by combining studies from LMICs into a large sample size (40 517 participants). Our findings are similar to a meta-analysis on MetS which included studies from around the world <sup>##REF##29787477##59##</sup>. In their analysis, postmenopausal women were 3.5 times more likely to develop MetS compared to premenopausal women.<sup>##REF##29787477##59##</sup> Furthermore, the higher prevalence of the individual components of MetS in post- than in premenopausal women observed in that study, corroborate our findings.</p>", "<p id=\"P50\">In longitudinal studies from HICs, menopause has been shown to have differential effects on CMD risk factors. In the SWAN study, MetS, total cholesterol, LDL-C, HDL-C, and apo-B lipoproteins were independently associated with menopause only in the first year after FMP.<sup>##REF##18663170##1##,##REF##20082925##2##</sup> The study also showed no influence of menopause on BMI, blood glucose, insulin, triglyceride, and blood pressure levels.<sup>##REF##20082925##2##</sup> In the Atherosclerosis Risk in Communities (ARIC) study, the progression of MetS was rapid during the MT but it decreased after the FMP, which was more prominent in African Americans than White women.<sup>##REF##18971793##60##</sup> In the Melbourne Women’s Midlife Health Project (MWMHP) study, HDL-C levels increased around the first year before FMP but decreased in the first year postmenopause.<sup>##REF##30097511##61##</sup> Other changes in blood lipids (triglycerides and LDL-C), BMI and diastolic blood pressure were only related to chronological ageing or one of the traditional risk factors.<sup>##REF##30097511##61##</sup> Furthermore, the Radiation Effects Research Foundation (RERF) study showed that total serum cholesterol levels increased from three years before FMP to one-year post-FMP whereas increased BMI and systolic blood pressure were associated with chronological ageing but not menopause.<sup>##REF##8964119##62##</sup> Guthrie <italic toggle=\"yes\">et al</italic>., observed that women gained an average of approximately 2.1 kilograms over five years, but these differences were not menopause related. However, the study showed that waist circumference and waist-hip ratio increased with MT.<sup>##REF##11910598##63##</sup> There are many possible reasons for these different outcomes across studies, as also observed in the current systematic review, including differences in sample size, ethnicity, and time points at which CMD risk factors were measured. However, it is interesting to note that in these studies changes in BMI were not related to the menopause but changes in waist and WHR were, and this was also observed in the current meta-analysis.</p>", "<p id=\"P30\">The differences in CMD risk factor levels between pre- and postmenopausal women may relate to hormonal changes during the MT. In the SWAN study, menopause was associated with increasing bioavailable T, and declining E2 and sex hormone binding globulin (SHBG) levels.<sup>##REF##18663170##1##</sup> The changes in testosterone and SHBG were associated with the MetS and its components. However, neither baseline E2 levels nor its decline during menopausal transition was associated with MetS.<sup>##REF##18663170##1##,##REF##18971793##60##</sup> In the age-adjusted analyses, the T:E2 ratio and free androgen index (FAI) increased by approximately 10% from baseline over the five years of follow-up. Supporting evidence from one meta-analysis study showed that women with type 2 diabetes mellitus had higher T but lower SHBG levels than controls <sup>##REF##16537739##64##</sup>. It is hypothesised that the association between SHBG and MetS is mediated by the inhibitory effect of insulin on the synthesis of SHBG.<sup>##REF##18165203##65##</sup> The association of sex hormone levels with CMD risk factors during menopause indicates that hormone therapy may be a useful intervention strategy for these diseases. However, the feasibility of using hormone therapy is debatable in under-resourced healthcare systems and very few studies have investigated its use in such environments. In a large cross-sectional study across 11 Latin American countries, the Collaborative Group for Research of the Climacteric in Latin America (REDLINC) showed that the current use of menopausal hormonal therapy (MHT) was associated with reduced risk of MetS.<sup>##REF##17453865##66##</sup> Furthermore, a study from Brazil showed that the use of MHT was associated with a lower risk for hypertension.<sup>##REF##35613189##67##</sup> However, these were cross-sectional studies and the use of MHT in these studies was low (12.5%).<sup>##REF##17453865##66##</sup></p>", "<title>Limitations and strengths</title>", "<p id=\"P31\">The present study has some limitations. Firstly, the number of identified articles per CMD risk factor in our analyses were small; thus, we could not investigate sources of heterogeneity further. Secondly, the studies assessed in the meta-analysis were dominated by large studies from China with none available from sub-Saharan Africa. Thirdly, our analyses were based on observational data and were therefore limited by study design as far as potential unmeasured confounders and direction of associations were concerned. Despite this, our study provides a comprehensive review of the current literature on this topic in LMICs and was guided by a registered protocol.</p>", "<title>Conclusions</title>", "<p id=\"P32\">The results of this systematic review and meta-analysis show that menopause is associated with an increased risk for CMD risk factor levels in LMICs. Therefore, it is important to focus on prevention strategies such as lifestyle and behavioural changes to mitigate the development of CMD in midlife women in these countries. However, it must be noted that this analysis included a small number of studies with high levels of heterogeneity. More studies are therefore required in LMICs to investigate the relationship of menopause with CMD risk factors and to develop cost-effective interventions for these diseases.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"P32\">The results of this systematic review and meta-analysis show that menopause is associated with an increased risk for CMD risk factor levels in LMICs. Therefore, it is important to focus on prevention strategies such as lifestyle and behavioural changes to mitigate the development of CMD in midlife women in these countries. However, it must be noted that this analysis included a small number of studies with high levels of heterogeneity. More studies are therefore required in LMICs to investigate the relationship of menopause with CMD risk factors and to develop cost-effective interventions for these diseases.</p>" ]
[ "<title>Importance</title>", "<p id=\"P1\">Menopause is an integral part of women’s health and studies in high income countries have shown an increase in CMD risk factors in post- compared to premenopausal women. However, to date no study has combined and assessed such studies across LMICs. This would better inform early monitoring and intervention strategies for reducing CMD risk factor levels in midlife women in these regions.</p>", "<title>Objective</title>", "<p id=\"P2\">To evaluate evidence from the literature on differences in CMD risk factors between pre- and post menopausal midlife women living in LMICs.</p>", "<title>Evidence Review</title>", "<p id=\"P3\">A systematic review with meta-analysis of original articles of all study designs from the databases PubMed, PubMed Central, Scopus, and ISI Web of Science was conducted from conception until April 24, 2023. Studies that met the inclusion criteria were included in the analysis. Quality assessment of the articles was done using the Newcastle-Ottawa Scale, adapted for each study design. The study protocol was registered with the International Prospective Register of Systematic Reviews and adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis. For the meta-analysis, fixed-effects models were used to pool the odds ratios (ORs), as measures of association.</p>", "<title>Findings</title>", "<p id=\"P4\">Our search identified 4,849 relevant articles; 44 for the systematic review and 16 for the meta-analysis, in accordance with our inclusion criteria. Compared with premenopausal women, the postmenopausal stage was associated with metabolic syndrome (OR=1.18 (95 % CI 1.11–1.27)), high waist-to-hip ratio (OR=1.22 (95% CI 1.12–1.32)), hypertension (OR=1.10 (95% 1.04–1.16)), elevated triglycerides (OR=1.16 (95% CI 1.11–1.21)) and elevated plasma glucose (OR=1.21 (95% CI 1.15–1.28)).</p>", "<title>Conclusions and Relevance</title>", "<p id=\"P5\">This study confirmed that CMD risk factors are present at higher levels in post- than premenopausal women. This demonstrates an urgent need for public health policies that focus on early monitoring and interventions targeted at reducing CMD risk and related adverse outcomes in midlife women in these nations.</p>" ]
[ "<title>Supplementary Material</title>" ]
[ "<title>Sources of funding</title>", "<p>PhD bursary from Shimadzu South Africa (Pty) Ltd.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><title>PRISMA flow chart of literature screening and selection. PRISMA - preferred reporting items for systematic reviews and meta-analysis</title></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><title>Meta-analysis of studies showing differences in cardiometabolic disease risk factors according to menopausal stage</title><p>High BMI (body mass index) (≥28 kg/m<sup>2</sup>), elevated cIMT (carotid intima media thickness) (≥0.70mm), elevated FG (fasting glucose) (≥6.1 mmol/L), low HDL-C (high density lipoprotein cholesterol) (&lt;1.29 mmol/L), Hyp (hypertension) 1 (systolic BP ≥140 mmHg and diastolic BP ≥90 mmHg), Hyp 2 (systolic BP ≥130 mmHg and diastolic BP ≥85 mmHg), MetS (metabolic syndrome) (NCEP ATP III definition), high TG (triglycerides) (≥1.69 mmol/L), high WC (waist circumference) 1 (≥80 cm), high WC2 (≥88 cm), and high WHR (waist-to-hip ratio) (≥0.85). Odds ratios presented as post- vs. premenopausal stage.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>CMD risk factors and corresponding articles examined</title></caption><table frame=\"box\" rules=\"all\"><thead><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">CMD risk factor</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Number of articles examined</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">17</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Obesity</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Blood lipids</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">WC and WHR</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Blood glucose and insulin levels</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">BP</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">cIMT</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Others</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Studies included in the qualitative analyses</title></caption><table frame=\"void\" rules=\"none\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">First Author, Year, Reference</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Country</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Study Type</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Age, y</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Sample Size</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Outcome</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Main Results</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Neto (2010)<sup>##REF##20658092##28##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">323</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influence of age on MetS was prevalent, and attenuated any menopausal differences</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Moreira (2020)<sup>##UREF##13##32##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">45–74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">419</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">No association between menopausal stage and MetS</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Jesmin (2013)<sup>##REF##23281703##56##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Bangladesh</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40.0±14.0</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1802</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS higher in post vs premenopausal women</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Belfki (2012)<sup>##REF##22883486##49##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Tunisia</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50.3±9.6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">961</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal stage was associated with higher risk of MetS</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Jeenduang (2014)<sup>##UREF##18##50##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Thailand</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">48.8±11.0</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">361</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">No association between menopausal stage and MetS</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ali (2014)<sup>##UREF##17##48##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Tunisia</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49.5±9.6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1311</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BP, obesity, glucose, and insulin resistance</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Only hyperglycemia was associated with postmenopausal stage</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ren (2019)<sup>##REF##30130291##16##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">56 (47–65)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8191</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, TGs, glucose, BP, WC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause associated with increased risk of higher BMI, hypertension, TGs, and WC</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">He (2012)<sup>##REF##22445219##21##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50.1±5.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4743</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, WHR, lipids, glucose, BP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Elevated total cholesterol, LDL-C, triglycerides, and waist hip ratio were the only risk factors associated with postmenopausal status.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Zhou (2014)<sup>##UREF##11##27##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">53.4±10.3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6324</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal status was a risk factor for hypertension</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ali (2016)<sup>##REF##27149156##47##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Tunisia</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">56.1±9.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">242</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, WC, BP, glucose, HOMA, lipids</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Waist circumference, HOMA, and apo B levels were associated with hypertension in postmenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Tehrani (2013)<sup>##REF##23895384##37##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longitudinal</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Baseline: 38.6±4.6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">675</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, glucose, lipids, WC, BP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Only LDL-C and total cholesterol were associated with postmenopause</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Zhou (2018)<sup>##REF##28980832##15##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">53.3±10.3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6022</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS was higher in postmenopausal women</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Chen (2020)<sup>##UREF##7##17##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">44.7±12.9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5373</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Obesity</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was a risk factor for central and visceral obesity but not general obesity</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Donato (2006)<sup>##REF##16645541##30##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–55</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">358</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">WC, WHR, BMI</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal women had higher WC &amp; WHR than premenopausal women</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ieamtairat (2019)<sup>##REF##29975281##51##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Thailand</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49.3±2.0</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">cIMT</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with increased cIMT levels</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Zhou (2015)<sup>##REF##25627797##18##</sup><sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-Sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2131</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">cIMT</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal had higher cIMT levels than premenopausal women</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Montazeri (2018)<sup>##REF##30929733##35##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longitudinal</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Baseline: 43±5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">929</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with increasing BMI.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Nazari (2003)<sup>##REF##26228673##36##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longitudinal</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Baseline: 30–74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3778</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HDL-C</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HDL-C associated with coronary heart disease in postmenopause</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Tehrani (2014)<sup>##REF##25267788##38##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longitudinal</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20–50</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">755</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipids</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dyslipidemia associated with lower AMH levels</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Heidari (2010)<sup>##UREF##15##39##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">45–70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1596</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was only associated with elevated triglycerides.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Maharlouei (2014)<sup>##REF##23157491##40##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">52.2±8.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">924</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with higher prevalence of MetS</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ainy (2007)<sup>##REF##17768019##41##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">45–65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2182</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with higher prevalence of MetS</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Sarrafzadega (2013)<sup>##REF##23432168##42##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30–60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4146</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">TGs, WC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was not associated with a high triglyceride/waist circumference phenotype</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yousefzadeh (2013)<sup>##REF##24575135##43##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Iran</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49.3±4.6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1538</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipids</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">LDL-C and total cholesterol levels were higher in post- than in premenopausal women</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Wang (2022)<sup>##UREF##10##25##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longitudinal</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Baseline: 50.9±10.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">281,319</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DM</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal women had higher risk of developing diabetes</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Zhou (2019)<sup>##REF##31601203##26##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49.4±8.1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">569</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">10-year risk of CVD in DM</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with 10-year risk of CVD</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yu (2021)<sup>##REF##33625107##20##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1352</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with higher prevalence of MetS</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Feng (2008)<sup>##REF##17675039##22##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">44.8±7.4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9097</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, WHR, glucose, insulin, lipids, BP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Only WHR, TGs, total cholesterol, HDL-C and LDL-C were higher in post- than in premenopausal women</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Wu (1990)<sup>##REF##2376439##23##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–54</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">598</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BP, TC, TGs, HDL-C</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal women had higher BP and lipid levels</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Li (2019)<sup>##UREF##9##24##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3227</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, WC, BP, glucose, lipids, TP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Waist circumference, systolic and diastolic blood pressure, triglycerides, ALT, TP, and BUN were risk factors for DM in postmenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Strand (2014)<sup>##UREF##8##19##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">China</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">440</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Prevalence of MetS was similar in pre- and postmenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Blümel (2001)<sup>##REF##11449080##57##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Chile</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longitudinal</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Baseline: 40–60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">271</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI independent of menopausal differences.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Theodoro (2012)<sup>##REF##22549168##29##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">617</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">WC, BMI</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopause was associated with increased general obesity but not abdominal obesity when compared to premenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Akl (2017)<sup>##UREF##12##31##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">47.7±5.8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">273</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">No association between menopausal status and metabolic syndrome.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Fonseca (2019)<sup>##REF##31706433##33##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49.6±8.5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1916</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipoprotein subfractions</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with TRL-C levels. Duration since menopause &lt;2 years had the highest association with higher TRL-C and VLDL3-C.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Mendes (2013)<sup>##UREF##14##34##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Brazil</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51.1±6.5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">551</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with high blood pressure and elevated glucose levels.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh (2010)<sup>##UREF##19##58##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">India</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25–65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">245</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, WC, WHR, total fat mass, fat free mass</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Increased total fat mass, free fat mass, WC, and WHR in post- than premenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh (2008)<sup>##REF##18776958##45##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">India</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30–65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS was higher in postmenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dasgupta (2012)<sup>##REF##22923976##44##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">India</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30–75</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">316</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Lipids, glucose, BP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Postmenopausal stage was associated with elevated glucose, total cholesterol, triglycerides, LDL-C, and BP.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dasgupta (2020)<sup>##UREF##16##46##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">India</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–55</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1400</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, BP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with higher BMI and BP.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Sanchez-Rodriguez (2012)<sup>##REF##21971210##52##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Mexico</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">374</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Oxidative stress</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with oxidative stress as measured by the high lipoperoxide biomarkers.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Muchanga (2014)<sup>##REF##24156784##53##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DRC</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BP</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Menopause was associated with prehypertension.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Setroame (2020)<sup>##REF##32420328##54##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghana</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">47.7±16.8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">185</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MetS</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Higher prevalence of metabolic syndrome in post- vs premenopausal women.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Jaff (2015)<sup>##REF##26031506##55##</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">South Africa</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cross-sectional</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40–60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">702</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BMI, WC, visceral fat, subcutaneous fat</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">No differences in BMI, WC, visceral, and subcutaneous fat between pre- and postmenopausal women.</td></tr></tbody></table></table-wrap>" ]
[]
[ "<boxed-text id=\"BX1\" position=\"float\"><caption><title>Key Points</title></caption><sec id=\"S29\"><title>Question/Objective</title><p>What is the association between menopause and cardiometabolic disease (CMD) risk factors in low- and middle-income countries (LMICs)?</p></sec><sec id=\"S30\"><title>Findings</title><p>Forty-four articles were included in the systematic review and sixteen in the meta-analysis. The results showed that compared to premenopausal women, postmenopausal women had higher levels of metabolic syndrome, waist-to-hip ratio, blood pressure, triglycerides, and blood glucose but not general obesity, high-density lipoprotein cholesterol and carotid intima media thickness.</p></sec><sec id=\"S31\"><title>Meaning</title><p>Menopause is associated with poor cardiometabolic health in LMICs; therefore, it is essential to increase public health awareness for monitoring and intervention of CMD risk factors in midlife women in these countries.</p></sec></boxed-text>" ]
[]
[]
[]
[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supplemental Data File (.doc, .tif, pdf, etc.)</label></supplementary-material>", "<supplementary-material id=\"SD2\" position=\"float\" content-type=\"local-data\"><label>Supplemental Video File</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P33\">Financial disclosures/Conflicts of interest:</p><p id=\"P34\">Nicole Jaff receives funding from the International Menopause Society and the South African Menopause Society. Tinashe Chikowore is an international training fellow supported by the Wellcome Trust grant (214205/Z/18/Z). The other authors have nothing to disclose.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><p id=\"P35\">BP-blood pressure, cIMT-carotid intima media thickness, CMD-cardiometabolic disease, DM-diabetes mellitus, MetS-metabolic syndrome, Others-Fat mass, visceral and subcutaneous adipose tissues, WC-waist circumference, and WHR-waist hip ratio.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN2\"><label>a</label><p id=\"P36\">Articles used in the quantitative meta-analysis, Age expressed as mean ± standard deviation or range, AMH-anti-mullerian hormone, BP-blood pressure, BMI-body mass index, cIMT-carotid intima media thickness, CVD-cardiovascular disease, DM-diabetes mellitus, HDL-C-high density lipoprotein-cholesterol, HOMA-homeostatic model assessment for insulin resistance, LDL-C-low density lipoprotein-cholesterol, MetS-metabolic syndrome,,TGs-triglycerides, TP-total protein, TRL-C- triglyceride-rich lipoprotein-cholesterol, VLDL3-C- very-low-density lipoprotein cholesterol subfraction 3, WC-waist circumference, WHR-waist-to-hip ratio.</p></fn></table-wrap-foot>" ]
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[{"label": ["4"], "person-group": ["\n"], "surname": ["Sowers", "Zheng", "Tomey"], "given-names": ["M", "H", "K"], "article-title": ["Changes in Body Composition in Women over Six Years at Midlife : Ovarian and Chronological Aging"], "source": ["J Clin Endocrinol Metab"], "year": ["2015"], "volume": ["92"], "issue": ["May"], "fpage": ["895"], "lpage": ["901"], "pub-id": ["10.1210/jc.2006-1393"]}, {"label": ["6"], "person-group": ["\n"], "surname": ["Tchernof", "Despr\u00e9s"], "given-names": ["A", "J"], "article-title": ["Pathophysiology of human visceral obesity: an update"], "source": ["Physiol Rev"], "fpage": ["359"], "lpage": ["404"], "pub-id": ["10.1152/physrev.00033.2011"], "comment": ["Published online 2013"]}, {"label": ["8"], "person-group": ["\n"], "surname": ["Ford", "Patel", "Narayan"], "given-names": ["ND", "SA", "KMV"], "article-title": ["Obesity in low-and middle-income countries: burden, drivers, and emerging challenges"], "source": ["Annu Rev Public Heal"], "year": ["2017"], "volume": ["38"], "fpage": ["145"], "lpage": ["164"], "pub-id": ["10.1146/annurev-publhealth-031816-044604"]}, {"label": ["11"], "person-group": ["\n"], "surname": ["Page", "Mckenzie", "Bossuyt"], "given-names": ["MJ", "JE", "PM"], "article-title": ["The PRISMA 2020 statement: An updated guideline for reporting systematic reviews"], "source": ["PLoS Med"], "year": ["2021"], "volume": ["18"], "issue": ["3"], "fpage": ["1"], "lpage": ["15"], "pub-id": ["10.1371/journal.pmed.1003583"]}, {"label": ["12"], "collab": ["The World Bank"], "source": ["World Bank list of economies (June 2020), World Bank list of economies (June 2020)"], "date-in-citation": ["Accessed September 7, 2023"], "comment": ["Published 2020"], "ext-link": ["www.hupo.org/resources/Documents/HPP/World%20Bank%20list%20of%20economies%20(June%202020).pdf"]}, {"label": ["13"], "person-group": ["\n"], "surname": ["Ouzzani", "Hammady", "Fedorowicz"], "given-names": ["Mourad", "H", "Z"], "article-title": ["Rayyan \u2014 a web and mobile app for systematic reviews"], "source": ["Syst Rev"], "volume": ["2016"], "fpage": ["1"], "lpage": ["10"], "pub-id": ["10.1186/s13643-016-0384-4"]}, {"label": ["14"], "person-group": ["\n"], "surname": ["Peterson", "Welch", "Losos"], "given-names": ["J", "V", "MTP"], "source": ["The Newcastle-Ottawa Scale (NOS) for Assessing the Quality of Nonrandomised Studies in Meta-Analyses; 2011"], "publisher-loc": ["Ottawa"], "publisher-name": ["Ottawa Hospital Research Institute"], "year": ["2011"], "volume": ["2"], "issue": ["1"], "fpage": ["1"], "lpage": ["2"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Chen", "Xi", "Ji"], "given-names": ["XY", "H", "L"], "article-title": ["Relationships between menstrual status and obesity phenotypes in women: a cross-sectional study in northern China"], "source": ["BMC Endocr Disord"], "year": ["2020"], "volume": ["20"], "issue": ["1"], "pub-id": ["10.1186/s12902-020-00577-6"]}, {"label": ["19"], "person-group": ["\n"], "surname": ["Strand", "Huseth-Zosel", "He", "Perry"], "given-names": ["MA", "A", "MZ", "J"], "article-title": ["Menopause and the risk of metabolic syndrome among middle-aged Chinese women"], "source": ["Fam Med Community Heal"], "year": ["2015"], "volume": ["3"], "issue": ["1"], "fpage": ["15"], "lpage": ["22"], "pub-id": ["10.15212/FMCH.2015.0106"]}, {"label": ["24"], "person-group": ["\n"], "surname": ["Li", "Wang", "Ni"], "given-names": ["Q", "X", "Y"], "article-title": ["Epidemiological characteristics and risk factors of T2DM in Chinese premenopausal and postmenopausal women"], "source": ["Lipids Health Dis"], "year": ["2019"], "volume": ["18"], "issue": ["1"], "pub-id": ["10.1186/s12944-019-1091-7"]}, {"label": ["25"], "person-group": ["\n"], "surname": ["Wang", "Gan", "Kartsonaki"], "given-names": ["M", "W", "C"], "article-title": ["Menopausal status, age at natural menopause and risk of diabetes in China: a 10-year prospective study of 300,000 women"], "source": ["Nutr Metab (Lond)"], "year": ["2022"], "volume": ["19"], "issue": ["1"], "pub-id": ["10.1186/s12986-022-00643-x"]}, {"label": ["27"], "person-group": ["\n"], "surname": ["Zhou", "Zhou", "Guo"], "given-names": ["Y", "X", "X"], "article-title": ["Prevalence and risk factors of hypertension among pre-and post-menopausal women: A cross-sectional study in a rural area of northeast China"], "source": ["Maturitas"], "fpage": ["1"], "lpage": ["6"], "pub-id": ["10.1016/j.maturitas.2014.12.001"], "comment": ["Published online 2014"]}, {"label": ["31"], "person-group": ["\n"], "surname": ["Akl", "Valadares", "Moraes de", "Pinto-Neto", "Lagrutta", "Costa-Paiva"], "given-names": ["LD", "ALR", "MJ", "AM", "B", "L"], "article-title": ["Metabolic syndrome in HIV-infected middle-aged women on antiretroviral therapy: prevalence and associated factors"], "source": ["Brazilian J Infect Dis"], "year": ["2017"], "volume": ["21"], "issue": ["3"], "fpage": ["263"], "lpage": ["269"], "pub-id": ["10.1016/j.bjid.2017.02.003"]}, {"label": ["32"], "person-group": ["\n"], "surname": ["Moreira", "da C\u00e2mara", "Fernandes", "Azevedo", "Cavalcanti Maciel"], "given-names": ["MA", "SMA", "SGG", "IG", "\u00c1C"], "article-title": ["Metabolic syndrome in middle-aged and older women: A cross-sectional study"], "source": ["Women\u2019s Health"], "year": ["2022"], "volume": ["18"], "pub-id": ["10.1177/17455065211070673"]}, {"label": ["34"], "person-group": ["\n"], "surname": ["Mendes", "Theodoro", "Rodrigues", "Busnello", "de Lorenzi"], "given-names": ["KG", "H", "AD", "F", "DROM"], "article-title": ["Menopausal Status and Metabolic Syndrome in Women in Climacteric Period Treated at a Clinic in Southern Brazil"], "source": ["Open J Endocr Metab Dis"], "year": ["2013"], "volume": ["2013"], "issue": ["February"], "fpage": ["31"], "lpage": ["41"]}, {"label": ["39"], "person-group": ["\n"], "surname": ["Heidari", "Sadeghi", "Talaei", "Rabiei", "Mohammadifard", "Sarrafzadegan"], "given-names": ["R", "M", "M", "K", "N", "N"], "article-title": ["Metabolic syndrome in menopausal transition: Isfahan Healthy Heart Program, a population based study"], "source": ["Diabetol Metab Syndr"], "year": ["2010"], "volume": ["2"], "issue": ["1"], "pub-id": ["10.1186/1758-5996-2-59"]}, {"label": ["46"], "person-group": ["\n"], "surname": ["Dasgupta", "Roy"], "given-names": ["D", "S"], "article-title": ["Does menopausal status have an effect on body mass index and blood pressure?"], "source": ["J Women Aging"], "year": ["2020"], "volume": ["00"], "issue": ["00"], "fpage": ["1"], "lpage": ["14"], "pub-id": ["10.1080/08952841.2020.1782700"]}, {"label": ["48"], "person-group": ["\n"], "surname": ["Ben Ali", "Belfki-Benali", "Aounallah-Skhiri"], "given-names": ["S", "H", "H"], "article-title": ["Menopause and metabolic syndrome in tunisian women"], "source": ["Biomed Res Int"], "year": ["2014"], "volume": ["2014"], "pub-id": ["10.1155/2014/457131"]}, {"label": ["50"], "person-group": ["\n"], "surname": ["Jeenduang", "Trongsakul", "Inhongsa", "Chaidach"], "given-names": ["N", "R", "P", "P"], "article-title": ["The prevalence of metabolic syndrome in premenopausal and postmenopausal women in Southern Thailand"], "source": ["Gynecol Endocrinol Off J Int Soc Gynecol Endocrinol"], "year": ["2014"], "volume": ["30"], "issue": ["8"], "fpage": ["573"], "lpage": ["576"], "pub-id": ["10.3109/09513590.2014.907261"]}, {"label": ["58"], "person-group": ["\n"], "surname": ["Ghosh", "Bhagat"], "given-names": ["A", "M"], "article-title": ["Anthropometric and body composition characteristics in pre-and postmenopausal Asian Indian Women: Santiniketan women study"], "source": ["Anthropol Anzeiger"], "year": ["2010"], "volume": ["68"], "issue": ["1"], "fpage": ["1"], "lpage": ["10"], "pub-id": ["10.1127/0003-5548/2010/0005"]}]
{ "acronym": [], "definition": [] }
67
CC BY
no
2024-01-13 00:07:36
Menopause. 2024 Jan 1; 31(1):77-85
oa_package/45/53/PMC7615510.tar.gz
PMC7615511
37408455
[]
[ "<title>Method</title>", "<title>Sample</title>", "<p id=\"P9\">The National Child Development Study (NCDS) is a prospective UK birth cohort study comprising 18 558 children born in England, Wales and Scotland on 3–9 March 1958. A research nurse visited participants aged 44–45 years for a face-to-face biomedical assessment, which included a detailed assessment of their physical health.</p>", "<p id=\"P10\">The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human subjects/patients were approved by South East Medical Research and Ethics Committee (NCDS Biomedical, Age 44, 2002, REC number 01/1/44). A comprehensive description of the NCDS study has been published.<sup>##REF##16155052##9##</sup> Informed parental consent was sought for the childhood measures and explicit written consent was obtained for the biomedical assessment.<sup>##UREF##0##11##</sup></p>", "<title>Measures</title>", "<title>Exposure variable: ADHD</title>", "<p id=\"P11\">When NCDS participants were aged 7, their mothers completed the (modified) Rutter A scale, which is a questionnaire measure of various child mental health problems, including ADHD, developed by Michael Rutter in the 1960s. The ADHD items have been validated against interview-assessed research diagnoses of ADHD.<sup>##UREF##1##12##</sup> The NCDS Rutter Scale includes items on whether the child ‘is squirmy or fidgety’ or ‘has difficulty in settling’. Response categories were: ‘never’, ‘sometimes’ and ‘frequently’.<sup>##UREF##2##13##</sup> Teacher reports on these two ADHD symptoms in the participants (‘squirmy, fidgety’ and ‘hardly ever still’) were also obtained at similar time points and derived from the Bristol Social Adjustment Guides (BSAG),<sup>##UREF##2##13##</sup> with response categories ‘don’t know’, ‘certainly applies’, ‘applies somewhat’ and ‘does not apply’. No measures of ADHD have been included in NCDS after childhood/adolescence.</p>", "<p id=\"P12\">Individuals were classified as having ‘ADHD problems’ if the parent endorsed both ADHD symptoms (regardless of frequency) and the teacher endorsed either of the ADHD symptoms as ‘certainly applies’, as a clinical diagnosis of ADHD requires difficulties across different settings. This approach generated a prevalence rate of 3% for ADHD problems, which is in line with global prevalence rates of DSM-IV/DSM-5 ADHD.<sup>##REF##25649325##1##</sup> Time trends research suggests that prevalence rates for ADHD have not changed over time.<sup>##REF##25496340##14##</sup></p>", "<title>Outcomes: CVD risk factors assessed in ‘mid-life’ (age 44–45)</title>", "<p id=\"P13\">For the purposes of these analyses, all outcomes were defined as continuous variables (apart from smoking status). To aid clinical interpretation, the four categorical outcomes using current UK guidelines for clinical action (e.g. hypertension, hyperlipidaemia) were also generated: <list list-type=\"simple\" id=\"L1\"><list-item><label>(a)</label><p id=\"P14\">Blood pressure (mmHg): three readings of systolic (SBP) and three readings of diastolic (DBP) blood pressure were available. A mean value for each (SBP and DBP) was derived. Given previous evidence,<sup>##REF##31492503##8##,##REF##10955386##15##</sup> systolic blood pressure was considered as the primary outcome.</p></list-item><list-item><label>(b)</label><p id=\"P15\">Lipid measurements (mmol/L): low-density lipoprotein (LDL) and triglyceride levels.</p></list-item><list-item><label>(c)</label><p id=\"P16\">Body mass index (BMI): height and weight were measured by nurses during home visits and BMI derived using the standard formula (weight/height, kg/m<sup>2</sup>). The principal measure of BMI was mid-life BMI, but we also checked whether substituting BMI at age 16 for mid-life BMI influenced the results.<sup>##REF##26315982##16##</sup></p></list-item><list-item><label>(d)</label><p id=\"P17\">Smoking status: this was based on self-report using a derived dichotomised item (‘Current smoker’, yes/no). Smoking is a well-established risk factor for CVD but also known to be strongly associated with ADHD.</p></list-item></list></p>", "<title>Analysis</title>", "<p id=\"P18\">There were three main steps in the analyses.</p>", "<title>Mid-life cardiovascular risk factors as outcome</title>", "<p id=\"P19\">Regression analyses were conducted with childhood ADHD as exposure and cardiovascular risk factors (SBP, DBP, BMI, current smoking status, triglycerides and LDL) as separate outcomes.</p>", "<title>Sensitivity analyses</title>", "<p id=\"P20\">As there is a well-recognised effect of sex (male/female) on systolic blood pressure and as ADHD is more prevalent in males, we added sex and an interaction term between sex and ADHD to the regression analysis of ADHD group on systolic blood pressure. We conducted similar sensitivity checks for birth socioeconomic status (SES) as a covariate and interaction term. SES at birth was classified using the Registrar General’s classification of the father’s social class, i.e. the social class the NCDS participant was ‘born into’. The categories were condensed into a dichotomised variable ‘higher SES’ (classes I, II and III non-manual/manual) and ‘lower SES’ (classes IV, V and unclassified).</p>", "<title>Missing data analysis: multiple imputation</title>", "<p id=\"P21\">Full descriptions of the process and results are provided in the ##SUPPL##0##Supplementary materials##, available at <ext-link xlink:href=\"https://dx.doi.org/10.1192/bjp.2023.90\" ext-link-type=\"uri\">https://dx.doi.org/10.1192/bjp.2023.90</ext-link>. We scored missing outcome measures (adult BMI, SBP, DBP, LDL cholesterol, current smoking status and triglycerides) for our analysis sample. All baseline data and outcomes reported in the main paper are unimputed. We performed multiple imputation using a multivariate normality assumption in STATA version 16.1 for Windows. We used: (a) variables in line with published recommendations for imputing missing data in the biomedical survey in the NCDS<sup>##UREF##3##17##</sup> (if they predicted missingness for all/most key mid-life outcomes); (b) earlier values of primary outcomes; (c) other key mid-life variables. A full list of variables used in the imputation is given in the ##SUPPL##0##Supplementary materials##.</p>" ]
[ "<title>Results</title>", "<p id=\"P22\">##FIG##0##Figure 1## is a flowchart of how the sample of participants included in our analysis was derived.</p>", "<title>Associations between childhood ADHD and mid-life cardiovascular risk factors</title>", "<p id=\"P23\">The values of mid-life cardiovascular risk factors for individuals with and without childhood ADHD problems and associations between childhood ADHD problems and mid-life cardiovascular risk factors are detailed in ##TAB##0##Table 1##.</p>", "<p id=\"P24\">Childhood ADHD problems were associated with BMI (<italic toggle=\"yes\">B</italic> = 0.92 kg/m<sup>2</sup>, s.d. = 0.27–1.56), systolic (3.5 mmHg, s.d. = 1.4–5.6) and diastolic (2.2 mmHg, s.d. = 0.8–3.6) blood pressure, triglyceride levels (0.24 mmol/l, s.d. = 0.02–0.46) and being a current smoker (odds ratio OR = 1.6, s.d. = 1.2–2.1) but not with LDL cholesterol levels.</p>", "<p id=\"P25\">In separate analyses childhood ADHD was not associated with BMI at age 16 (ADHD group: mean 20.6 kg/m<sup>2</sup>; non-ADHD group: mean also 20.6 kg/m<sup>2</sup>) nor with BMI at age 11 (ADHD group: mean 17.3 kg/m<sup>2</sup>; non-ADHD group: mean 17.5 kg/m<sup>2</sup>).</p>", "<p id=\"P26\">Using current UK cut-offs for clinical action for mid-life hypertension, overweight, obesity and high LDL and high triglycerides (these vary across countries), odds ratios comparing the ADHD with the non-ADHD group were calculated (##TAB##1##Table 2##). Rates of those who met these cut-offs were higher in the ADHD group (OR &gt; 1) but differences were robust only for obesity.</p>", "<title>Sensitivity analyses</title>", "<p id=\"P27\">As expected, sex was associated both with ADHD and systolic blood pressure in this sample (##SUPPL##0##Supplementary eTable 1##). Including sex as a covariate in the regression analysis of ADHD exposure and systolic blood pressure outcome, the association between ADHD and systolic blood pressure was attenuated. The interaction term between sex and ADHD did not significantly predict systolic blood pressure. Similarly, SES at birth attenuated the association between ADHD and systolic blood pressure (##SUPPL##0##Supplementary eTable 2##) and the interaction term did not achieve conventional levels of statistical significance.</p>", "<title>Multiple imputation</title>", "<p id=\"P28\">We imputed missing data for the 8016 individuals in our core group (##FIG##0##Fig. 1##). The patterns of missing data are shown in ##SUPPL##0##Supplementary eFigs 1 and 2##; bivariate analyses with childhood ADHD as exposure and individual cardiovascular risk factors as outcomes using original and imputed data are shown in ##SUPPL##0##Supplementary eTable 4##. Results for imputed data were similar to those found using the original data-set.</p>" ]
[ "<title>Discussion</title>", "<p id=\"P29\">We found that individuals with childhood ADHD problems (‘squirmy, fidgety’ and ‘hardly ever still’) showed higher levels of multiple cardiovascular risk factors by age 44. These included higher blood pressure (both systolic and diastolic), triglyceride levels and BMI; they were also more likely to be current cigarette smokers. Similar results were obtained using the imputed data-set. When outcomes were examined categorically, to help inform clinicians as to how worthwhile cardiovascular risk profiling in psychiatry might be, out of 100 people with broadly defined ADHD aged 44–45, 14 in our sample met the UK threshold for treating hypertension and 33 for elevated triglycerides.</p>", "<p id=\"P30\">When we defined cardiovascular risk factors categorically, using current UK cut-offs for clinical intervention, we observed a higher odds ratio for each risk factor in those with childhood ADHD compared with those without. However, apart from obesity, we could not reject the null hypothesis; that is, the possibility that there is no difference in cardiovascular risk categories. The threshold for treating different cardiovascular risk factors (e.g. with antihypertensive medication, statins) varies across countries and is, for example, lower in the USA.</p>", "<p id=\"P31\">Given that adult psychiatrists are likely to be following up those with persistent ADHD rather than broadly defined childhood ADHD, these observations are probably an underestimate and now need to be examined in clinical adult psychiatry settings.</p>", "<title>Associations with BMI and cigarette smoking</title>", "<p id=\"P32\">There is a well-established association between ADHD and obesity (obesity defined as BMI ≥30 kg/m<sup>2</sup>).<sup>##REF##26315982##16##</sup> Genetic overlaps between ADHD and BMI have also been well documented<sup>##REF##32373164##18##</sup> and a recent study using two-sample Mendelian randomisation suggests, using genetic variants, a bidirectional causal relationship between ADHD and childhood obesity.<sup>##REF##33324987##7##</sup> We found a robust association between childhood ADHD problems and obesity but not between ADHD problems and overweight status. Interestingly, we only found elevated BMI in mid-life, not in childhood and adolescence. This suggests that adult psychiatrists need to monitor obesity status in particular.</p>", "<p id=\"P33\">Childhood ADHD was also associated with cigarette smoking in mid-life. Again, this link between smoking and ADHD is well established, with evidence suggesting that associations over the longer term may arise because of nicotine dependence linked to self-medication<sup>##REF##29039714##19##</sup> and increased likelihood of experiencing nicotine withdrawal effects such as irritability and difficulty concentrating on stopping.<sup>##REF##12834657##20##</sup> Genetic studies also have found evidence for genetic overlap between substance use disorder (including cigarette smoking) and ADHD.<sup>##UREF##4##21##</sup> This is the first study to report mid-life smoking outcomes for individuals with ADHD symptoms assessed in childhood.</p>", "<title>Associations with blood pressure</title>", "<p id=\"P34\">Hypertension is the most important risk factor for coronary artery disease<sup>##REF##31492503##8##</sup> and the risk seems most pronounced for elevated systolic blood pressure.<sup>##REF##10955386##15##</sup> To our knowledge, no prospective studies have followed up children with ADHD or ADHD symptoms to examine blood pressure in mid-life. As medication used to treat ADHD has the propensity to cause elevated blood pressure, several studies have followed up children with ADHD on medication into early adulthood to check whether blood pressure becomes elevated; their findings are mixed, but a recent review has concluded that there is evidence for a slight elevation (3–8 mmHg) in systolic blood pressure.<sup>##REF##30531411##22##</sup> The individuals in our study were born in 1958. Medication for childhood ADHD was first approved in the 1960s but UK prescribing was relatively low until around 2000.<sup>##REF##27145886##23##</sup> Most individuals in our study would therefore not have been prescribed any medication for ADHD during childhood and adolescence. It is intriguing that, given our study findings, probably untreated childhood ADHD is also associated with a modest elevation of blood pressure in mid-life (age 44). Our study further elaborates recent studies on younger populations which found that untreated ADHD is associated with low blood pressure in late adolescence<sup>##UREF##5##24##</sup> but that this finding does not persist into young adulthood.<sup>##REF##33672943##25##</sup> This would suggest the need to examine mechanisms that lead to the increase in blood pressure through adulthood for individuals with ADHD and the need for caution in interpreting any findings from more recent cohorts that focus on examining the long-term effects of medication for ADHD on blood pressure.</p>", "<title>Associations with cardiovascular risk and disease</title>", "<p id=\"P35\">Although there is growing recognition of the importance of cardiovascular disease and CVD risk factors in those with psychiatric disorders, especially depression, neurodevelopmental disorders such as ADHD that typically first manifest in childhood have often been ignored. Recent large-scale patient registry and clinical studies have suggested that clinically diagnosed ADHD is associated with cardiovascular diseases, but we do not know whether this is because of obesity and cigarette smoking alone. Our prospective study suggests that childhood ADHD problems in the general population are associated with multiple cardiovascular risk factors in mid-life. The findings from this study when taken together with clinical and registry-based studies highlight the importance of monitoring cardiovascular risk profiles in those with ADHD to prevent later CVD. Few psychiatrists will be trained in using CVD risk calculators and primary care physicians may not recognise people with ADHD as benefitting from repeated physical health checks.</p>", "<title>Systolic blood pressure, sex and SES</title>", "<p id=\"P36\">ADHD and systolic blood pressure are known to be associated with both sex and socioeconomic status. Sensitivity checks were conducted to examine males and females separately and higher and lower SES at birth. These suggested that the relationship between ADHD and systolic blood pressure is attenuated by sex; we observed the same attenuation for SES at birth. We did not detect robust evidence that the association between ADHD and systolic blood varied by sex or SES at birth. However, given the small sample size, there is a need for caution in interpreting these results, especially given robust evidence of elevated cardiovascular disease rates in those with ADHD in larger patient registry data, regardless of sex and SES.<sup>##UREF##6##26##</sup></p>", "<title>Strengths and limitations</title>", "<p id=\"P37\">Although this study benefits from using a population-based birth cohort followed up until mid-life with repeated direct assessments there are some limitations. In common with most population cohorts, there is some loss to follow-up, which leads to some missing data. However, results using multiple imputation were very similar to those for the primary analysis, although data on which to base imputations were limited, as were detailed data on medications and healthcare use. Another limitation was that ADHD assessment during childhood in the 1960s was limited to the Rutter Scale. The NCDS includes only two questions, with a focus more on the hyperactive rather than the inattentive domain – a decision that might have seemed appropriate at that time. However, both parent and teacher ratings were available and used for the categorisation, the questionnaires have been validated against diagnoses and our prevalence rate of broadly defined ADHD (3%) is not dissimilar to rates observed using diagnostic interviews. It is now known that childhood ADHD symptoms and impairment persist in most individuals into adult life and that adult ADHD requires clinical attention.<sup>##REF##27783340##27##</sup> However, the NCDS like many other population cohorts and volunteer cohorts, such as UK Biobank, has never included any measures of adult ADHD. Other limitations are that until information from this cohort at an older age is available, it is not possible to replicate recent health registry findings on ADHD diagnosis and CVD associations. However, the registry studies are unable to directly examine CVD risk factors, which we were able to do. Moreover, triangulation of findings on CVD and CVD risk from patient-based registry studies, genetic studies<sup>##UREF##7##28##</sup> and now a population cohort study provides greater confidence in conclusions across these different designs.</p>", "<title>Implications</title>", "<p id=\"P38\">As noted, childhood ADHD problems are associated with higher levels of multiple risk factors in mid-life that are particularly associated with the development of CVD.<sup>##UREF##7##28##</sup> However, individuals with ADHD may be less likely to consult a primary care physician about their physical health and may therefore be less likely to have the opportunity to discuss the benefits of preventive strategies, including lifestyle measures and interventions such as treating hypertension and hyperlipidaemia. Also, monitoring physical health may not be a priority for adult mental health services. For example, guidance on managing ADHD from the UK’s National Institute for Health and Care Excellence does not mention the need for physical health screening in adulthood unless related to medication.<sup>##UREF##8##29##</sup> This study adds to the literature in highlighting the importance of considering physical and mental health together with neurodevelopmental disorders. Taking all the findings across different studies together, CVD risk monitoring and early prevention of CVD should be part of follow-up for individuals with a history of ADHD, given that CVD risk factors are amenable to intervention.</p>" ]
[]
[ "<title>Background</title>", "<p id=\"P1\">It is well-known that childhood attention-deficit hyperactivity disorder (ADHD) is associated with later adverse mental health and social outcomes. Patient-based studies suggest that ADHD may be associated with later cardiovascular disease (CVD) but the focus of preventive interventions is unclear. It is unknown whether ADHD leads to established cardiovascular risk factors because so few cohort studies measure ADHD and also follow up to an age where CVD risk is evident.</p>", "<title>Aims</title>", "<p id=\"P2\">To examine associations between childhood ADHD problems and directly measured CVD risk factors at ages 44/45 years in a UK population-based cohort study (National Child Development Study) of individuals born in 1958.</p>", "<title>Method</title>", "<p id=\"P3\">Childhood ADHD problems were defined by elevated ratings on both the parent Rutter A scale and a teacher-rated questionnaire at age 7 years. Outcomes were known cardiovascular risk factors (blood pressure, lipid measurements, body mass index and smoking) at the age 44/45 biomedical assessment.</p>", "<title>Results</title>", "<p id=\"P4\">Of the 8016 individuals assessed both during childhood and at the biomedical assessment 3.0% were categorised as having childhood ADHD problems. ADHD problems were associated with higher body mass index (<italic toggle=\"yes\">B</italic> = 0.92 kg/m<sup>2</sup>, s.d. = 0.27–1.56), systolic (3.5 mmHg, s.d. = 1.4–5.6) and diastolic (2.2 mmHg, s.d. = 0.8–3.6) blood pressure, triglyceride levels (0.24 mol/l, s.d. = 0.02–0.46) and being a current smoker (odds ratio OR = 1.6, s.d. = 1.2–2.1) but not with LDL cholesterol.</p>", "<title>Conclusions</title>", "<p id=\"P5\">Childhood ADHD problems predicted multiple cardiovascular risk factors by mid-life. These findings, when taken together with previously observed associations with cardiovascular disease in registries, suggest that individuals with ADHD could benefit from cardiovascular risk monitoring, given these risk factors are modifiable with timely intervention.</p>" ]
[ "<p id=\"P6\">Attention-deficit hyperactivity disorder (ADHD) is a common neurodevelopmental condition with an estimated prevalence in children and adolescents of 3–5%.<sup>##REF##25649325##1##</sup> It typically first manifests during childhood, although most individuals continue to have symptoms and impairment in adolescence and adult life.<sup>##REF##27866355##2##</sup> As there are multiple barriers to accessing specialist healthcare, even in high-income countries, many people with ADHD fail to receive a diagnosis and appropriate treatment. This is despite evidence that ADHD is associated with multiple adverse mental health, educational and social outcomes.<sup>##REF##25726514##3##–##REF##35157020##5##</sup> More recently there is growing evidence that ADHD may be a risk factor for physical ill health and premature mortality.<sup>##REF##36073682##6##</sup> Recent studies of patient populations including large Scandinavian registry-based studies have confirmed risks between clinically recognised ADHD and a diagnosis of cardiovascular disease (CVD).<sup>##REF##36073682##6##</sup> A two-sample Mendelian randomisation approach also found that genetic liability for ADHD may be causal for coronary artery disease.<sup>##REF##33324987##7##</sup> As ADHD is known to be associated with higher rates of cigarette smoking and elevated body mass index (BMI), these risk exposures could explain elevated rates of CVD in people with ADHD. However, observed associations could also be explained by additional CVD risk factors.</p>", "<title>Cardiovascular risk factors</title>", "<p id=\"P7\">Major CVD risk factors include elevated blood lipids (particularly low-density lipoprotein (LDL) cholesterol and triglycerides), elevated blood pressure, cigarette smoking and obesity/elevated body mass index (BMI), as well as genetic liability.<sup>##REF##31492503##8##</sup> CVD can be prevented by targeting many of these risk factors (e.g. hypertension, high lipids) for intervention and CVD risk prediction tools have therefore been adopted in primary care settings in many countries but not in psychiatry clinics.</p>", "<title>The need for population-based studies</title>", "<p id=\"P8\">Although clinical studies and registry data are invaluable, detailed data on lifestyle and cardiovascular risks are not routinely available. Also, given known referral biases, all clinical studies are subject to observed associations arising from ascertainment and collider bias, especially as many people with ADHD are never clinically recognised. Another challenge is that findings, particularly for systolic blood pressure, may be confounded owing to widespread use, in recent clinical cohorts, of ADHD medications known to have cardiovascular side-effects.<sup>##REF##33324987##7##</sup> Prospective population-based studies have different strengths compared with clinical studies. However, studies to date that have examined links between ADHD and physical health have been cross-sectional or only examined outcomes into early adult life. To the best of our knowledge, there are no prospective population-based studies that span birth to mid-life and that have allowed examination of links between childhood ADHD problems and CVD risk factors in mid-life. We used prospective data from a UK birth cohort, the National Child Development Study, in which participants were recruited at birth in 1958.<sup>##REF##16155052##9##</sup> This study collected information on childhood ADHD symptoms using the most widely validated measure at that time and followed up individuals into mid-life (aged 44–45 years), when biomedical data were collected. As ADHD behaves as a risk dimension, broader definitions of ADHD show associations with the same risk factors, correlates and outcomes as an interview-derived DSM-5/DSM-IV or ICD-10 diagnosis.<sup>##REF##26386541##10##</sup> Our aim was to examine whether ADHD problems in childhood were associated with specified CVD risk factors at age 44.</p>", "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgement</title>", "<p>We are extremely grateful to all the families who took part in the NCDS study.</p>", "<title>Funding</title>", "<p>A.K.T., S.C., L.R. and A.T. work in centres funded by the Wolfson Foundation and UK Medical Research Council. R.B., E.S., G.D.S. and K.T. work in a unit that receives funding from the University of Bristol and the UK Medical Research Council (MC_UU_00011/1 and MC_UU_00011/3). This research was conducted while funded by the Wellcome Trust (204895/Z/16/Z).</p>", "<title>Data availability</title>", "<p id=\"P39\">The data that supports the findings of this study are available from the UK Data Service-National Child Development Study (<ext-link xlink:href=\"https://ukdataservice.ac.uk/help/datatypes/longitudinal-data-studies/\" ext-link-type=\"uri\">https://ukdataservice.ac.uk/help/datatypes/longitudinal-data-studies/</ext-link>). Restrictions apply to the availability of these data, which were used under licence for this study.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Fig. 1</label><caption><title>Flowchart of participant numbers.</title><p>NCDS, National Child Development Study; ADHD, attention-deficit hyperactivity disorder. ‘ADHD’ indicates the presence of childhood ADHD problems (defined using two symptoms: ‘squirmy, fidgety’ and ‘hardly ever still’), rather than clinical diagnosis of ADHD (the reference to all four symptoms is for both the parent and the teacher reports).</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Childhood attention-deficit hyperactivity disorder (ADHD) and individual mid-life cardiovascular risk factors: descriptive details and comparisons with non-ADHD group</title></caption><table frame=\"box\" rules=\"groups\"><thead><tr style=\"background-color:#9CB6D6\"><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" colspan=\"2\" rowspan=\"1\">Mean 95% CI</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Outcome</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Exposure</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean (s.d., n)</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">F</italic> (degrees of freedom)</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Upper</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Lower</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">P</italic>\n</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Effect size, <italic toggle=\"yes\">d</italic></th></tr></thead><tbody><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mid-life BMI, kg/m<sup>2</sup></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">28.3 (5.7), 234</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.8 (1,7871)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">29.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.005</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.20</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27.3 (5.0), 7639</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27.2</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SBP, mmHg</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">129.9 (17.4), 233</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10.3 (1,7892)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">127.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">132.2</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.001</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.21</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">126.4 (16.3), 7661</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">126.1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">126.8</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DBP, mmHg</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">80.9 (10.7), 233</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9.7 (1,7892)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">79.5</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">82.3</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.002</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.21</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78.7 (10.7), 7661</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78.9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">LDL, mmol/L</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.5 (0.97), 184</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.45 (1,6329)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.33</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.61</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.504</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.11</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.4 (0.91), 6147</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.40</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.45</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Triglycerides, mmol/L</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.27 (1.89), 202</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.7 (1,6675)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.54</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.029</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.16</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.03 (1.53), 6475</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.00</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.07</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Outcome</bold>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold><italic toggle=\"yes\">n</italic> (%) yes</bold>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">χ<sup>2</sup> (d.f.)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>OR (95% CI)</bold>\n</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Current smoker</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">232 (38.4%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.0 (1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.001</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.60 (1.22–2.1)</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7519 (28%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Comparison of individuals with and without childhood attention-deficit hyperactivity disorder (ADHD) problems using UK-recommended cardiovascular risk factors thresholds for intervention</title></caption><table frame=\"box\" rules=\"groups\"><thead><tr style=\"background-color:#9CB6D6\"><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" colspan=\"2\" rowspan=\"1\">95% CI for OR</th></tr><tr><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Outcome (cut-off), <italic toggle=\"yes\">n</italic></th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADHD group, % (<italic toggle=\"yes\">n</italic>)</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-ADHD group, % (<italic toggle=\"yes\">n</italic>)</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Odds ratio (OR)</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Upper</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Lower</th></tr></thead><tbody><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Hypertension (blood pressure ≥140/90 mmHg), 7894</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13.7 (233)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11.3 (7661)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.25</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.86</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.83</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Overweight (BMI ≥25 kg/m<sup>2</sup>), 7873</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">65.4 (234)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">65.4 (7639)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.77</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.31</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Obese (BMI ≥30 kg/m<sup>2</sup>), 7873</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">30.3 (234)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24.2 (7639)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.36</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.03</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.81</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated triglycerides (≥1.7 mmol/L<sup><xref rid=\"TFN2\" ref-type=\"table-fn\">a</xref></sup>), 6677</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">53.0 (202)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">49.5 (6475)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.15</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.87</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.52</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Elevated triglycerides (≥2.3 mmol/L<sup><xref rid=\"TFN3\" ref-type=\"table-fn\">b</xref></sup>), 6677</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">35.1 (202)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">30.9 (6475)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.21</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.62</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supplementary file</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"con\"><p id=\"P40\">\n<bold>Author contributions</bold>\n</p><p id=\"P41\">A.K.T. and A.T. formulated the research questions. A.K.T. and A.T. designed the study. A.K.T., K.T., E.S., S.C. and L.R. analysed the data/advised on data analysis. All authors contributed to writing the article.</p></fn><fn id=\"FN2\" fn-type=\"COI-statement\"><p id=\"P42\">\n<bold>Declaration of interest</bold>\n</p><p id=\"P43\">None.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><p id=\"P44\">BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL, low-density lipoprotein.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN2\"><label>a</label><p id=\"P45\">Recommended fasting level (National Institute for Health and Care Excellence).</p></fn><fn id=\"TFN3\"><label>b</label><p id=\"P46\">Recommended non-fasting level (HEART UK).</p></fn></table-wrap-foot>" ]
[ "<graphic xlink:href=\"EMS192899-f001\" position=\"float\"/>" ]
[ "<media xlink:href=\"EMS192899-supplement-Supplementary_file.docx\" id=\"d64e494\" position=\"anchor\"/>" ]
[{"label": ["11"], "collab": ["National Child Development Study"], "source": ["Ethical Review and Consent"], "publisher-name": ["Centre for Longitudinal Studies, Institute of Education, University of London"], "year": ["2014"], "date-in-citation": ["accessed 23 Jun 2023"], "comment": ["\n"], "ext-link": ["https://cls.ucl.ac.uk/wp-content/uploads/2017/07/NCDS-Ethical-review-and-Consent-2014.pdf"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Rutter", "Tizard", "Whitmore"], "given-names": ["M", "J", "K"], "source": ["Education, Health and Behavior"], "publisher-name": ["Longman Group"], "year": ["1970"]}, {"label": ["13"], "person-group": ["\n"], "surname": ["Stott", "Sykes"], "given-names": ["DH", "EG"], "article-title": ["The social adjustment of children: the Bristol Social Adjustment Guides"], "source": ["AMA Arch Gen Psychiatry"], "year": ["1959"], "volume": ["1"], "issue": ["5"], "fpage": ["556"]}, {"label": ["17"], "person-group": ["\n"], "surname": ["Silverwood", "Narayanan", "Dodgeon", "Ploubidis"], "given-names": ["R", "M", "B", "G"], "source": ["Handling Missing Data in the National Child Development Study: User Guide (Version 2)"], "publisher-name": ["UCL Centre for Longitudinal Studies"], "year": ["2021"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Vilar-Rib\u00f3", "S\u00e1nchez-Mora", "Rovira", "Richarte", "Corrales", "Fadeuilhe"], "given-names": ["L", "C", "P", "V", "M", "C"], "article-title": ["Genetic overlap and causality between substance use disorder and attention-deficit and hyperactivity disorder"], "source": ["Am J Med Gen Part B"], "year": ["2021"], "volume": ["186"], "fpage": ["140"], "lpage": ["50"]}, {"label": ["24"], "person-group": ["\n"], "surname": ["Garcia-Argibay", "du Rietz", "Hartman", "Lichtenstein", "Chang", "Larsson"], "given-names": ["M", "E", "CA", "P", "Z", "H"], "article-title": ["Cardiovascular risk factors in attention-deficit/hyperactivity disorder: a family design study of Swedish conscripts"], "year": ["2022"], "volume": ["31"], "issue": ["4"], "elocation-id": ["e1930"]}, {"label": ["26"], "person-group": ["\n"], "surname": ["Bowerman", "O\u2019Connell"], "given-names": ["BL", "RT"], "source": ["Linear Statistical Models: An Integrated Approach"], "publisher-name": ["Duxbury Press"], "year": ["1990"]}, {"label": ["28"], "person-group": ["\n"], "surname": ["Wilson"], "given-names": ["PWF"], "source": ["Overview of Established Risk Factors for Cardiovascular Disease"], "year": ["2023"], "date-in-citation": ["accessed 21 Feb 2023"], "comment": ["\n"], "ext-link": ["https://www.uptodate.com/contents/overview-of-established-risk-factors-for-cardiovascular-disease"]}, {"label": ["29"], "collab": ["National Institute for Health and Care Excellence"], "part-title": ["Recommendations"], "source": ["Attention deficit Hyperactivity Disorder: Diagnosis and Management (NICE Guideline NG87)"], "publisher-name": ["NICE"], "date-in-citation": ["accessed 9 Mar 2022"], "comment": ["\n"], "ext-link": ["https://www.nice.org.uk/guidance/ng87/chapter/recommendations"]}]
{ "acronym": [], "definition": [] }
29
CC BY
no
2024-01-13 00:12:46
Br J Psychiatry. 2023 Oct 1; 223(4):472-477
oa_package/df/70/PMC7615511.tar.gz
PMC7615513
38213397
[ "<title>Introduction</title>", "<p id=\"P2\">People spend almost 90% of their time indoors. Therefore, the indoor thermal environment is closely related to occupant comfort, well-being, and health [##REF##34419205##1##–##REF##33049930##3##]. Extreme heat and cold exposure poses health risks, particularly for the elderly, children, and those with chronic illness [##UREF##1##4##–##UREF##2##6##]. Jevons et al. and Tham et al. investigated the lower and upper indoor temperature thresholds through systematic literature review [##REF##27106281##7##,##REF##31707154##8##]. They concluded that negative health effects in the general population start to occur when the indoor temperature is lower than 18 °C and higher than 26 °C-32 °C. They also reported the insufficient data and heterogeneity of the studies reviewed. It is difficult to obtain indoor environment data since the indoor climates of individual dwellings vary with complex factors, including the outdoor climate, envelope quality, air-conditioning equipment, occupant’s behavior, etc. [##UREF##3##9##–##UREF##6##12##].</p>", "<p id=\"P3\">A common approach to obtain indoor air temperature data is to install thermometers at target places to measure the air temperature directly. Morey et al. conducted a temperature monitoring survey of 122 houses to investigate the extent of overheating risk in summer and found that significant proportions of dwellings were uncomfortably warm in summer, especially in bedrooms and living rooms [##UREF##7##13##]. The researchers have concluded that the overheating risk will increase owing to climate change, especially for houses with highly insulated envelope [##UREF##8##14##,##UREF##9##15##]. A cold indoor environment in winter is also a concern. Many studies have indicated that an indoor environment with a low temperature has a notable impact on the health of vulnerable people. Wang et al. investigated 292 households with children and collected 877 air samples from bedrooms and living rooms. They found a significant correlation between low indoor temperature and the prevalence of the common cold among children [##UREF##10##16##]. Similarly, Han et al. conducted a prospective cohort study of 285 community-dwelling elderly individuals. They collected indoor temperature and acute respiratory illness incidence data continuously and found a negative correlation between indoor temperature and incidence [##REF##32297941##17##]. Furthermore, Miguel-Bellod et al. reported a relationship between poverty and indoor temperature in winter. They conducted a survey on 112 multifamily dwellings, and the principal findings showed that families with financial constraints spent more time below 18 °C, which may seriously affect their thermal comfort and health [##UREF##11##18##]. Therefore, large-scale investigation of indoor temperature of housing is important for understanding the comfort, health, and living conditions of local residents. However, it is difficult to obtain residential indoor temperature data in large-scale. There exit two practical issues conducting large-scale indoor temperature survey. First, placing temperature sensors house-by-house in region-, or even city-scale results in large instrument and labor costs. Second, direct temperature measurement requires entering each household to place sensors, which interferes with the daily lives of the residents.</p>", "<p id=\"P4\">Infrared thermography, a nonintrusive technique for measuring temperature, might be a breakthrough for large-scale indoor temperature investigation. As a prominent tool for detecting infrared energy emitted by object surfaces, infrared thermography has been widely applied in building diagnostics, such as determination of the overall thermal conductivity (U-value) of exterior walls [##UREF##12##19##–##UREF##14##21##] and windows [##UREF##15##22##–##UREF##17##24##] and the detection of heat loss points [##UREF##18##25##–##UREF##20##27##], air leakages [##UREF##21##28##–##UREF##23##30##], and moisture [##UREF##24##31##–##REF##35590872##33##] of the building envelope. Several attempts have been made to employ infrared thermography to measure indoor temperature. Lee et al. proposed a method to measure the indoor mean radiant temperature by using a pan-tilt camera to detect the interior surface temperatures [##UREF##26##34##]. Fokaides et al. used infrared camera to measure the indoor air temperature by placing mock target material interior the test room [##UREF##27##35##,##UREF##28##36##]. They experimentally proofed that the surface temperature of the mock target can present indoor air temperature when thermal equilibrium is reached. Porras-Amores et al. developed a method of using infrared cameras to measure the distribution of indoor air temperature by setting highly reflective screens inside the room. [##UREF##29##37##].</p>", "<p id=\"P5\">Previous studies on indoor temperature measurement using infrared thermography were conducted interior the rooms. Practically, it is difficult and time consuming to enter the rooms and place materials for indoor temperature measurement house by house during the indoor temperature survey. In this regard, we were inspired by the question that, based on the remote sensing property of infrared thermography, is there any way to detect the indoor temperature from building outside, without the need of interrupting the occupants?</p>", "<p id=\"P6\">In this paper, a novel method is proposed to use infrared thermography to identify the indoor temperature remotely by opening window hole from outside the building. Different from previous studies, one could obtain indoor temperature data from building outside by applying this method. This method, if successful, could lay a foundation for obtaining room temperature data from remote view infrared images. In this manuscript, an experimental study was conducted to explore the feasibility and accuracy of this method. The experimental results were analyzed and limitations of the proposed method were discussed.</p>" ]
[ "<title>Methodology</title>", "<p id=\"P7\">From the infrared images of building envelopes, it was observed that the detected temperature at the window opening gap differs from the temperature of other building envelope surface areas when windows are occasionally opened. Little attention has been paid to obtaining information about the temperature at the window opening. As shown in ##FIG##0##Fig. 1##, because rooms in multistory buildings (the most common building type in city centers) are usually connected with corridors or conditioned adjacent rooms, a relatively stable indoor thermal condition can be considered in these residential buildings. This means that the room interior surface temperature is similar to the room air temperature. Therefore, the hypothesis was made that the infrared temperature detected at the window opening gap reflects the indoor thermal environment to a certain level. Based on this assumption, an experimental study was conducted to explore the following questions: <list list-type=\"bullet\" id=\"L1\"><list-item><p id=\"P8\">Does the detected infrared temperature reflect the room temperature?</p></list-item><list-item><p id=\"P9\">To what extent can the infrared temperature reflect the room temperature?</p></list-item><list-item><p id=\"P10\">What influences the accuracy of this method?</p></list-item></list></p>", "<p id=\"P11\">To answer these questions, an experiment in a conditioned test room was conducted. Purpose of the experiment was to explore the possibility and feasibility of remotely sensing the indoor thermal environment by using infrared camera outside the building.</p>", "<title>Experiment facility</title>", "<p id=\"P12\">The room used in the experiment is located on the campus of Tsinghua University in Beijing, China. This southerly oriented room has interior dimensions of 2.7 × 2.7 × 2.7 m. All the walls of the room are exterior walls made of 30-cm-thick concrete. As shown in ##FIG##1##Fig. 2(a)##, the room has a large exterior window on the south wall. During the experiment, the right side window remained open to form a hole through which the infrared camera could detect the interior condition. To simulate a room condition with an interior wall, a lightweight partition wall made of plastic board was built inside the room (##FIG##1##Fig. 2 (b)##). The indoor thermal condition of the test room could be controlled by fan coil cooling and radiator heating. The inlet and outlet pipes of the fan coil and radiator were connected to a thermostat water tank located in the equipment room behind the test room. Different temperature sensors were installed in the room to monitor the indoor thermal environment. As shown in ##FIG##2##Fig. 3##, three air temperature sensors (T1–T3) were positioned along the room central line with heights of 0.08, 1.3, and 1.8 m, respectively. Four air temperature sensors (T4–T7) were arranged at the four corners at a height of 1.3 m. One globe thermometer was installed at the center of the room at a height of 1.3 m. The temperatures of the six interior surfaces were monitored by corresponding surface temperature sensors (B1–B6). In addition, two temperature sensors (P1 and P2) were positioned on the front surface of the partition wall at a height of 1 m. All the probes of the temperature sensors were wrapped in tinfoil to prevent the interference of short-wave solar radiation and long-wave radiation from the environment. Viewed from the horizontal direction outside the room, the measurement points that could be directly seen from the window opening were the air temperature sensor T4 and surface temperature of the partition wall P1 (see ##FIG##2##Fig. 3(a)## and ##FIG##3##4(a)##). All temperature sensors were Pt 100 with an accuracy of ± 0.2 °C. The measurement data were recorded in an Agilent data logger at time intervals of 10 s.</p>", "<title>Experimental setup</title>", "<p id=\"P13\">The experiment was conducted in June 2021. The infrared camera VarioCAM HiRes produced by Jenoptik was used. The infrared camera was placed 5 m from the target opening window. The height of the infrared camera tripod was adjusted so that camera lens maintained a horizontal shooting view to the target window. To avoid the influence of solar radiation on the infrared photography, experiments were conducted at night from approximately 19:30 to 20:30. During the experimental period, the infrared images were recorded at time intervals of approximately 60 s. To investigate the effect of the window opening size on the remote-sensing accuracy, the window opening grade gradually decreased from 100% to 10% during each filming time. The opening grade is defined as the ratio of the visible opening area from the horizontal view to the maximal opening area. To investigate the possible influence of the temperature difference between the indoor and outdoor environments on this method, the indoor thermal environment was controlled by a different operation mode during each experiment. As illustrated in ##TAB##0##Table 1##, three operation modes were employed separately. When the room was in heating mode, hot water was supplied to the radiator to heat the room. In cooling mode, cold water was supplied to the fan coil under the ceiling. The fan coil sent cold air to cool the room. In the transitional mode, the cooling and heating loops were shut down. To keep the room temperature as stable as possible during the night filming period, the radiator heating or fan coil cooling was performed 10 h before the experiment started to ensure that the temperature fluctuation of each interior measuring point did not exceed 1 °C during the experimental period [##UREF##30##38##,##UREF##31##39##].</p>", "<title>Validation process</title>", "<p id=\"P14\">The purpose of the experimental study is to validate the feasibility and accuracy of this method. Since the infrared image provides the spatial distribution information of indoor temperatures, and numbers of temperature sensors were arranged at different locations inside the test room. For the purpose of quantitative validation, the infrared temperatures at specific locations were extracted and compared with the measured temperatures of sensors at the corresponding locations. The validation process is detailed in ##FIG##3##Fig. 4##, infrared images with the window opening grade (OG) ranging from 100% to 10% were imported into MATLAB. The written program reads the red–green–blue (RGB) value of each pixel in the images and generates the corresponding temperature matrices by comparing the RGB value of each pixel with the temperature color scale. The identified infrared temperatures at specific spatial locations were extracted and compared with the measured room temperatures at corresponding locations.</p>" ]
[ "<title>Results</title>", "<title>Infrared temperature identification</title>", "<p id=\"P15\">##FIG##4##Fig. 5(a)## shows a full-size infrared image taken when the room was in heating mode with the side window fully open. The infrared temperature of the window opening hole was significantly higher than those of the other areas because the average room air temperature was approximately 5.3 °C higher than the ambient temperature during the test. Owing to the high reflectance of the tinfoil, air temperature sensor T4 (height: 1.3 m) and partition wall surface temperature P1 (height: 1.0 m) in the sight area was bright white. For the fully open window, the indoor temperature information of a rectangular area with a width of 0.5 m and height of 0.55–1.9 m could be obtained from outside the room. By converting the rectangular region into a temperature matrix using MATLAB, the spatial distribution of the detected indoor infrared temperature was obtained, as shown in ##FIG##4##Fig. 5(c)##. The room temperature shows a stable vertical layering under radiator heating.</p>", "<p id=\"P16\">##FIG##5##Fig. 6(a)## shows the horizontal infrared temperature distribution at heights of 0.8, 1.3, and 1.8 m, respectively. The infrared temperature distribution along the height (W = 0.18 m) is presented in ##FIG##5##Fig. 6(b)##. The indoor infrared temperature has an almost uniform distribution along the horizontal direction and a gradually accelerating temperature rise along the vertical direction. The infrared temperature increased 1.6 °C when the height increased from 0.8 to 1.3 m and 2.0 °C when the height increased from 1.3 to 1.8 m.</p>", "<p id=\"P17\">The same method was used to analyze the infrared image in the transitional and cooling modes. ##FIG##6##Fig. 7## shows the infrared temperature matrix in the transitional mode with the window fully open. As ##FIG##6##Fig. 7(b)## reveals, the infrared temperature had a relatively uniform distribution in the horizontal direction under natural conditions. A slight temperature rise was observed in the vertical direction. The temperature at a height of 1.3 m was 0.5 °C higher than the one at a height of 0.8 m and 0.3 °C lower than the one at height of 1.8 m.</p>", "<p id=\"P18\">##FIG##7##Fig. 8## illustrates the detected infrared temperature matrix in cooling mode with the window fully open. The infrared temperature distribution is in a circulation shape — warmer near the room center and cooler near the edge. The infrared temperature distribution trend is consistent with the jet flow formed by the cold air supplied by the ceiling fan coil. Influenced by the cold jet flow, the infrared temperature distribution is non-uniform from the vertical and horizontal directions (see ##FIG##7##Fig. 8 (c) and (d)##).</p>", "<p id=\"P19\">Analyzing three operation conditions (radiator heating, natural condition, and fan coil cooling) reveals that the infrared temperature data at the window opening gap provide the spatial distribution information of the room temperature to a certain extent. To investigate the degree of accuracy with which the detected infrared temperature reflects the indoor thermal environment, the infrared temperature distribution was compared with the measured temperature data in the next section.</p>", "<title>Comparison between infrared temperature and measured data</title>", "<title>Heating mode</title>", "<p id=\"P20\">##FIG##8##Fig. 9## shows the temperature sensor data of different locations during the camera-screening period when room was in heating mode. In order to explore the effect of window opening grade (OG) on the identify accuracy, the window opening was gradually transiting from fully opening (OG = 100%) to almost closing (OG = 10%) during the experiment, and the infrared images at certain opening levels were filmed. The vertical dotted lines indicate the time scales for taking photos with different window opening grades. The status changes of window opening were controlled manually and kept in interval around 1 min. The surface temperature points are shown by dotted lines of different colors, the air temperature points are shown by bubbles of different colors, and the globe temperature at the center of the room is represented by black bubbles. The experiment was completed in 10 min by taking infrared images with a decreasing window opening grade at 1-min intervals. During the test, the indoor thermal environment was relatively stable — the fluctuation of the measured temperature did not exceed 0.5 °C. The highest temperature was at the ceiling surface (red dotted line) with a value of approximately 34 °C, owing to the double effect of direct solar radiation and natural convection flow driven by radiator heating. The next-highest were the surface temperatures of the west wall (green dotted line) and south wall (blue dotted line). The floor had the lowest surface temperature (~28.5 °C). Influenced by the interior surface temperature difference and natural buoyancy flow, the indoor air points along the room central line (at 0.08, 1.3, and 1.8 m) showed a stable vertical temperature gradient. The globe temperature (1.3 m) was approximately 1 °C higher than the air temperature at a nearby point. The reason could be that the globe temperature was affected by the warm wall and ceiling surfaces.</p>", "<p id=\"P21\">The measured temperature points distributed by height at the camera capture moment of a window opening grade of 100% (19:33) are shown in ##FIG##9##Fig. 10##. The temperature points of the room interior surfaces are in green rhombi, air temperature points are in blue bubbles, surface temperature points of the partition wall are in orange pentagrams, and globe temperature is in a black bubble. The air and surface temperatures show a consistent monotonic temperature rise in the vertical spatial distribution, excluding the surface temperatures of the west and east walls (1.3 m), which are strongly affected by solar radiation. By fitting the values of the measured points at different heights, i.e., air temperature (T1–T7), partition wall surface temperature (P1 and P2), and surface temperature of floor and ceiling (B1 and B6), the trend line shown in the figure (blue dotted line) was obtained. For comparison, the vertical distribution of the infrared temperature is also plotted in ##FIG##9##Fig. 10## in small black points (W = 0.18 m, i.e., the column of values near measuring points T4 and P1). The vertical spatial distribution of the infrared temperature matches the measured room temperature well. At a height of 1.0 m, the average difference between the infrared temperature and the two surface temperatures of the partition wall was 0.2 °C. At a height of 1.3 m, the average difference between the infrared temperature and five air temperatures was 0.5 °C. At a height of 1.8 m, the difference between the infrared and air temperatures was 0.2 °C.</p>", "<title>Transitional mode</title>", "<p id=\"P22\">##FIG##10##Fig. 11## shows the measured room temperature when the room was under natural conditions without heating air conditioning devices operation. A slight vertical temperature gradient existed under natural conditions. During the camera filming period, the average air temperature difference between the points at 0.08 m (green bubbles) and 1.3 m (red bubbles) was 1.1 °C, and the difference between the points at 1.3 m (red bubbles) and 1.8 m (blue bubbles) was 0.5 °C. The interior surface temperature difference between the ceiling (red dotted line) and floor (navy dotted line) was approximately 2.8 °C. The globe temperature on average was 0.3 °C higher than the nearby air temperature, influenced by the warm ceiling and wall surfaces.</p>", "<p id=\"P23\">The spatial distribution of the measured temperatures at 19:25 (window opening grade of 100%) in the transitional mode is plotted in ##FIG##11##Fig. 12##. The blue dotted line represents the vertical distribution trend of the air temperature (T1–T7), partition wall surface temperature (P1–P2), and surface temperature of floor and ceiling (B1 and B6). In the lower height range, the vertical distribution of the detected infrared temperatures is consistent with the trend line of the measured room temperatures, and the trend line slightly deviates when the height exceeds 1.3 m. The deviation in a higher height range is caused by the warm ceiling, which is heated by solar radiation in daytime. However, in the visible height range of the window opening gap, the infrared temperature agrees well with the measured air and surface temperatures of the partition wall. At a height of 1.0 m, the difference between the infrared and average partition wall surface temperatures is 0.2 °C. At a height of 1.3 m, the difference between infrared and average air temperatures is 0.5 °C. At a height of 1.8 m, the difference between infrared and air temperatures is 0.2 °C.</p>", "<title>Cooling mode</title>", "<p id=\"P24\">##FIG##12##Fig. 13## shows the transient data of the measured room temperature during the cooling period. When the room was in cooling mode, the fan coil positioned beneath the ceiling provided a cold jet flow to cool the room. Therefore, the ceiling had the coldest temperature compared with the other interior surfaces. Relatively high surface temperatures were detected at the south and west walls owing to solar radiation heating. Because the indoor air was well mixed by the circulating jet flow, the air temperatures at the various heights had similar and stable values within the range of 20.5 °C–21.5 °C.</p>", "<p id=\"P25\">As discussed in <xref rid=\"S7\" ref-type=\"sec\">Section 3.1</xref>, unlike the heating and transitional modes in which the room temperature showed a relatively stable vertical gradient, the indoor temperature in cooling mode had a circulating form driven by the cold jet flow. Therefore, the room temperature in cooling mode was asymmetrically distributed in the horizontal and vertical directions. In this situation, the T4 air temperature and P1 partition wall surface temperature (points directly seen in infrared image) and the nearby infrared temperature were plotted for comparison. As ##FIG##13##Fig. 14(a)## shows, at a height of 1.0 m, the infrared temperature nearby was 0.9 °C higher than the measured P1 surface temperature. At a height of 1.3 m, the infrared temperature approaches the air temperature with a temperature difference of 0.2 °C. Unlike the heating and transitional modes, where the detected infrared temperature at the window opening gap matched the indoor room temperature well, in the cooling condition, the infrared temperature considerably overestimated the indoor thermal condition. Two replication experiments were conducted on June 17 and June 24 to verify the results. The corresponding data are plotted in ##FIG##13##Fig. 14(b) and (c)##. The results of the original experiment and two duplication experiments are similar. In the duplication experiments, the infrared temperature was approximately 1.2–1.3 °C higher than the measured surface temperature at 1.0 m and 0.2 °C higher than the measured air temperature at 1.3 m. A supposed reason is that the infrared camera had a higher measurement error when used to detect a surface colder than the environment in which the camera was located. A detailed explanation can be found in <xref rid=\"S15\" ref-type=\"sec\">Section 4.2</xref>.</p>", "<p id=\"P26\">The absolute measurement errors between infrared temperature and measured room temperatures under three operation modes are summarized in ##FIG##14##Fig. 15##. As shown, the identified infrared temperatures were relatively lower than the measured room temperatures at target positions under heating and transitional modes. The measurement errors under heating and transitional modes ranged from −0.5 °C to −0.1 °C and from −0.3 °C to −0.1 °C, respectively. When the room was under cooling mode, the infrared temperatures were higher than the measured room temperature, the absolute measurement error ranged from 0.2 °C to 1.3 °C.</p>", "<title>Effect of the window opening grade</title>", "<p id=\"P27\">In practical application, occupants do not often open the windows fully. Therefore, it is necessary to explore the effect of the window opening grade on the accuracy of this method. Considering this, the window opening grade was gradually decreased in each HVAC operation mode, and the infrared images of various window opening size were analyzed. The detected infrared images with decreasing window opening size in the heating, transitional, and cooling modes are presented in ##TAB##1##Tables 2##–##TAB##3##4## respectively.</p>", "<p id=\"P28\">As the window opening grade decreased, less information was obtained from the infrared image at the window opening gap. When the window opening grade decreased to 10%, only a column of temperature information near the window frame was visible in the infrared image. Because there was less information acquisition, to analyze the effect of the window opening on the infrared recognition accuracy, the vertical distribution of infrared temperature near the window frame (0.06 m) from images with various window opening grades was analyzed. As plotted in ##FIG##15##Fig. 16##, the sets of identified infrared temperature data at W = 0.06 m are almost identical. The deviations of the temperature points are within 0.5 °C. Since the room temperature remained relatively stable during the quick tests (less than 15 min), it can be concluded that the window opening grade did not have much influence on the accuracy of infrared temperature identification.</p>" ]
[ "<title>Discussion</title>", "<title>Relationship of three temperatures</title>", "<p id=\"P29\">The detected infrared temperature at the window opening gap reflects the spatial distribution of the air and partition wall surface temperatures to certain extent when the room is under a relatively stable thermal condition. The physical principle of the remote measurement is shown in ##FIG##16##Fig. 17##. The working principle of the infrared camera is to receive the infrared rays emitted by the object surface and obtain an image by photoelectric conversion. Therefore, the infrared temperature detected through the window opening gap indicates the surface temperature of partition wall. Assuming that the effects of heat conduction and heat capacity of the partition wall can be ignored, the surface temperature of the partition wall is a weighted average of the air temperature adjacent to the partition and radiant temperature from the interior surrounding wall surface. When the room is in a relatively stable thermal condition and the target wall connected to adjacent room or corridor has a similar temperature, the surface temperature of the partition wall, the adjacent air temperature and the radiant temperature of interior surrounding are close to each other through convective and radiant heat exchange. Therefore, as ##FIG##16##Fig. 17 (b)## shows, analyzing the relationship between infrared temperature, surface temperature of the partition wall and the indoor air temperature reveals that the infrared temperature at the window opening gap reflects the spatial distribution of the indoor air temperature when certain conditions are met. In practical applications, for conventional multistory houses, the surface temperature of the interior partition wall is usually not much different from the indoor air temperature. Therefore, it is feasible to detect the indoor air temperature through infrared remote sensing from the window opening gap.</p>", "<title>Influence of HVAC operation condition and window opening grade</title>", "<p id=\"P30\">As illustrated in <xref rid=\"S8\" ref-type=\"sec\">Section 3.2</xref>, the infrared sensing has a relatively high accuracy when the room is under radiator heating or a natural condition. Infrared image at the window opening gap can obtain the vertical air temperature gradient with an absolute error almost within 0.5 °C. However, when the room is under fan coil cooling, although the infrared image at the window opening gap can obtain the cold circulation flow blown by the ceiling fan coil, the infrared temperature significantly overestimates the room temperature (exceeding 1 °C). Considering the working principle of the infrared camera, the supposed reason for the relatively high error for a cold surface is that, when the surface of the object is colder than the surroundings, the emitted radiant intensity is lower than that of the surroundings according to Planck’s law. The resulting disturbance might cause overestimation of the object surface temperature. Maley et al. also reported an overestimation of skin temperature by infrared sensing when the skin was colder than the surroundings after immersion in cold water [##UREF##32##40##].</p>", "<p id=\"P31\">In the practical applications, the window can be partially or fully opened depending on user behavior. When the window opening area is smaller, less information about the indoor thermal environment can be obtained by infrared sensing. The analysis in <xref rid=\"S16\" ref-type=\"sec\">Section 4.3</xref> verified that the accuracy of infrared detection remained almost unchanged with decreasing opening area. The practical significance of this finding is that an infrared camera can remotely sense the room temperature, even if the window is only slightly open.</p>", "<title>Limitations and future studies</title>", "<p id=\"P32\">This study was conducted under laboratory conditions with a well-controlled indoor thermal environment. Following factors should be concerned in the future study. <list list-type=\"simple\" id=\"L2\"><list-item><label>(1)</label><p id=\"P33\">The thermal condition of the test room was relatively stable when the window was open. This might differ from the thermal condition in a real room.</p></list-item><list-item><label>(2)</label><p id=\"P34\">The experiment was conducted in an empty test room without heat sources such as occupants and electric devices, which have influence on the surrounding air temperature.</p></list-item><list-item><label>(3)</label><p id=\"P35\">The partition wall in the test room was made of lightweight plastic board. The surface temperature of the partition wall was highly consistent with the ambient air temperature. The interior wall and thermal environment of a real building might or might not be similar with the experimental condition.</p></list-item><list-item><label>(4)</label><p id=\"P36\">The experiment was conducted with an infrared camera fixed with a horizontal view at a constant distance of 5.2 m. The influence of the camera distance and angle should also be investigated in the future.</p></list-item></list></p>", "<p id=\"P37\">Overall, this experimental study preliminarily validated the feasibility and accuracy of the method by using an infrared camera to detect the indoor thermal environment through the window opening gap. In the further study, this method should be further validated in real buildings considering such practical issues as the thermal stability of a real building (especially with opening windows), influence of interior heat sources, camera distance and angle, sight interference caused by trees and window nets, etc.</p>" ]
[ "<title>Conclusions</title>", "<p id=\"P38\">This study proposed a novel method to measure indoor temperatures remotely from outside the building by using infrared camera. Compared with the traditional method by placing temperature sensors house by house, this remote sensing method could directly measure indoor temperature without the need to enter the room. Moreover, it could even possibly reflect the spatial distribution of indoor temperatures that is impossible to obtain by point measurements. The feasibility of this assumption was verified through an experiment conducted in a cement room under radiator heating, transitional, and fan coil cooling conditions. The results showed that the remote sensing of the indoor air temperature by an infrared camera is feasible when the indoor thermal environment is relatively stable. The main conclusions of this experimental study are as follows. <list list-type=\"simple\" id=\"L3\"><list-item><label>1)</label><p id=\"P39\">The temperature matrix identified by an infrared image taken at a fully opened window can quantitatively describe the vertical temperature gradient of the interior room environment when the room is under radiator heating or a natural condition, as well as the circulating cold air flow pattern resulting from fan coil cooling.</p></list-item><list-item><label>2)</label><p id=\"P40\">The vertical distribution of infrared temperature was highly consistent with the distribution tendency of the measured air and surface temperatures at different heights in heating and transitional mode. The absolute deviations between the identified infrared temperature and the measured room temperatures at different heights were within 0.5 °C. While in cooling mode, higher deviations were observed between the infrared and partition wall surface temperatures, with values ranging from 0.9 to 1.3 °C at 1.0 m during the original and two duplication experiments.</p></list-item><list-item><label>3)</label><p id=\"P41\">Less information about the indoor thermal environment can be obtained with a decreased window opening grade. However, the accuracy of infrared recognition does not change in the identifiable opening area.</p></list-item><list-item><label>4)</label><p id=\"P42\">These conclusions were obtained in a laboratory study with a well-controlled and stable thermal condition and light-weight partition wall. The feasibility and accuracy of this remote-sensing method in real buildings should be investigated further.</p></list-item></list></p>" ]
[ "<p id=\"P1\">Investigation of housing indoor temperature is important for understanding the comfort, health and living conditions of the local residents. The traditional method to measure indoor temperature is to place sensors at the target places, which is not only expensive but also inconvenient for indoor temperature investigation, especially for the investigation at community and city scale. In this study, a novel method was proposed to obtain the indoor temperatures remotely from outside the building through window opening area using and infrared camera. Compared with the traditional contact measurement method, the proposed remote sensing method could detect the indoor temperature without entering the room. Moreover, the infrared image could reflect the spatial distribution information of indoor temperature. To verify the feasibility and accuracy of this method, an experiment was conducted in a test room under heating, transitional, and cooling conditions with various window opening grades. It was found that the infrared images at the window opening area could reflect the spatial distribution of indoor temperature with an accuracy within 0.5 °C under stable heating and transitional conditions. In the fan coil cooling condition, however, although the infrared image can reflect the cold air flow pattern, the deviations between the infrared temperature and the measured room temperature exceeded 1.0 °C. The effect of window opening grade on the recognition accuracy kept within 0.5 °C.</p>" ]
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[ "<title>Acknowledgments</title>", "<p>This work was supported by the Pathways to Equitable Healthy Cities grant from the Wellcome Trust (209376/Z/17/Z), the National Natural Science Foundation of China (51838007), the Beijing Heating Group Co., Ltd. commissioned project (20212001787) and Beijing Key Laboratory of Indoor Air Quality Evaluation and Control. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any author accepted manuscript version arising from this submission.</p>", "<title>Data availability</title>", "<p id=\"P43\">Data will be made available on request.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Fig. 1</label><caption><title>Hypothesis of detecting the indoor thermal environment through an infrared image at the window opening gap.</title></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Fig. 2</label><caption><p>Photograph of the test room: (a) outside view and (b) inside view.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Fig. 3</label><caption><p>Positions of air temperature sensors T1–T7, surface temperature sensors of room interior surfaces B1–B6, and surface temperature sensors of partition walls P1 and P2.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Fig. 4</label><caption><title>Validation progress (OG is the window opening grade).</title></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Fig. 5</label><caption><p>Infrared image of room in heating mode at 100% window opening grade: (a) original image, (b) target opening gap, and (c) detected temperature spatial distribution matrix.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Fig. 6</label><caption><p>Detected infrared temperature (IRT) in heating mode at a window opening grade of 100%: (a) horizontal distribution and (b) vertical distribution.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Fig. 7</label><caption><p>Infrared image in transitional mode at window opening grade of 100%: (a) infrared image of the window opening gap, (b) detected temperature spatial distribution matrix, (c) horizontal temperature distribution, and (d) vertical temperature distribution.</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Fig. 8</label><caption><p>Infrared image in room cooling mode at window opening grade of 100%: (a) infrared image of the window opening gap, (b) detected temperature spatial distribution matrix, (c) horizontal temperature distribution, and (d) vertical temperature distribution.</p></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Fig. 9</label><caption><p>Measured data of indoor air temperature, globe temperature, and surface temperature of interior room surfaces in heating mode (June 14, 2021): OG is the window opening grade (100%–10%).</p></caption></fig>", "<fig position=\"float\" id=\"F10\"><label>Fig. 10</label><caption><p>Comparison between the vertical distribution of the measured temperature data and vertical distribution of the infrared temperature (IRT) at a window width of 0.18 m in heating mode at a window opening grade of 100% (June 14, 2021, 19:33).</p></caption></fig>", "<fig position=\"float\" id=\"F11\"><label>Fig. 11</label><caption><p>Measured data of indoor air temperature, globe temperature, and surface temperature of interior room surfaces in transitional mode (June 16, 2021): OG is the window opening grade (100%–10%).</p></caption></fig>", "<fig position=\"float\" id=\"F12\"><label>Fig. 12</label><caption><p>Comparison of the vertical distributions of measured temperature data and infrared temperature (IRT) at window width of 0.18 m in transitional mode at window opening grade of 100% (June 16, 19:25).</p></caption></fig>", "<fig position=\"float\" id=\"F13\"><label>Fig. 13</label><caption><p>Measured data of indoor air temperature, globe temperature, and surface temperature of interior room surfaces in transitional mode (June 15, 2021): OG is the window opening grade (100%–10%).</p></caption></fig>", "<fig position=\"float\" id=\"F14\"><label>Fig. 14</label><caption><p>Comparison of the vertical distributions of measured temperature data and infrared temperature (IRT) at window width of 0.18 m in transitional mode at window opening grade of 100%: (a) experiment on June 15, (b) replication experiment I on June 17, and (c) replication experiment II on June 24.</p></caption></fig>", "<fig position=\"float\" id=\"F15\"><label>Fig. 15</label><caption><title>Absolute measurement errors between infrared temperature and measured room temperatures at the window opening grade of 100% under three operation conditions.</title></caption></fig>", "<fig position=\"float\" id=\"F16\"><label>Fig. 16</label><caption><p>Comparison of infrared temperature at window width of 0.06 m with the window opening grade (OG) ranging from 100% to 10%: (a) heating mode, (b) transitional mode, and (c) cooling mode.</p></caption></fig>", "<fig position=\"float\" id=\"F17\"><label>Fig. 17</label><caption><title>Relationship between infrared temperature at window opening gap, wall surface temperature, and indoor air temperature.</title></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Experimental conditions of three operation modes.</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Heating mode</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Transitional mode</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Cooling mode</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Date</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6/14/2021</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6/16/2021</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6/15/2021</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Camera filming time</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19:30–19:45</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19:25–19:37</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19:51–20:02</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Filming interval</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">60 s</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">60 s</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">60 s</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Ambient temperature</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">25.7 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.00 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">25.1 °C</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Globe temperature</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31.7 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24.6 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">21.7 °C</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean temperature of interior surfaces</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">32.2 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24.9 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.1 °C</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean air temperature at 0.08 m</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">29.0 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23.3 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20.6 °C</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean air temperature at 1.3 m</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31.3 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24.4 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">21.3 °C</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean air temperature at 1.8 m</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">33.1 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24.8 °C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">21.4 °C</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Infrared images in heating mode with the window opening grade ranging from 75% to 10%.</title></caption><table frame=\"void\" rules=\"none\"><tbody><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><title>Infrared images in transitional mode with the window opening grade ranging from 75% to 10%.</title></caption><table frame=\"void\" rules=\"none\"><tbody><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n\n</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><title>Infrared images in cooling mode with the window opening grade ranging from 75% to 10%.</title></caption><table frame=\"void\" rules=\"none\"><tbody><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n\n</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P44\">\n<bold>Declaration of Competing Interest</bold>\n</p><p id=\"P45\">The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><p id=\"P46\">Note: All temperatures in this table are average values during the experimental period.</p></fn></table-wrap-foot>" ]
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[{"label": ["[2]"], "person-group": ["\n"], "surname": ["Vergerio", "Becchio"], "given-names": ["G", "C"], "article-title": ["Pursuing occupants\u2019 health and well-being in building management: Definition of new metrics based on indoor air parameters"], "source": ["Build Environ"], "year": ["2022"], "volume": ["223"], "pub-id": ["10.1016/j.buildenv.2022.109447"]}, {"label": ["[4]"], "person-group": ["\n"], "surname": ["Mitchell", "Heaviside", "Vardoulakis", "Huntingford", "Masato", "Guillod", "Frumhoff", "Bowery", "Wallom", "Allen"], "given-names": ["D", "C", "S", "C", "G", "BP", "P", "A", "D", "M"], "article-title": ["Attributing human mortality during extreme heat waves to anthropogenic climate change"], "source": ["Environ Res Lett"], "year": ["2016"], "volume": ["11"], "issue": ["7"], "pub-id": ["10.1088/1748-9326/11/7/074006"]}, {"label": ["[6]"], "person-group": ["\n"], "surname": ["Yang", "Zhang", "Qin", "Liu"], "given-names": ["Z", "W", "M", "H"], "article-title": ["Comparative study of indoor thermal environment and human thermal comfort in residential buildings among cities, towns, and rural areas in arid regions of China"], "source": ["Energy Build"], "year": ["2022"], "volume": ["273"], "pub-id": ["10.1016/j.enbuild.2022.112373"]}, {"label": ["[9]"], "person-group": ["\n"], "surname": ["Feng", "Zhou", "Li"], "given-names": ["J", "Z", "W"], "article-title": ["Associating indoor air temperature with building spatial design and occupancy features: A statistical analysis on university classrooms"], "source": ["Build Environ"], "year": ["2022"], "volume": ["216"], "pub-id": ["10.1016/j.buildenv.2022.109009"]}, {"label": ["[10]"], "person-group": ["\n"], "surname": ["Kong", "Chang", "Li", "Li", "Li"], "given-names": ["X", "Y", "N", "H", "W"], "article-title": ["Comparison study of thermal comfort and energy saving under eight different ventilation modes for space heating"], "source": ["Build Simul"], "year": ["2022"], "volume": ["15"], "issue": ["7"], "fpage": ["1323"], "lpage": ["1337"], "pub-id": ["10.1007/s12273-021-0814-7"]}, {"label": ["[11]"], "person-group": ["\n"], "surname": ["Thapa", "Bansal", "Panda"], "given-names": ["S", "AK", "GK"], "article-title": ["Adaptive thermal comfort in the residential buildings of north east India\u2014An effect of difference in elevation"], "source": ["Build Simul"], "year": ["2018"], "volume": ["11"], "issue": ["2"], "fpage": ["245"], "lpage": ["267"], "pub-id": ["10.1007/s12273-017-0404-x"]}, {"label": ["[12]"], "person-group": ["\n"], "surname": ["Su", "Yang", "Zhou", "Wang", "Li"], "given-names": ["W", "B", "B", "F", "A"], "article-title": ["A novel convection and radiation combined terminal device: Its impact on occupant thermal comfort and cognitive performance in winter indoor environments"], "source": ["Energy Build"], "year": ["2021"], "volume": ["246"], "pub-id": ["10.1016/j.enbuild.2021.111123"]}, {"label": ["[13]"], "person-group": ["\n"], "surname": ["Morey", "Beizaee", "Wright"], "given-names": ["J", "A", "A"], "article-title": ["An investigation into overheating in social housing dwellings in central England"], "source": ["Build Environ"], "year": ["2020"], "volume": ["176"], "pub-id": ["10.1016/j.buildenv.2020.106814"]}, {"label": ["[14]"], "person-group": ["\n"], "surname": ["Baba", "Ge", "Wang", "Zmeureanu"], "given-names": ["FM", "H", "L", "R"], "article-title": ["Do high energy-efficient buildings increase overheating risk in cold climates? Causes and mitigation measures required under recent and future climates"], "source": ["Build Environ"], "year": ["2022"], "volume": ["219"], "elocation-id": ["109230"]}, {"label": ["[15]"], "person-group": ["\n"], "surname": ["Bo", "Chang", "Yu", "Xu", "Guo"], "given-names": ["R", "W-S", "Y", "Y", "H"], "article-title": ["Overheating of residential buildings in the severe cold and cold regions of China: The gap between building policy and performance"], "source": ["Build Environ"], "year": ["2022"], "volume": ["225"], "pub-id": ["10.1016/j.buildenv.2022.109601"]}, {"label": ["[16]"], "person-group": ["\n"], "surname": ["Wang", "Yan", "Yang", "Ye", "Fan", "Liao", "Zhang", "Yang", "Li", "Yao", "Wan"], "given-names": ["J", "X", "W", "D", "L", "Y", "Y", "Y", "X", "X", "L"], "article-title": ["Association between indoor environment and common cold among children aged 7\u20139 years in five typical cities in China"], "source": ["Environ Sustain Indic"], "year": ["2020"], "volume": ["6"], "pub-id": ["10.1016/j.indic.2020.100033"]}, {"label": ["[18]"], "person-group": ["\n"], "surname": ["San Miguel-Bellod", "Gonz\u00e1lez-Mart\u00ednez", "S\u00e1nchez-Ostiz"], "given-names": ["J", "P", "A"], "article-title": ["The relationship between poverty and indoor temperatures in winter: Determinants of cold homes in social housing contexts from the 40s\u201380s in Northern Spain"], "source": ["Energy Build"], "year": ["2018"], "volume": ["173"], "fpage": ["428"], "lpage": ["442"], "pub-id": ["10.1016/j.enbuild.2018.05.022"]}, {"label": ["[19]"], "person-group": ["\n"], "surname": ["Fokaides", "Kalogirou"], "given-names": ["PA", "SA"], "article-title": ["Application of infrared thermography for the determination of the overall heat transfer coefficient (U-Value) in building envelopes"], "source": ["Appl Energy"], "year": ["2011"], "volume": ["88"], "issue": ["12"], "fpage": ["4358"], "lpage": ["4365"], "pub-id": ["10.1016/j.apenergy.2011.05.014"]}, {"label": ["[20]"], "person-group": ["\n"], "surname": ["Nardi", "Paoletti", "Ambrosini", "de Rubeis", "Sfarra"], "given-names": ["I", "D", "D", "T", "S"], "article-title": ["U-value assessment by infrared thermography: A comparison of different calculation methods in a Guarded Hot Box"], "source": ["Energy Build"], "year": ["2016"], "volume": ["122"], "fpage": ["211"], "lpage": ["221"], "pub-id": ["10.1016/j.enbuild.2016.04.017"]}, {"label": ["[21]"], "person-group": ["\n"], "surname": ["Lu", "Memari"], "given-names": ["X", "A"], "article-title": ["Application of infrared thermography for in-situ determination of building envelope thermal properties"], "source": ["J Build Eng"], "year": ["2019"], "volume": ["26"], "pub-id": ["10.1016/j.jobe.2019.100885"]}, {"label": ["[22]"], "person-group": ["\n"], "surname": ["Aversa", "Palumbo", "Donatelli", "Tamborrino", "Ancona", "Galietti", "Luprano"], "given-names": ["P", "D", "A", "R", "F", "U", "VAM"], "article-title": ["Infrared thermography for the investigation of dynamic thermal behaviour of opaque building elements: Comparison between empty and filled with hemp fibres prototype walls"], "source": ["Energy Build"], "year": ["2017"], "volume": ["152"], "fpage": ["264"], "lpage": ["272"], "pub-id": ["10.1016/j.enbuild.2017.07.055"]}, {"label": ["[23]"], "person-group": ["\n"], "surname": ["Maroy", "Carbonez", "Steeman", "Van Den Bossche"], "given-names": ["K", "K", "M", "N"], "article-title": ["Assessing the thermal performance of insulating glass units with infrared thermography: Potential and limitations"], "source": ["Energy Build"], "year": ["2017"], "volume": ["138"], "fpage": ["175"], "lpage": ["192"], "pub-id": ["10.1016/j.enbuild.2016.10.054"]}, {"label": ["[24]"], "person-group": ["\n"], "surname": ["Albatici", "Tonelli"], "given-names": ["R", "AM"], "article-title": ["Infrared thermovision technique for the assessment of thermal transmittance value of opaque building elements on site"], "source": ["Energy Build"], "year": ["2010"], "volume": ["42"], "issue": ["11"], "fpage": ["2177"], "lpage": ["2183"], "pub-id": ["10.1016/j.enbuild.2010.07.010"]}, {"label": ["[25]"], "person-group": ["\n"], "surname": ["Fran\u00e7ois", "Ibos", "Feuillet", "Meulemans"], "given-names": ["A", "L", "V", "J"], "article-title": ["In situ measurement method for the quantification of the thermal transmittance of a non-homogeneous wall or a thermal bridge using an inverse technique and active infrared thermography"], "source": ["Energy Build"], "year": ["2021"], "volume": ["233"], "pub-id": ["10.1016/j.enbuild.2020.110633"]}, {"label": ["[26]"], "person-group": ["\n"], "surname": ["Martin Oca\u00f1a", "Ca\u00f1as Guerrero", "Gonzalez Requena"], "given-names": ["S", "I", "I"], "article-title": ["Thermographic survey of two rural buildings in Spain"], "source": ["Energy Build"], "year": ["2004"], "volume": ["36"], "issue": ["6"], "fpage": ["515"], "lpage": ["523"]}, {"label": ["[27]"], "person-group": ["\n"], "surname": ["Vavilov"], "given-names": ["VP"], "article-title": ["A pessimistic view of the energy auditing of building structures with the use of infrared thermography"], "source": ["Russ J Nondestruct Test"], "year": ["2010"], "volume": ["46"], "issue": ["12"], "fpage": ["906"], "lpage": ["910"], "pub-id": ["10.1134/S1061830910120065"]}, {"label": ["[28]"], "person-group": ["\n"], "surname": ["Zhou", "Wen", "Su"], "given-names": ["R", "Z", "H"], "article-title": ["Automatic recognition of earth rock embankment leakage based on UAV passive infrared thermography and deep learning"], "source": ["ISPRS J Photogramm Remote Sens"], "year": ["2022"], "volume": ["191"], "fpage": ["85"], "lpage": ["104"], "pub-id": ["10.1016/j.isprsjprs.2022.07.009"]}, {"label": ["[29]"], "person-group": ["\n"], "surname": ["Ye", "Zhou", "Tu", "Xiao", "Yan", "Wu", "Wu", "Zheng", "Tittel"], "given-names": ["W", "B", "Z", "X", "J", "T", "F", "C", "FK"], "article-title": ["Leakage source location based on Gaussian plume diffusion model using a near-infrared sensor"], "source": ["Infrared Phys Technol"], "year": ["2020"], "volume": ["109"], "pub-id": ["10.1016/j.infrared.2020.103411"]}, {"label": ["[30]"], "person-group": ["\n"], "surname": ["Dudi\u0107", "Ignjatovi\u0107", "\u0160e\u0161lija", "Blagojevi\u0107", "Stojiljkovi\u0107"], "given-names": ["S", "I", "D", "V", "M"], "article-title": ["Leakage quantification of compressed air using ultrasound and infrared thermography"], "source": ["Measurement"], "year": ["2012"], "volume": ["45"], "issue": ["7"], "fpage": ["1689"], "lpage": ["1694"], "pub-id": ["10.1016/j.measurement.2012.04.019"]}, {"label": ["[31]"], "person-group": ["\n"], "surname": ["Liu", "Zhang", "Yang", "Hu", "Luo", "Li", "Dong"], "given-names": ["Z", "R", "C", "B", "X", "Y", "C"], "article-title": ["Research on moisture content detection method during green tea processing based on machine vision and near-infrared spectroscopy technology"], "source": ["Spectrochim, Acta Part A Mol Biomol Spectrosc"], "year": ["2022"], "volume": ["271"], "pub-id": ["10.1016/j.saa.2022.120921"]}, {"label": ["[32]"], "person-group": ["\n"], "surname": ["Edis", "Flores-Colen", "de Brito"], "given-names": ["E", "I", "J"], "article-title": ["Passive thermographic detection of moisture problems in fa\u00e7ades with adhered ceramic cladding"], "source": ["Constr Build Mater"], "year": ["2014"], "volume": ["51"], "fpage": ["187"], "lpage": ["197"], "pub-id": ["10.1016/j.conbuildmat.2013.10.085"]}, {"label": ["[34]"], "person-group": ["\n"], "surname": ["Lee", "Jo"], "given-names": ["D-S", "J-H"], "article-title": ["Application of IR camera and pyranometer for estimation of longwave and shortwave mean radiant temperatures at multiple locations"], "source": ["Build Environ"], "year": ["2022"], "volume": ["207"], "pub-id": ["10.1016/j.buildenv.2021.108423"]}, {"label": ["[35]"], "person-group": ["\n"], "surname": ["Fokaides", "Jurelionis", "Gagyte", "Kalogirou"], "given-names": ["PA", "A", "L", "SA"], "article-title": ["Mock target IR thermography for indoor air temperature measurement"], "source": ["Appl Energy"], "year": ["2016"], "volume": ["164"], "fpage": ["676"], "lpage": ["685"], "pub-id": ["10.1016/j.apenergy.2015.12.025"]}, {"label": ["[36]"], "person-group": ["\n"], "surname": ["Georgiou", "Stasiuliene", "Valancius", "Seduikyte", "Jurelionis", "Fokaides"], "given-names": ["L", "L", "R", "L", "A", "PA"], "article-title": ["Investigation of the performance of mock-target IR thermography for indoor air temperature measurements under transient conditions"], "source": ["Measurement"], "year": ["2023"], "volume": ["208"], "elocation-id": ["112461"]}, {"label": ["[37]"], "person-group": ["\n"], "surname": ["Porras-Amores", "Mazarr\u00f3n", "Ca\u00f1as"], "given-names": ["C", "FR", "I"], "article-title": ["Using quantitative infrared thermography to determine indoor air temperature"], "source": ["Energy Build"], "year": ["2013"], "volume": ["65"], "fpage": ["292"], "lpage": ["298"], "pub-id": ["10.1016/j.enbuild.2013.06.022"]}, {"label": ["[38]"], "collab": ["Ministry of Housing and Urban-Rural Development of the People\u2019s Republic of China"], "source": ["GB/T 50785-2012 Evaluation standard for indoor thermal environment in civil buildings [in Chinese]"], "year": ["2012"]}, {"label": ["[39]"], "collab": ["General Administration of Quality and Construction Inspection and Quarantine of the People\u2019s Republic of China"], "source": ["GB/T 30801-2014: Hydrothermal performance of building materials and products \u2013 Determination of water-vapor transmission properties \u2014 Box method [in Chinese]"], "year": ["2014"]}, {"label": ["[40]"], "person-group": ["\n"], "surname": ["Maley", "Hunt", "Bach", "Eglin", "Costello"], "given-names": ["MJ", "AP", "AJ", "CM", "JT"], "article-title": ["Infrared cameras overestimate skin temperature during rewarming from cold exposure"], "source": ["J Therm Biol"], "year": ["2020"], "volume": ["91"], "pub-id": ["10.1016/j.jtherbio.2020.102614"]}]
{ "acronym": [], "definition": [] }
40
CC BY
no
2024-01-13 00:12:45
Energy Build. 2023 May; 286:112975
oa_package/e9/3f/PMC7615513.tar.gz
PMC7615514
38213413
[ "<title>Introduction</title>", "<p id=\"P2\">Multiple Sclerosis (MS) is a chronic, auto-immune disease caused by inflammatory damage to myelin in the central nervous system (##REF##19289565##Coles, 2009##). MS symptoms typically manifest as the involvement of motor, sensory, visual and autonomic systems depending on the area in the central nervous system that is affected (##REF##18970977##Compston and Coles, 2008##). In collaboration with Public Health England, the ##UREF##1##MS Society (2020)## estimate that there are over 130 000 people with MS in the UK, and that each year nearly 7000 people are newly diagnosed, with the prevalence more than double in females compared to males.</p>", "<p id=\"P3\">Approximately 85% of people with MS are initially diagnosed with relapsing remitting MS (RRMS) (##UREF##5##National MS Society, 2022##), defined by periods of new or worsening neurological symptoms, lasting longer than 24 hours, and in the absence of any other cause (##UREF##2##National Institute for Health and Care Excellence, 2016##). A relapse is characterised by symptoms that commence slowly, stabilise over days or weeks and resolve in a gradual way, either completely or in-part (##REF##19289565##Coles, 2009##). Management of RRMS includes treatment with disease modifying therapies (DMTs) to reduce disease activity and progression (##REF##26374509##Gallo et al, 2015##). There are a wide range of DMTs available for use as either a pill, injection or intravenous infusion, each with a different mechanism of action. In 2015, a Cochrane systematic review was undertaken to compare the benefit and acceptability of DMTs (immunomodulators and immunosuppressants) for the treatment of people with RRMS (##REF##26384035##Tramacere et al, 2015##).</p>", "<p id=\"P4\">This commentary aims to critically appraise the methods used within the Cochrane systematic review and to discuss the findings in the context of more recent clinical evidence and the implications for clinical practice.</p>" ]
[ "<title>Methods</title>", "<p id=\"P5\">A multiple database search was undertaken from the date of inception to September 2014. Additional screening of included studies and relevant systematic review citations was undertaken. Only randomised controlled trials (RCTs) that compared one or more immunomodulators or immunosuppressants (all types) to different active therapies or a placebo were included. RCTs that completed the follow-up in less than six months were excluded, as were those trials that evaluated combination treatments, comparison of same drug regimens, non-pharmacological treatments and over the counter drugs.</p>", "<p id=\"P6\">Participants from the included trials were aged 18 years or over, with a diagnosis of RRMS according to diagnostic criteria. There were no exclusions of the participants based on sex, degree of disability or disease duration.</p>", "<p id=\"P7\">Screening, data extraction and assessment of bias (using the Cochrane collaboration criteria), were undertaken independently by two reviewers, with arbitration by a third reviewer. The primary outcomes for the review were the proportion of participants experiencing relapse (new or worsening symptoms), disability worsening (irreversible worsening at 3 months) and acceptability (number of participants who withdrew because of an adverse event). Secondary outcomes were the total number of serious adverse events. Pairwise conventional meta - analyses were undertaken for all primary outcomes using a random effects model. A network meta-analysis was also undertaken for relapses, disability worsening and acceptability outcomes, and a surface under the cumulative ranking curve (SUCRA) score (0-100%) was calculated to enable the ranking of interventions. To evaluate the presence of statistical inconsistency, a loop specific method was used for local networks and ‘design by treatment’ for the entire network. A classification of confidence in the estimated effect was given using grading of recommendations assessment, development and evaluation (GRADE) for network meta-analysis. Risk of bias and inconsistencies were taken into consideration when grading the overall confidence in each outcome.</p>" ]
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[ "<p id=\"P1\">Disease modifying therapies are available for the treatment of relapse remitting multiple sclerosis, making relapses less common and severe. A Cochrane systematic review was undertaken to compare their benefit and acceptability. This article summarises and appraises the review evidence.</p>" ]
[ "<title>Main Review Findings</title>", "<p id=\"P8\">A total of 39 studies, comprising 25 113 randomised participants diagnosed with MS were included in the review. Of these studies, 24 (60%) were controlled by a placebo group, whereas 15 (40%) were a head-to-head comparison. Most of the trials assessed short-term outcomes (median follow up of 24 months). The following DMTs were evaluated: Interferon beta-1b (Betaseron), Interferon beta-1a (Avonex and Rebif), Glatiramer acetate, Natalizumab, Mitoxantrone, Fingolimod, Teriflunomide, Dimethyl fumarate, Alemtuzumab, Pegylated interferon beta-1a, Daclizumab, Laquinimod, Azathioprine and Immunoglobulins.</p>", "<p id=\"P9\">The risk of bias for the included studies was mixed with 51% of studies judged as high, 41% as moderate and 8% as low risk of bias. The main areas of high risk were non-blinding of participants and investigators (15 studies, 38%), incomplete outcome data (14 studies, 36%) and other biases (33 studies, 85%) such as the role and influence of the study sponsor in authorship. Over a 24-month period, local statistical inconsistency was observed in the network meta-analysis of relapse and disability worsening. There was no indication of global inconsistency within any network. Owing to the limited number of studies per comparison and statistical inconsistency, the quality of evidence was downgraded in most comparisons.</p>", "<p id=\"P10\">Over the 12- and 24-month periods, the network meta-analysis showed that the most effective therapies for reducing the risk of relapse in RRMS, when compared to a placebo, were Alemtuzumab, Mitoxantrone, Natalizumab and Fingolimod (##TAB##0##Table 1## and ##TAB##1##2##). All four therapies demonstrated a clinical and statistical reduction in the risk of experiencing a relapse. Over the 12- and 24-month period, excluding Mitoxantrone and Natalizumab, Alemtuzumab showed a significantly lower proportion of participants who experienced new relapses.</p>", "<p id=\"P11\">Over a 24-month period, when compared to a placebo, the most effective therapies for reducing the risk of disability worsening were Mitoxantrone, Alemtuzumab, Natalizumab and Azathioprine (##TAB##2##Table 3##). All four therapies demonstrated a clinical and statistical reduction in the risk of disability worsening. Over the 24-month period, Alemtuzumab also demonstrated a significant reduction in the risk of disability worsening compared to all other therapies excluding Mitoxantrone, which showed a significant reduction in the proportion of participants who experienced disability worsening compared to a placebo and five other therapies.</p>", "<p id=\"P12\">The network meta-analysis showed that over a 12-month period several therapies had a significantly increased risk of treatment withdrawal because of adverse events, when compared to a placebo group. These were Teriflunomide, Peg-interferon beta-1a, Interferon beta-1a (Avonex), Interferon beta-1a (Rebif) and Fingolimod (##TAB##3##Table 4##). Over 24 months, only fingolimod had a significantly higher risk of treatment withdrawal when compared to the placebo group (risk ratio 1.69, 95% confidence interval 1.32-2.17). There was limited and unclear evidence for the difference in risk of serious adverse events when comparing all active therapies to a placebo. There was no statistically significant difference for any of the subgroup or sensitivity analyses undertaken.</p>", "<title>Quality of Systematic Review</title>", "<p id=\"P13\">The Amstar2 tool was used to assess the quality of the Cochrane systematic review, scoring it 16 out of 16 (##REF##28935701##Shea et al, 2017##). Therefore, this systematic review provides an accurate and comprehensive summary of the available studies that address the question of interest.</p>", "<p id=\"P14\">However, other factors should be taken into consideration when interpreting the findings from this review. For example, there were limited numbers of trials that directly compared DMTs. It should also be considered that all the therapies were evaluated within the short-term (24-month follow-up period). Therefore, identifying benefit beyond this timeframe is uncertain and thus, limits the implications for practice for what is a life-long disease. Furthermore, the short-term nature of these trials does not provide enough data on serious adverse events and so makes it difficult to assess the risk related to these therapies.</p>", "<title>Implications for practice</title>", "<p id=\"P15\">The most effective DMTs for preventing clinical relapses in RRMS based on the quality of evidence required (moderate to high quality) and limited to the first 24 months of treatment are Alemtuzumab, Natalizumab and Fingolimod. Given the quality of evidence identified for the proportion of participants experiencing disability worsening (low to moderate), there is inconsistent evidence to recommend which therapies are most effective in preventing the worsening of disability. However, there was moderate confidence in the estimate of effect that Natalizumab statistically and clinically reduces the risk of disability worsening compared to a placebo at 24 months. Although most therapies were associated with a higher risk of treatment withdrawal because of an adverse event at 12 months, Fingolimod was the only treatment that had a significant risk at 24 months.</p>", "<p id=\"P16\">As the Cochrane review is now seven years old, it is important to consider more recent evidence in order to improve the understanding of the efficacy and safety of DMTs when treating RRMS. Further network meta-analyses have found Natalizumab and Alemtuzumab to consistently demonstrate a high ranking of efficacy across clinical outcomes including rate of relapse and disability progression (##REF##27645339##Fogarty et al, 2016##; ##REF##30014314##Lucetta et al, 2018##; ##REF##31129710##Li et al, 2020##; ##REF##33878488##Liu et al, 2021##). Ocrelizumab has also been shown to have high efficacy among DMTs with a similar safety profile (##REF##30014314##Lucchetta et al, 2018##; ##REF##30677733##McCool et al, 2019##; ##REF##31129710##Li et al, 2020##), and Ofatumumab was found to be superior to or as effective as other highly efficacious DMTs (##REF##33090003##Samjoo et al, 2020##; ##REF##33878488##Liu et al, 2021##). Therefore, more recent findings support the results of the Cochrane review regarding the efficacy of Natalizumab and Alemtuzumab and go on to suggest that other DMTs, such as Ocrelizumab and Ofatumumab, may also be beneficial for reducing relapses and disability progression.</p>", "<p id=\"P17\">It should be noted that since the Cochrane review was published, Alemtuzumab has been reported by the European Medicines Agency to have rare but serious side effects, including cardiovascular and immune-related disorders, leading to restrictions on its use. Healthcare professionals are now advised that ‘<italic toggle=\"yes\">Lemtrada (alemtuzumab) should only be used to treat relapsing-remitting multiple sclerosis if the disease is highly active despite treatment with at least one disease-modifying therapy or if the disease is worsening rapidly. Lemtrada must also no longer be used in patients with certain heart, circulation or bleeding disorders or in patients who have autoimmune disorders other than multiple sclerosis</italic> (##UREF##0##European Medicines Agency, 2020##). Therefore, recommendations for the use of specific DMTs should be based on an individualised approach to the benefits and harms for each patient, referring to ##UREF##3##National Institute for Health and Care Excellence (2022a)## guidelines and ##UREF##4##National Institute for Health and Care Excellence (2022b)## technology appraisal guidance for individual products.</p>", "<p id=\"P18\">When deciding on the use of DMTs, it is essential to consider all the relevant variables including efficacy, safety information (as described above), contraindications and how well patients will adhere to taking the therapy. In addition to individual contraindications, it has also been noted that live vaccinations may be contraindicated in people with MS who are being treated with DMTs and, thus, should be discussed with the patient (##UREF##3##National Institute for Health and Care Excellence, 2022a##). Medication adherence is also relevant to the decision-making process as adverse reactions, intolerance and disease activity ultimately result in treatment withdrawal (##REF##30590239##Yoon and Cheong, 2019##). A study of medication adherence among patients with MS also identified that adherence to treatment was better with oral DMTs when compared with injectable DMTs (##REF##32559310##Lahdenperä et al. 2020##).</p>", "<p id=\"P19\">A number of DMTs have been identified as beneficial for reducing the recurrence of relapses in RRMS. Most of the comparisons within this network meta-analysis however were based on a low number of trials and as such the results should be interpreted with caution. As the treatment of RRMS can be given for multiple decades, it is important that future trials of DMTs should ensure participants are followed up long-term. Given the limited evidence available for serious adverse events in this analysis, future trials should also ensure that a clear and transparent safety profile is captured and presented clearly.</p>" ]
[ "<title>Acknowledgement</title>", "<p>This report is independent research partly-funded by the National Institute for Health and Care Research Applied Research Collaboration North West Coast (NIHR ARC NWC). The views expressed in this publication are those of the author(s) and not necessarily those of the National Institute for Health and Care Research, the NHS, or the Department of Health and Social Care.</p>" ]
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[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Most effective therapies against the recurrence of relapses in relapsing-remitting multiple sclerosis during the first 12 months of treatment versus placebo</title></caption><table frame=\"box\" rules=\"all\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ranking of effectiveness</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunomodulator/immunosuppressant</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Risk ratio 95% confidence interval (CI)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surface under the cumulative ranking curve</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Confidence in the evidence</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alemtuzumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.40, 95% CI: 0.31-0.51</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">97%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mitoxantrone</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.40, 95% CI: 0.20-0.76</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">93%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Natalizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.56, 95% CI: 0.43-0.73</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">85%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fingolimod</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.63, 95% CI: 0.53-0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">80%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Most effective therapies against the recurrence of relapses in relapsing-remitting multiple sclerosis during the first 24 months of treatment versus placebo</title></caption><table frame=\"box\" rules=\"all\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ranking of effectiveness</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunomodulator/immunosuppressant</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Risk ratio 95% confidence interval (CI)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surface under the cumulative ranking curve</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Confidence in the evidence</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alemtuzumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.46, 95% CI: 0.38-0.55.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">96%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mitoxantrone</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.47, 95% CI: 0.27-0.81</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">92%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Very low</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Natalizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.56, 95% CI: 0.47-0.66</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">88%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fingolimod</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.72, 95% CI: 0.64-0.81</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">71%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><title>Most effective therapies against worsening disability during the first 24 months of treatment</title></caption><table frame=\"box\" rules=\"all\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ranking of effectiveness</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunomodulator/immunosuppressant</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Risk ratio 95% confidence interval (CI)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surface under the cumulative ranking curve</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Confidence in the evidence</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mitoxantrone</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.20, 95% CI: 0.05-0.84</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">96%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alemtuzumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.35, 95% CI: 0.26-0.48</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">94%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Natalizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.64, 95% CI: 0.49-0.85</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">74%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">moderate</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Azathioprine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.64 95% CI: 0.30-1.37</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">64%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Very low</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><title>Proportion of patients who withdrew from treatment (acceptability) because of an adverse event compared to placebo over 12 months</title></caption><table frame=\"box\" rules=\"all\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ranking of risk (1=greatest risk)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunomodulator/immunosuppressant</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Risk ratio 95% confidence interval (CI)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fingolimod</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.26 95% CI: 3.25-20.97</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interferon beta-1a (Rebif)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.83 95% CI: 2.59-9.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interferon beta-1a (Avonex)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.36 95% CI: 1.98-9.60</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peg-interferon beta 1a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.80 95% CI: 1.39-5.64</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Teriflunomide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.24 95% CI: 1.50-3.34</td></tr></tbody></table></table-wrap>" ]
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[ "<boxed-text id=\"BX1\" position=\"float\"><caption><title>Key points</title></caption><list list-type=\"bullet\" id=\"L1\"><list-item><p>The most effective disease modifying therapies for preventing relapses in relapse-remitting multiple sclerosis, based on moderate to high evidence, are alemtuzumab, natalizumab and fingolimod.</p></list-item><list-item><p>Reports of rare but serious side effects of alemtuzumab have led to restrictions on its use.</p></list-item><list-item><p>There is inconsistent quality of evidence to state which therapies are most effective in preventing disability worsening.</p></list-item><list-item><p>Several therapies had a significantly higher risk of treatment withdrawal because of an adverse event at 12 months.</p></list-item><list-item><p>Safety information should always be considered alongside efficacy when determining the benefits and harm to an individual patient.</p></list-item></list></boxed-text>" ]
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[{"collab": ["European Medicines Agency"], "source": ["Measures to minimise risk of serious side effects of multiple sclerosis medicine Lemtrada"], "year": ["2020"], "date-in-citation": ["accessed 12 July 2022"], "comment": ["\n"], "ext-link": ["https://www.ema.europa.eu/en/documents/referral/lemtrada-article-20-procedure-measures-minimise-risk-serious-side-effects-multiple-sclerosis_en-0.pdf"]}, {"collab": ["MS Society"], "source": ["MS in the UK"], "year": ["2020"], "date-in-citation": ["accessed 21 July 2022"], "comment": ["\n"], "ext-link": ["https://www.mssociety.org.uk/sites/default/files/2020-08/MS-in-the-UK_2020.pdf"]}, {"collab": ["National Institute for Health and Care Excellence"], "source": ["Multiple sclerosis: quality standard"], "year": ["2016"], "date-in-citation": ["accessed 12 July 2022"], "comment": ["\n"], "ext-link": ["https://www.nice.org.uk/guidance/qs108/resources/multiple-sclerosis-pdf-75545244362437"]}, {"collab": ["National Institute for Health and Care Excellence"], "source": ["Multiple sclerosis in adults: management"], "year": ["2022a"], "date-in-citation": ["accessed 21 July 2022"], "comment": ["\n"], "ext-link": ["https://www.nice.org.uk/guidance/ng220"]}, {"collab": ["National Institute for Health and Care Excellence"], "source": ["Multiple Sclerosis Products"], "year": ["2022b"], "date-in-citation": ["accessed 21 July 2022"], "publisher-name": ["National Institute for Health and Care Excellence"], "comment": ["\n"], "ext-link": ["https://www.nice.org.uk/guidance/conditions-and-diseases/neurological-conditions/multiple-sclerosis/products?GuidanceProgramme=TA"]}, {"collab": ["National MS Society"], "source": ["Relapsing-remitting MS (RRMS)"], "year": ["2022"], "date-in-citation": ["accessed 21 July 2022"], "comment": ["\n"], "ext-link": ["https://www.nationalmssociety.org/What-is-MS/Types-of-MS/Relapsing-remitting-MS"]}]
{ "acronym": [], "definition": [] }
20
CC BY
no
2024-01-13 00:12:45
Br J Neurosci Nurs. 2022 Jul 1; 18(Sup3):S16-S19
oa_package/b8/5f/PMC7615514.tar.gz
PMC7615515
38214012
[ "<title>Introduction</title>", "<p id=\"P2\">The identification of the small ubiquitin-like protein Atg8 as a core autophagy protein came from early studies of essential autophagy-related (ATG) proteins in the budding yeast <italic toggle=\"yes\">Saccharomyces cerevisiae</italic> [##REF##24366340##1##]. Autophagosome formation is induced by the activation of two kinase complexes, i. e. Atg1/ULK (unc-51 like autophagy activation kinase 1) and PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type-3), and it also depends on Atg9-containing vesicles [##UREF##0##2##]. Their activation results in a local formation of phosphatidylinositol-3-phosphate (PI3P) at a pre-autophagosomal structure or phagophore-assembly site (PAS) close to ER, and a double membrane structure named phagophore, or isolation membrane, is formed. A complex containing Atg18/WIPI and Atg2 is recruited to mediate phagophore expansion directly by the lipid transferase activity of Atg2 [##REF##31271352##3##], and to aid Atg9 vesicles in the delivery of phospholipids from the ER to the phagophore. ATG9A promotes phagophore expansion by acting as a lipid scramblase [##REF##33106659##4##,##REF##33106658##5##]. Autophagosome formation also relies on two different conjugation systems. The ubiquitin like protein Atg12 is conjugated to Atg5, and the Atg12-Atg5 forms a complex with Atg16 that is recruited to PAS. This complex acts as an E3 ligase for the conjugation of Atg8 to the phagophore membrane. Atg7 is the E1 in both conjugation reactions, while Atg10 and Atg3 works as E2 for Atg12 and Atg8, respectively [##REF##31901645##6##]. On the phagophore membrane, Atg8 acts as a scaffold for the recruitment of core autophagy proteins to the phagophore and this facilitates phagophore expansion. A closure of the expanded phagophore results in autophagosome formation, and cytoplasmic material engulfed by the autophagosome is degraded upon fusion of the autophagosome with a lysosome [##UREF##0##2##,##REF##32372019##7##].</p>", "<p id=\"P3\">The role of Atg8 in macroautophagy is evolutionary conserved, but the number of Atg8 orthologues vary between species from a single family member in yeast to more than 10 variants in higher plants. In metazoans, the family members are divided into two subgroups, i. e. microtubule-associated protein 1 light chain 3 (LC3) and γ-amino-butyric acid receptor-associated protein (GABARAP). The GABARAP subgroup is evolutionary conserved, while the LC3 subgroup is metazoan specific [##REF##31901645##6##,##REF##21867568##8##]. Atg8 orthologues are in all species produced as a pro-form lacking a C-terminal glycine residue needed for the conjugation reaction. The pro-form (pro-Atg8) is activated by proteolytic cleavage by cysteine proteases of the Atg4 family. The resulting I form with a C-terminal glycine residue exposed can then be lipidated to phosphatidylethanolamine (PE) on the outer membrane surface of the phagophore membrane, and this creates the II form that is an adaptor for the recruitment of other proteins involved in phagophore expansion, including other core autophagy proteins. The presence of Atg8 proteins is believed to be most important at the rim of the phagophore since this is where phagophore growth occurs. The closure of the expanded phagophore correlates with a partial de-lipidation of the Atg8 coat by Atg4 family proteases [##REF##31901645##6##]. However, some Atg8 proteins remain on the completed autophagosome to facilitate recruitment of proteins involved in transport or fusion of the autophagosome with a lysosome. Atg8 proteins are also lipidated to the inner membrane surface of the phagophore where they act as adaptors for the binding of selective autophagy receptors (SARs)[##REF##31310766##9##]. In selective autophagy, a cargo selected for degradation by autophagy must first be identified and bound by a SAR. Different types of SARs have been identified that associate with different types of cargos, but a common feature is the direct binding of the SAR to Atg8 proteins attached to the inner membrane surface of the growing phagophore. This way, the SAR is responsible for the tight docking of the selected cargo to the inner phagophore membrane [##REF##31310766##9##].</p>", "<p id=\"P4\">Most studies on Atg8 proteins have focused on their role(s) in macroautophagy, but several recent studies have shown that Atg8 proteins can be lipidated to single membranes to participate in non-conventional autophagy pathways, secretory autophagy, or endocytic processes like LC3-associated phagocytosis (LAP), entosis, micropinocytosis or endocytosis. The term “atg8ylation” has been coined to emphasize the analogy to ubiquitin since proteins can be atg8ylated by being covalently bound to the C-terminal glycine of Atg8 proteins [##UREF##1##10##], and ubiquitin can be conjugated to PE [##REF##36044902##11##]. Atg8ylation is used as a response to membrane stress and membrane remodeling activities both in autophagy and in other processes involving atg8ylation to single membranes including lysosomal damage responses [##UREF##1##10##,##UREF##2##12##]. There is also increasing evidence for that unlipidated Atg8 proteins may have functional roles, including reported nuclear roles displayed by nuclear pools of these proteins [##REF##26524528##13##–##REF##32989246##16##]. Of note, a mammalian Atg8 protein, GABARAPL2 (also known as GATE-16), was identified first as a SNARE-interacting protein, before its engagement in autophagy became appreciated. GABARAPL2, in association with the Golgi SNARE GOSR1 (GOS-28) and the SNARE complex-unfolding protein NSF [##REF##9446628##17##–##UREF##3##20##], controls intra-Golgi transport, protein trafficking to the plasma membrane, and post-mitotic Golgi reassembly. Expanding upon this early work, recent studies show direct interactions of all LC3 and GABARAP proteins with an assortment of other SNARE proteins, including SNAREs that regulate autophagosome and lysosome biogenesis [##REF##31625181##21##,##REF##29420192##22##].</p>", "<p id=\"P5\">The aim of this review is to summarize the knowledge we have on binding motifs and interaction surfaces utilized by Atg8 orthologues and interacting proteins. Most characterized interactions depend on a small sequence motif in the interacting protein that was initially named LC3 interacting region (LIR) following its discovery in human p62/SQSTM1 [##REF##17580304##23##]. Later studies revealed that this motif is evolutionary conserved, and often referred to as Atg8 interacting motif (AIM) in yeast, fungi and plants [##REF##20083108##24##]. The LIR/AIM motif docks into a so-called LIR docking site (LDS) that is conserved in all Atg8 orthologues. An overwhelming majority of known interactions with Atg8 proteins involve LIR motifs [##REF##31310766##9##]. The LIR-LDS interaction will therefore be discussed in detail in this review, but we will also discuss other known interactions relevant for the function of Atg8 proteins.</p>" ]
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[ "<title>Conclusions - future perspectives</title>", "<p id=\"P54\">Since the discovery of the LIR/AIM motifs a large number of Atg8-interacting proteins have been identified in all model organisms used for autophagy studies as well as in humans. The approach of identifying Atg8-interacting proteins has had a major impact on autophagy research particularly in studies of selective autophagy. We have learned a lot on the nature of the LIR-LDS interactions from structural studies combined with mutagenesis and protein-protein interaction analyses. New tools for predicting LIR-Atg8 interactions have emerged along with databases of LIR motifs and LIR-containing proteins in many species. This will aid in a more rapid discovery of remaining unidentified LIR-containing proteins, also those containing non-canonical motifs. The power in combining artificial intelligence aided 3D modeling and docking with evolutionary conservation and binding analyses using mutants in the LIR motifs and in the LDS will aid both in discovery and validation of new LIR-containing proteins. Other interaction modes, are beginning to be described. Hence, it will be important to obtain structural data on interactions involving the UDS and the N-terminal arm to understand these better. This will likely aid us in identifying new Atg8-interacting proteins that use these interaction surfaces. We will likely also discover more combined motifs as already exemplified by the UFIM and Atg8 interaction motifs found in UBA5 and C53.</p>" ]
[ "<p id=\"P1\">The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, <italic toggle=\"yes\">Arabidopsis, C. elegans</italic> and <italic toggle=\"yes\">Drosophila</italic> to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like “half-LIRs” and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.</p>" ]
[ "<title>Yeast Atg8</title>", "<p id=\"P6\">As mentioned above, Atg8 was first identified in <italic toggle=\"yes\">S. cerevisiae</italic> as a protein required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway [##REF##14536056##25##]. Atg8 has in its core a ubiquitin-like fold with an N-terminal extension (##FIG##0##Figure 1A##). In this organism, Atg8 is synthesized as a proform with a single arginine (Arg117) at the C terminus, which is immediately removed by the cysteine proteinase Atg4 [##REF##11038174##26##]. The newly exposed C-terminal glycine (Gly116) is activated by the E1 enzyme Atg7 with consumption of ATP and forms a thioester bond with the cysteine residue (Cys507) of Atg7. Then, the Gly116 of Atg8 is transferred to the cysteine residue (Cys234) of the E2 enzyme Atg3 and finally forms an amide bond with the amino group in the hydrophilic head of PE [##REF##11100732##27##]. In this final step of Atg8 lipidation, the Atg12-Atg5-Atg16 complex serves as an E3 enzyme, which stimulates the transfer of Atg8 from Atg3 to PE and thereby confines the reaction to the PAS and phagophore where the complex localizes [##REF##17986448##28##–##REF##11689437##30##]. Atg8 conjugation to PE is reversible; the amide bond in Atg8-PE is cleaved by Atg4, releasing Atg8 from membranes [##REF##11038174##26##]. This deconjugation reaction maintains a reservoir of unlipidated Atg8 and promotes autophagosome formation [##REF##28704456##31##–##REF##22652539##34##].</p>", "<p id=\"P7\">The molecular functions of Atg8, especially its lipidated form, have been studied using in vitro reconstitution system [##REF##24485455##35##–##REF##23064152##40##]. Initial reconstitution experiments were performed using Atg7, Atg3, processed form of Atg8, small unilamellar vesicles (SUVs) containing high content of PE, and MgATP, which were shown to be sufficient for Atg8-PE formation <italic toggle=\"yes\">in vitro</italic> [##REF##15277523##41##]. Later, addition of the Atg12-Atg5 conjugate to this reconstitution system was shown to dramatically accelerate Atg8-PE formation, even when using SUVs with physiological content of PE, establishing that the Atg12-Atg5 conjugate is the E3 enzyme for the Atg8 system [##REF##17986448##28##]. In the reconstitution system, progression of lipidation reaction caused multimerization of Atg8, which induced tethering and hemi-fusion of SUVs to each other. Moreover, mutations inhibiting these Atg8 activities partially or completely resulted in generation of smaller or no autophagosomes in yeast, respectively. These observations suggested that Atg8-PE possesses membrane tethering and hemi-fusion activities proposed to represent its function in autophagosomal membrane expansion [##REF##17632063##39##]. When giant unilamellar vesicles (GUVs) were used instead of SUVs, the Atg12-Atg5 conjugate required Atg16 to function as the E3 enzyme for Atg8-PE formation as observed <italic toggle=\"yes\">in vivo</italic>, confirming that Atg16 is also a critical component of the E3 enzyme of the Atg8 system [##REF##23064152##40##]. Monitoring the time-course of GUV targeting revealed that Atg8-PE on GUVs has the activity to recruit the Atg12-Atg5-Atg16 complex to GUVs, which further promotes Atg8-PE formation as a positive feedback loop [##REF##24485455##35##]. In addition to the monitoring of protein localization, utilization of GUVs enabled the monitoring of membrane morphology upon Atg8-PE formation. Formation of Atg8-PE caused membrane tubulation in addition to tethering of spherical GUVs [##REF##25522362##36##]. When non-spherical, prolate GUVs were used, Atg8-PE caused dramatic shape change of prolate GUVs into a sphere without-bud. This shape change was dependent on the membrane-perturbation activity of Atg8-PE to increase area difference between outer and inner leaflets of GUVs. The mutations that impaired this <italic toggle=\"yes\">in vitro</italic> activity resulted in smaller and fewer autophagosomes in yeast, indicating its importance in autophagosome formation [##REF##34239122##37##].</p>", "<p id=\"P8\">Besides its lipidation-dependent roles in autophagy, Atg8 is known to play lipidation-independent roles that are not related to autophagy. In yeasts such as <italic toggle=\"yes\">S. cerevisiae, Schizosaccharomyces pombe</italic>, and <italic toggle=\"yes\">Komagataella phaffii</italic>, Atg8 plays a critical role in maintaining the vacuole morphology independently of conjugating enzymes [##UREF##4##42##–##REF##21045113##44##]. In <italic toggle=\"yes\">S. cerevisiae</italic> and <italic toggle=\"yes\">S. pombe</italic>, Atg8 was recruited to the vacuole via interaction with the vacuolar membrane protein Hfl1 and they collaboratively contributed to the fragmented vacuolar morphology under stress such as dithiothreitol treatment [##UREF##4##42##]. This non-autophagic role was shown to require the membrane perturbation activity of Atg8 [##REF##34239122##37##]. In <italic toggle=\"yes\">Drosophila melanogaster</italic> lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. Additionally, high expression of non-lipidated Atg8b in the male germline is required for fertility (see also the section on <xref rid=\"S5\" ref-type=\"sec\">Atg8s in <italic toggle=\"yes\">Drosophila melanogaster</italic></xref>)[##REF##33249988##45##].</p>", "<p id=\"P9\">In addition to autophagosome formation, Atg8 also plays an important role in cargo sequestration into autophagosomes during selective autophagy in yeasts as well as other organisms. In <italic toggle=\"yes\">S. cerevisiae</italic>, Atg8 binds to the AIMs/LIRs in the autophagy receptors Atg19, Atg34, Atg32, Atg36, Atg39, Atg40, and Cue5 [##REF##19061865##46##–##REF##21343297##55##]. While Atg19 recognizes vacuolar enzymes (Cvt cargos), α-mannosidase and Ty1 virus-like particles, Atg34 specifically interacts with α-mannosidase. Atg32, Atg36, Atg39 and Atg40 localize to mitochondria, peroxisomes, the nucleus, the endoplasmic reticulum (ER), and initiate autophagic degradation of these organelles, respectively. Cue5 mediates sequestration of polyQ proteins and inactive proteasomes[##REF##19061865##46##–##REF##21343297##55##]. In selective autophagy of peroxisomes (pexophagy) in <italic toggle=\"yes\">K. phaffii</italic> and <italic toggle=\"yes\">Candida boidinii</italic>, Atg8 cooperates with the pexophagy receptor Atg30 [##REF##18331717##56##,##REF##21966472##57##]. In <italic toggle=\"yes\">S. pombe</italic>, selective sequestration of mitochondria and the ER into autophagosomes is mediated by the interaction of Atg8 with Atg43 and Epr1, respectively [##UREF##5##58##,##REF##32735772##59##]. <italic toggle=\"yes\">S. cerevisiae</italic> Atg8 also directly (not via autophagy receptors) binds to selective autophagy cargos such as the endocytic protein Ede1, fatty acid synthase, and nuclear pore complexes [##REF##32029894##60##–##REF##33207182##63##].</p>", "<p id=\"P10\">In <italic toggle=\"yes\">S. cerevisiae</italic>, the expression of Atg8 was shown to be transcriptionally upregulated upon nitrogen starvation or the inactivation of mechanistic target of rapamycin complex 1 (mTORC1) [##REF##22733735##64##,##REF##10525546##65##]. The Rpd3-Sin3-Ume6 histone deacetylase complex binds to the promoter region of the <italic toggle=\"yes\">ATG8</italic> gene to repress its transcription under nutrient rich conditions. A similar mechanism is likely to work in mammalian cells [##REF##22733735##64##]. Increased levels of Atg8 result in an increase in the size of autophagosomes [##REF##22733735##64##,##REF##18508918##66##].</p>", "<title>Plant Atg8s</title>", "<p id=\"P11\">Like the yeast Atg8 orthologue, plant Atg8s also contain a ubiquitin fold, the N-terminal extension, and the C-terminal glycine that serves as a recognition site for Atg4 protease [##REF##27458016##67##]. However, unlike yeast, plants have multiple Atg8 homologs (##FIG##0##Figure 1B## and ##FIG##1##Figure 2##), ranging from 2 homologs in the emerging model system <italic toggle=\"yes\">Marchantia polymorpha</italic> to 9 homologs in <italic toggle=\"yes\">Arabidopsis thaliana</italic> and up to 22 homologs in the <italic toggle=\"yes\">Arabidopsis</italic> relative plant <italic toggle=\"yes\">Capsella rubella</italic> and <italic toggle=\"yes\">Brassica napus</italic> [##REF##28038982##68##,##REF##31379911##69##]. These homologs form two well-supported evolutionary clades as seen for the <italic toggle=\"yes\">Arabidopsis</italic> Atg8s in ##FIG##0##Figure 1B##, where Clade I is closely related to fungal Atg8s, whereas Clade II groups, represented by AtATG8i and -h, cluster together with metazoan Atg8 orthologues [##REF##28038982##68##]. Surprisingly, some of the homologs in Clade II lack any residues after the C-terminal glycine (##FIG##1##Figure 2##), but they still associate with Atg4, consistent with recent finding suggesting that Atg4 can promote autophagosome formation, independent of its protease activity [##REF##33773106##70##–##REF##31329577##72##]. Another interesting feature of plant Atg8 phylogenetic tree is the formation of family specific Atg8 subclades. For example, <italic toggle=\"yes\">Brassicaceae</italic> family that contains <italic toggle=\"yes\">Arabidopsis</italic> forms 9 monophyletic subclades, whereas <italic toggle=\"yes\">Poaceae</italic> family that contains wheat forms 4 subclades. Each subclade contains fixed polymorphisms, suggesting they maybe functionally diversified [##REF##28038982##68##].</p>", "<p id=\"P12\">Similar to the metazoan Atg8 homologs, recent studies have shown that plant Atg8s are also functionally specialized [##REF##31329577##72##,##REF##33249982##73##]. Domain swap analysis and interactome studies performed using potato Atg8s have shown that the N terminal β-strand that forms a part of the hydrophobic pocket 1 (HP1) at the LIR docking site (see “LIR-LDS interaction” below) underpins specialization towards a pathogen effector protein and plant proteins. Further genetic studies are necessary to show if Clade I or Clade II Atg8 homologs have specific functions in macroautophagy or non-canonical forms of autophagy.</p>", "<title>Atg8s in <italic toggle=\"yes\">Caenorhabditis elegans</italic></title>", "<p id=\"P13\"><italic toggle=\"yes\">C. elegans</italic> contains two Atg8 homologs, LGG-1 and LGG-2, that have differential functions in autophagy. They are also structurally different. LGG-1 is similar to the GABARAP subfamily and LGG-2 more similar to the LC3 subfamily (##FIG##0##Figure 1B##) [##REF##26687600##74##]. LGG-1 is synthesized as a 123-amino acid precursor, whose C-terminal seven amino acids are cleaved by ATG-4 to expose the glycine for PE conjugation. In embryonic extracts, the unlipidated processed form (LGG-1-I) and the lipidated processed form (LGG-1-II) are detected, while the LGG-1 precursor is absent [##REF##22767594##75##]. The LGG-1 precursor is processed by the two Atg4 homologs in <italic toggle=\"yes\">C. elegans</italic>, ATG-4.1 and ATG-4.2. ATG-4.1 cleaves LGG-1 precursors about 100-fold more efficiently than ATG-4.2 in <italic toggle=\"yes\">in vitro</italic> cleavage assays [##REF##22767594##75##]. Compared to wild-type animals, LGG-1 is properly processed and lipidated in <italic toggle=\"yes\">atg-4.2</italic> mutants, while in <italic toggle=\"yes\">atg-4.1</italic> mutants, LGG-1 precursors accumulate dramatically, lipidated LGG-1-II is present at a similar level, but unlipidated LGG-1-I is absent [##REF##22767594##75##]. In LGG-2, the glycine for conjugation is directly exposed (##FIG##1##Figure 2##). However, the unlipidated form of LGG-2 is predominant in embryonic extracts [##REF##26687600##74##].</p>", "<p id=\"P14\">LGG-1 and LGG-2 form spatiotemporally dynamic punctate structures during <italic toggle=\"yes\">C. elegans</italic> embryogenesis. Loss of function of autophagy genes acting at different steps of the autophagy pathway results in characteristic levels of lipidated LGG-1 and formation of LGG-1 puncta. In mutants of the conjugation systems, including <italic toggle=\"yes\">atg-3, atg-7, atg-5</italic> and the <italic toggle=\"yes\">atg-4.1; atg-4.2</italic> double mutant, the lipidated forms of LGG-1 and LGG-2 are not detected and formation of LGG-1 and LGG-2 puncta is abolished [##REF##22767594##75##,##REF##20550938##76##]. ATG-16 is not required for LGG-1 lipidation, but is essential for formation of LGG-1 puncta in <italic toggle=\"yes\">C. elegans</italic> [##REF##24185444##77##]. In UNC-51/EPG-1/EPG-9 Atg1 complex mutant embryos, LGG-1-I accumulates and LGG-1 puncta are largely absent except in a few cells which contain large aggregates [##REF##22885670##78##]. In <italic toggle=\"yes\">epg-8</italic> (encoding <italic toggle=\"yes\">C. elegans</italic> Atg14 homolog) or <italic toggle=\"yes\">bec-1</italic> mutants, levels of LGG-1-I and LGG-1-II are elevated but LGG-1 puncta are weaker [##REF##21116129##79##]. <italic toggle=\"yes\">atg-9</italic> mutant embryos have larger but fewer LGG-1 puncta than wild-type embryos [##REF##23530068##80##]. In loss-of-function mutants of genes downstream of autophagosome initiation, including <italic toggle=\"yes\">epg-3, epg-4, epg-6, atg-2</italic> and <italic toggle=\"yes\">epg-5</italic>, lipidated LGG-1 and LGG-1 puncta accumulate [##REF##20550938##76##,##REF##21802374##81##]. <italic toggle=\"yes\">lgg-2</italic> mutants exhibit a wild-type pattern of LGG-1 punctum formation, while loss of <italic toggle=\"yes\">lgg-1</italic> dramatically increases the number of LGG-2 puncta [##REF##26687600##74##]. Modulating the lipidation of one Atg8 homolog by another provides a novel mechanism for regulating their differential functions in the pathway.</p>", "<p id=\"P15\">LGG-1 and LGG-2 differentially bind to autophagy cargos such as SQST-1, SEPA-1 and AIN-1 [##REF##26687600##74##]. The scaffold proteins EPG-7 and EPG-2, which mediate degradation of SQST-1 and PGL granules, respectively, bind strongly to LGG-1, but show no or weak interaction to LGG-2 [##REF##26687600##74##]. The Atg1 complex components UNC-51 and EPG-1 preferentially interact with LGG-1, while LGG-2, but not LGG-1, directly interacts with the LGG-3/ATG-5/ATG-16 complex [##REF##26687600##74##]. EPG-5, which acts as a tethering factor to promote fusion of autophagosomes with late endosomes/lysosomes, directly interacts with LGG-1 [##REF##27588602##82##]. Their differential interactions with autophagy proteins may contribute to hierarchical recruitment of Atg proteins in the autophagy pathway.</p>", "<title>Atg8s in <italic toggle=\"yes\">Drosophila melanogaster</italic></title>", "<p id=\"P16\">Contrary to the significantly more extended and diverse family of ubiquitin-like ATG8 proteins in mammals, the <italic toggle=\"yes\">Drosophila</italic> Atg8 group is represented by two members: Atg8a and Atg8b. Both are structurally more similar to GABARAP than LC3, they are upregulated in response to autophagy induction, colocalize to autophagosomes and share at least some degree of redundancy as seen from loss-of-function alleles that present milder autophagy-mutant phenotypes than otherwise expected for loss of this central Atg protein [##REF##18083103##83##–##REF##15296714##86##]. Despite these similarities, the Atg8a isoform shows the more ubiquitous expression, while Atg8b is mostly relegated to the adult male testis [##REF##33249988##45##]. As such, the bulk of assays that monitor autophagy in <italic toggle=\"yes\">Drosophila</italic>, use Atg8a as the reference marker [##REF##27557573##87##–##REF##20713604##89##]. In terms of ontology, Atg8b most probably originated from a retrotransposition event of Atg8a during the emergence of fruit flies, as it lacks the intron regions of the latter [##REF##12466289##90##]. The tissue-specific enrichment of Atg8b in the testis supports this theory, as it is in line with how the process of late spermatogenesis and inactivation of the X chromosome, that occurs in both <italic toggle=\"yes\">Drosophila</italic> and mammals, can result in the generation of autosomal retrogenes from X-linked genes (such as Atg8a) with male germline-specific localization [##REF##12466289##90##]. Closely related to the process of spermatogenesis, in the fruit fly male testis, Atg8b has mostly foregone its critical requirement in autophagy for a non-autophagic, lipidation-independent role, as it is essential for viable sperm production and regulation of male fertility [##REF##33249988##45##]. Of note here, in this setting, overexpression of Atg8a in Atg8b-null mutants was able to restore male fertility, suggesting that both fly Atg8 proteins can mediate their non-autophagic effects with regards to spermatogenesis [##REF##33249988##45##].</p>", "<p id=\"P17\">An added layer of complexity in the mammalian system is that the LC3 and GABAPAP subfamilies have further evolved to perform distinct functions in autophagy and by extension their members are encountered at different stages of the process [##REF##20418806##91##]. In contrast Atg8a, is found throughout all stages of autophagy in <italic toggle=\"yes\">Drosophila</italic>, which can at times simplify the monitoring of autophagy in this model organism compared to others.</p>", "<title>Human, mammalian and other metazoan Atg8s</title>", "<p id=\"P18\">Humans express seven ATG8 orthologues of which three are in the GABARAP subfamily. This subfamily is again separated into two subgroups, i. e. one consisting of GABARAP and GABARAPL1 and the other by GABARAPL2. The LC3 subfamily is similarly divided into two subgroups, i. e. one consisting of LC3A (two splice-isoforms with differing N-terminals), LC3B and LC3B2, and one consisting of LC3C (##FIG##0##Figure 1B##). Intriguingly, rodents lack LC3C [##UREF##7##92##]. All human ATG8s are formed as precursors that must be cleaved by ATG4 to expose the glycine for PE lipidation. Four different ATG4 cysteine proteases (A-D) exist in human cells, and they differ in specificity related to the processing or delipidation of human ATG8 orthologues. The binding between ATG4 and ATG8 orthologues is interesting since it involves two different interactions. One involves the catalytic domain in ATG4 [##REF##19322194##93##], and the other a LIR motif at the C-terminus of ATG4 [##REF##28287329##94##]. A number of other human core autophagy proteins also have a LIR motif. Unlike the motif in ATG4B which has a broad binding specificity, the motifs in other core autophagy proteins have a binding preference for GABARAP and GABARAPL1 [##REF##23043107##95##–##REF##31053714##100##], suggesting an important role for this subgroup of ATG8s in autophagosome formation. This is also the subgroup that has the highest similarity to protist and yeast Atg8s. Hence, it comes as no surprise that this subgroup is important for autophagosome formation. CRISPR/CAS9 knockout studies show that autophagosomes are formed even without mammalian Atg8s, although the autophagy is very inefficient, and LC3/GABARAP proteins are needed for efficient autophagosome-lysosome fusion [##REF##27864321##101##]. Rescue experiments underscored the importance of the GABARAP subfamily for selective autophagy and starvation induced autophagy [##REF##27864321##101##,##REF##29038162##102##]. Knock down studies suggested the LC3 subfamily to be dispensable, while the GABARAP subfamily is required for bulk autophagy [##REF##25684710##103##]. The LC3 subfamily is suggested to be more important as an adaptor for the docking of cargos to the phagophore membrane in selective autophagy, and it may also be involved in the transport of endosomes and autophagosomes whereas GABARAPs are involved in fusion between autophagosomes and lysosomes. However, the interplay between the different human ATG8s is complex and remains poorly understood, and there may exist functional redundancy between them [##REF##31310766##9##].</p>", "<p id=\"P19\">During evolution, the four unique Atg8 subgroups in mammals appear to be derived from an early metazoan lineage split of Atg8 orthologues. For example, a primitive animal like Hydra, a freshwater <italic toggle=\"yes\">Hydrozoa</italic> of the <italic toggle=\"yes\">Cnidarium</italic> phylum, has four Atg8 orthologues that are homologous with each of the subgroups found in mammals, i. e. GABARAP, GABARAPL2, LC3A/B and LC3C [##UREF##8##104##]. A similar conservation of Atg8 subgroups is not seen throughout the metazoans. Due to divergent evolution, two important model organisms, i. e. the fruit fly <italic toggle=\"yes\">Drosophila</italic> and the nematode <italic toggle=\"yes\">C. elegans</italic> have retained only two Atg8 homologs representing only one and two of the subgroups, respectively [##REF##33249988##45##].</p>", "<title>The LIR – LDS interaction</title>", "<p id=\"P20\">The LIR/AIM motif binds to LDS and is universally used for Atg8 protein interactions throughout eukaryotic evolution. To describe the LIR/AIM-LDS interaction, we will initially focus on mammals and yeast since the vast majority of identified LIRs are from mammals and most structures are on mammalian and yeast LIR/AIM-LDS interactions.</p>", "<p id=\"P21\">The LIR in mammals and AIM in yeast were described in pioneering biochemical [##REF##17580304##23##] and structural [##REF##18524774##105##,##REF##19021777##106##] works, as short polypeptide sequences containing ~10 residues. Early structural studies revealed that the core LIR/AIM (LIR hereafter) sequence contains a W-X-X-L motif (where X is any residue). The LIR polypeptide of p62/SQSTM1, for instance, adopts a β-stranded conformation, forming an intermolecular parallel β-sheet with the β-strand β2 of LC3B, while the sidechains of W and L residues occupy the two hydrophobic pockets - HP1 (also known as W-site) and HP2 (L-site) - on LC3B surface, stabilizing the complex (##FIG##2##Figure 3A##). Extensive studies in past years provided a more general core consensus, which can be described as Θ<sub>0</sub>-X<sub>1</sub>-X<sub>2</sub>-Γ<sub>3</sub>(or positions 0, +1, +2 and +3), where Θ is an aromatic (W/F/Y) and Γ is a hydrophobic (L/I/V) residue (##FIG##2##Figure 3B##). Investigations of the residues which could occupy the Θ and Γ positions (either by analyzing the sequences of the hitherto known canonical LIR motifs [##REF##31310766##9##] or by mutational 2D peptide arrays [##REF##23043107##95##,##REF##30767700##96##,##REF##31053714##100##,##REF##28253953##107##] revealed a very high conservation of the three aromatic residues in Θ. As expected from the hydrophobicity profile of HP1, a much higher abundance of solely non-polar Trp and Phe was observed in the native canonical LIRs. In contrast, partially polar Tyr residues are found in only a minority of canonical LIR motifs. Trp is the most energetically favorable residue for the Θ position. Mutation of the Tyr732 to a Trp increases the NBR1 LIR affinity to GABARAPL1 8-fold, while the Y732F mutant showed the same affinity [##REF##21620860##108##]. The Phe-containing OPTN LIR shows an 8-fold increase in affinity to LC3B when Phe178 is substituted to a Trp [##REF##23805866##109##]. Of note, the lower affinity of Tyr- and Phe-containing canonical LIR motifs in both aforementioned cases might be associated with the ability of NBR1 and OPTN to regulate autophagic functions [##REF##21620860##108##,##REF##23805866##109##]. The Γ position is a bit less conserved and tolerates large hydrophobic residues, including canonical L/V/I and aromatic residues, except His. Apparently, smaller hydrophobic residues, such as Ala, Pro or Met, do not have enough volume to fill the HP2, while aromatic residues can be too big to be docked (but still could be present at the Γ position (+3)) [##REF##27402840##110##–##REF##29867141##112##]. There is a Met residue in this position in a very few LIRs [##REF##32620754##113##]. Also, for some LIRs, e.g. PCM1, ULK1, or Atg14, aromatic residues are likely able to dock to HP2 as Phe in that position results in good binding in peptide arrays [##REF##30767700##96##,##REF##31053714##100##].</p>", "<p id=\"P22\">Another functionally important feature of LIR sequences is the presence of negatively charged (Glu/Asp) or phosphorylatable (Ser/Thr) residues prior to the core motif at position -1 or at positions -2 or -3 (##FIG##2##Figure 3B##). Mutational analyses have shown that their presence strongly enhances the affinity of LIR interactions with Atg8/LC3/GABARAP [##REF##31310766##9##,##REF##17580304##23##].</p>", "<title>Structural features of the LIR-LDS interaction</title>", "<p id=\"P23\">The main structural difference between Atg8/LC3/GABARAP proteins and other UBLs, which also determines the specific role of Atg8/LC3/GABARAP in autophagy, is the presence of two additional α-helices located N-terminally to the ubiquitin core (##FIG##0##Figure 1A##). This was revealed with the determination of the first Atg8 structure, the one of GABARAPL2/GATE-16 [##REF##10856287##114##]. This N-terminal α-helical subdomain significantly varies in the amino-acid content among the different members of the Atg8/LC3/GABARAP family, and structural studies indicate that it displays a dynamic behavior, participating in a conformational exchange [##REF##11779480##115##–##REF##11875056##117##]. Consequently, this structural adaptation of Atg8/LC3/GABARAP is reflected in a set of new functions not observed for other UBLs. For instance, the N-terminal α-helices are essential for tubulin binding and oligomerization [##REF##11779480##115##], strengthening tethering of lipid bilayers upon autophagosome maturation [##REF##17632063##39##,##REF##21497758##118##], and recognition of mitochondrial phospholipids [##REF##24036476##119##].</p>", "<p id=\"P24\">Despite their flexibility, the N-terminal α-helices are specifically aligned to the ubiquitin-like core, forming the deep HP1, (also termed W-site) together with residues of the β-strand β2 (##FIG##2##Figure 3A##). This pocket binds preferentially indole-based substances, albeit with low affinity [##REF##18567048##120##], and usually accommodates large sidechains of non-polar aromatic residues within the LIRs. HP1 is formed by residues D19, I23, P32, I34, K51, L53, and F108 in LC3B [##REF##19021777##106##]. HP2 (L-site), is built by hydrophobic residues of the central α-helix α3 and β-strand β2 (F52, V54, P55, L63, I66, and I67). These two pockets form the LDS and mediate a vast majority of known-to-date interactions between SARs, adaptors and scaffolding proteins with Atg8/LC3/GABARAPs [##REF##31310766##9##].</p>", "<p id=\"P25\">Besides the LDS, Atg8/LC3/GABARAP possess an interaction site at the opposite molecular surface. This site, called the UIM-docking site (UDS) (##FIG##2##Figure 3C##) is similar to the well-known hydrophobic patch (L8-I44-V70) of ubiquitin [##REF##8107144##121##], and contains a number of surface-exposed hydrophobic residues around F79 and L81 in LC3A and LC3B corresponding to L76 and F78 in GABARAPs. The UDS is used by components of the UPS machinery (such as RPN10) and during intracellular trafficking (such as Ataxin-3 and EPS15) [##REF##30955882##122##]. The unlipidated form of yeast Atg8 also utilizes the UDS (named Y-site since it accommodates Tyr) in addition to LDS for binding the non-canonical LIR of Hfl1, which collaboratively regulates vacuolar morphology under stress as mentioned above (##FIG##2##Figure 3D##)[##UREF##4##42##]. Interactions involving the UDS are further discussed in separate sections below.</p>", "<p id=\"P26\">The structural differences within the human Atg8-family proteins are rather small - the backbone of the core regions of human LC3/GABARAP proteins can be overlaid with a RMSD of 1.2 Å. Nevertheless, structural differences exist and reflect mostly differences in Atg8/LC3/GABARAP sequences (##FIG##1##Figure 2##), therefore, it is important to further analyze these sequence deviations to define and understand the structural differences, which may confer functional differences as well. There are substantial differences in the sequences not only between the subfamilies but also between the individual subfamily members. This was proposed to lead to a functional segregation of the LC3 and GABARAP proteins. Indeed, the LC3 and GABARAP proteins were first identified in different compartments of human cells (microtubules for LC3 [##REF##3803603##123##] and synaptic membranes for GABARAP [##REF##9892355##124##], suggesting different functions for each subfamily. It could subsequently be shown that, upon starvation-induced autophagy, LC3-subfamily proteins are responsible for the elongation of the autophagosomal membranes, while GABARAPs are acting downstream, participating in the maturation and closure steps of the autophagosome formation [##REF##20418806##91##]. Recent studies showed that only GABARAP-subfamily members are important for the activation of the phagophore-priming ULK1-ATG13-ATG101-FIP200 complex [##REF##31208283##125##,##REF##26687599##126##]. The centriolar satellites protein PCM1 binds unconjugated GABARAP and LC3C proteins to mediate their localization at the pericentriolar material and control autophagic degradation of centriolar satellites and GABARAPs [##REF##31519908##127##,##REF##28712572##128##]. Another example of the selective function of individual LC3/GABARAPs is the recruitment of LC3C to invading bacteria (<italic toggle=\"yes\">Salmonella typhimurium</italic>) via the specific SAR, NDP52, important for autophagy-mediated restriction of bacterial growth. Depletion of both (NDP52 and LC3C) proteins is followed by an inability of the cell to defend the cytosol against invasion by <italic toggle=\"yes\">S. typhimurium</italic>, while depletion of all other LC3/GABARAPs does not affect it [##REF##23022382##129##]. Investigations of the molecular mechanisms behind these selective functions (e.g., linkage between residues at specific positions within LC3/GABARAP proteins and their functions) are only at their beginning.</p>", "<p id=\"P27\">In plants, the number of Atg8 orthologs varies from 1 in algae to 22 in angiosperms [##REF##28038982##68##]; however, the diversity of plant Atg8 proteins could be significantly higher due to multiple gene duplications in order to adapt to various adverse conditions where Atg8 proteins play a crucial role [##REF##32596242##130##]. The plant Atg8 proteins also reveal significant selectivity in interaction with their interaction partners, originated from the sequence difference between Atg8 homologs in different species [##REF##31329577##72##]. Of note, all the key residues, participating in the HP1, HP2 [##REF##19021777##106##] and UDS [##REF##30955882##122##] are conserved, as well as the key lysine residues involved in regulating LIR binding: K49 and K51 in LC3A and LC3B (K46 and K49 in GABARAPs). The K49 performs a gatekeeper function, regulating the entrance of the aromatic residues of canonical LIRs into the HP1 [##REF##24290141##131##]. Interestingly, K49A mutation significantly enhances binding of canonical LIRs to LC3B protein [##REF##24290141##131##,##REF##28127051##132##], while K51A decreased or abolished it. Drosophila Atg8a-LDS (K48A/Y49A) mutant flies exhibit strong accumulation of LIR-motif containing proteins like Ref(2)P and Kenny [##REF##35649355##133##,##REF##29097655##134##].</p>", "<p id=\"P28\">The N-terminal α-helices show significantly less conservation, which agrees with the hypothesis that these helices predetermine the selectivity of the interactions between Atg8 proteins and LIR motifs in SARs [##REF##21497758##118##] and thus should be different in amino acid content for each individual family member. The few conserved residues within these α-helices participate either in folding of Atg8 proteins or in the formation of HP1. As expected, the loop regions are significantly less conserved, the relatively long loops L1, L2 and L3 show almost no identical or similar residues. Most conserved are regions of all β-strands, indicating their pivotal role in Atg8-protein folding and in the formation of HP1 and HP2.</p>", "<title>Unique features of GABARAP and LC3 subfamilies</title>", "<p id=\"P29\">The most significant consequence of the alignment shown in ##FIG##1##Figure 2##, is the clear separation of all Atg8 proteins in LC3B and Atg8/GABARAP subtypes based on a few positions within their sequence [##UREF##9##135##]. This separation is also strongly supported by phylogenetic analyses of the nucleotide sequences of 36 Atg8 genes from 14 eukaryotic species (##FIG##0##Figure 1B##). The first conserved difference between LC3B and Atg8/GABARAP subtypes of proteins is the switch between the intramolecular electrostatic contacts for residues at positions 8 and 47 in Atg8/GABARAP (positions 10 and 50 in LC3B, respectively). For all Atg8/GABARAPs, position 8 is occupied by a negatively charged or polar residues which are able to play a role as hydrogen bond acceptors (Glu, Asp, Gln, Asn, Ser, and Thr), while position 47 is consistently used for electropositive residues (Lys and Arg), serving as the hydrogen bond donors. In contrast, in the LC3 subfamily there are electropositive residues (donors) at position 10 and electronegative residues (acceptors) at position 50. These residues come close to each other and form intramolecular hydrogen bonds or salt bridges to stabilize the Atg8/LC3/GABARAP structure and ensure a proper orientation of the N-terminal α-helical subdomain (##FIG##2##Figure 3E##). Importantly, these residues also form intermolecular contacts to residues in LIR motifs and, therefore, also contribute to the selectivity of Atg8 interactions with other proteins. Accordingly, phosphorylation of T50 by STK3, STK4, PKCζ and NEK9 [##REF##31857374##136##,##REF##25544559##137##], as well as introducing the phosphomimetic mutation T50E in LC3B strongly reduce both LC3B binding to several LIR-containing proteins, including FYCO1, and lipidation of LC3B [##REF##31857374##136##]. FYCO1 is involved in directional transport of autophagosomes and blockade of T50 phosphorylation by STK4 decreases the starvation-induced perinuclear positioning of autophagosomes and their colocalization with lysosomes.</p>", "<p id=\"P30\">Another difference is consistently shorter long loops in Atg8 and GABARAPs. The loops between β-strands β1 and β2 (L1) and between β-strand β3 and α-helix α4 (L3) display no conservation; however, they undergo significant dynamic modulations in the free and LIR-bound forms of Atg8/LC3/GABARAPs, as was observed by NMR experiments [##REF##28655748##99##,##REF##23805866##109##]. Therefore, lack of one residue could in principle affect their dynamics and thus modulate selectivity to a specific LIR [##REF##30990354##138##].</p>", "<p id=\"P31\">Although yeast Atg8 is more similar to the GABARAP subfamily in both sequence and structure, the extreme N-terminal region is more similar to the LC3 subfamily [##REF##26687600##74##]. In Atg8 and LC3 subfamily proteins, the N-terminal arm is away from the ubiquitin fold and has a flexible open conformation, whereas in the GABARAP subfamily it has a closed conformation and tightly interacts with the ubiquitin fold. This structural difference is caused by the type of amino acid present at position 3 and 108 of Atg8: The GABARAP subfamily conserves Ala108 that forms hydrophobic interactions with Met1 and the aromatic residue (Phe/Trp) at position 3 in the N-terminal arm. Whereas in Atg8 and LC3 proteins position 3 is occupied by non-aromatic residues and position 108 is occupied by Val or Thr, impairing the interaction between the N-terminal arm and the ubiquitin fold. This structural difference is also observed between LGG-1 and LGG-2 in <italic toggle=\"yes\">C. elegans</italic>, and may contribute to subfamily-specific functions.</p>", "<title>Selectivity determinants in LIR motifs</title>", "<p id=\"P32\">In species that express functionally distinct Atg8s, selectivity is regulated by subfamily or subset specific salt bridges, hydrogen bonds or hydrophobic interactions. Selectivity determinants in human Atg8 proteins are only partially characterized, but mutational analysis has shown that certain LIR core sequences possess an increased affinity to the GABARAP versus LC3 subfamily. This allowed the definition of a broad consensus for the GABARAP-interacting motif (GIM), conforming to the core sequence (W/F)-(V/I)-X-V [##REF##28655748##99##]. A similar consensus sequence has not been made for LC3 binding, and there are also exceptions of GABARAP-selective LIR motifs (e.g. FIP200 and ATG14 LIR motifs) lacking a GIM. The residue in the +2 position in core LIR has a stronger effect on LC3 subfamily interactions than GABARAP subfamily interactions [##REF##31053714##100##]. A combined use of mutational analysis, affinity measurements and X-ray crystallography revealed a tendency of the +2 residue to clash with LDS residues Q26, H27 and K30 in LC3B, while the corresponding residues in GABARAP (K24, Y25, R28) enables a more robust conformation that is less affected by the residue in the +2 position [##REF##31053714##100##]. Y25 (invariant in all GABARAPs) participates frequently in the formation of intermolecular hydrogen bonds with positively charged or polar residues at position +1 of LIR motifs. The favorable conformation of Y25 is stabilized via cation-π interactions with a guanidinium moiety of invariant R28 (##FIG##2##Figure 3E##). The distinct conformation of Y25 and R28 makes the intermolecular hydrogen bonds more energetically favorable and thus increases the affinity of the LIR binding to the GABARAPs. In the LC3-subfamily, there are His or Phe residues at the position of GABARAPs Y25. Therefore, the favorable conformation of aromatic rings cannot be stabilized by cation-π interactions and effective intermolecular hydrogen bonds cannot form. How this can create selectivity is illustrated by the GABARAP selective LIR motifs of PCM1, ULK1 and FIP200 that are blocked in LC3 binding because they contain a Lys (PCM1) or Met (ULK1, FIP200) in position +2. For all these proteins, mutation of the +2 residue to Ile, Leu, Val or Phe resulted in LC3 binding, but no other residues gave LC3 binding [##REF##31053714##100##]. Conversely, substitution of the +2 residue with Arg impairs LC3 binding of the AnkG LIR motif and renders it highly selective for GABARAPs [##REF##29867141##112##].</p>", "<p id=\"P33\">The selectivity of a LIR motif can also be regulated by residues N-terminal to the core LIR. The above mentioned electropositive R10 and R11 residues (R16 and K17 in LC3C) on helix α1 are unique for the LC3 subfamily. They form important electrostatic interactions with acidic residues located in positions -2, -3 or -4 of a LIR motif [##REF##18524774##105##,##REF##19021777##106##,##REF##26468287##139##]. Examples are the FYCO1 LIR (LC3 selective) that is stabilized by an electrostatic interaction formed between R10 in LC3B and D1277 in the -3 position of FYCO1 LIR [##REF##26468287##139##], and the p62 LIR that is similarly stabilized by interactions formed between R10 and R11 in LC3B, respectively, with D337 and D338 in the -3 to -2 positions in the LIR of p62 [##REF##17580304##23##,##REF##18524774##105##,##REF##18653543##140##]. However, acidic residues in position -1 and -2 are also crucial for GABARAP binding and the importance of N-terminal residues in creating selectivity is therefore restricted to the -3 and -4 positions [##REF##31053714##100##].</p>", "<p id=\"P34\">More recent studies have revealed that residues C-terminal to the core motif can have a strong impact both on the selectivity and affinity of some LIR motifs. Many LIR motifs contain a negatively charged residue in the +7 position [##REF##27246247##141##], and this enables the formation of an electrostatic interaction with the R70 residue in LC3B (R67 in GABARAP). Since this residue is conserved in all Atg8 proteins, the presence of a negative +7 residue does not affect the selectivity, but the affinity is normally increased. In some LIR motifs including the FYCO1 LIR [##REF##26468287##139##,##REF##27246247##141##], the +7 position marks the start of a short amphipathic α-helix that strengthens the interaction with Atg8 proteins. In LIR motifs of AnkG, AnkB, and FAM134B, this α-helix is extended resulting in very strong Atg8 binding affinity [##REF##29867141##112##]. Studies of the LC3 selective LIR in FYCO1 and the GABARAP selective LIR in ALFY (autophagy-linked FYVE protein) identified position +5 as an important molecular selectivity determinant in LIR sequences [##REF##26468287##139##,##REF##24668264##142##]. In FYCO1, the D1285 residue in the +5 position provides specificity by binding to the H57 residue in LC3A or LC3B [##REF##26468287##139##]. The corresponding residue in LC3C (Glu) or GABARAP (Asp) leads to charge repulsion and the selectivity is therefore directed towards LC3A and LC3B. The LIR motif in ALFY has a Tyr residue in the +5 position that clashes with the H57 residues in LC3A/B leading to repulsion of the interaction [##REF##24668264##142##]. The Tyr residue instead binds to the corresponding Asp residue in GABARAPs (D54). Obviously, selectivity can be achieved in different ways, and the exact mechanism may vary between LIR motifs and often involve more than a single selectivity determinant. For the GABARAP selective LIR in ULK1, mutating the N-terminal -3 position (T to E), the +2 position in the core LIR (M to I) and the C-terminal +4 position (P to D) resulted in strong LC3B binding, and this illustrates the combined effect of three different selectivity determinants that all prevent the LC3A/B interaction [##REF##31053714##100##].</p>", "<p id=\"P35\">Equally important as binding selectivity is the availability of individual Atg8s in a cellular context. The strong co-localization consistently seen between p62 and LC3B suggests that LC3B is a preferred interaction partner for p62, but we have also observed that most LIR proteins have low affinity for LC3B leading to less competition for binding this Atg8. A LIR motif occasionally overlaps with other binding motifs, and this similarly results in competition for binding. A relevant motif to mention here is FIR (FIP200 interacting region) that binds to the CLAW domain in RB1CC1/FIP200 [##UREF##10##143##]. FIR is identified in several proteins involved in autophagy, including p62 [##UREF##10##143##], OPTN [##REF##33692357##144##], CCPG1 [##REF##29290589##145##], TBK1 adaptor proteins [##UREF##11##146##], and ATG16L1 [##REF##23262492##147##,##REF##23392225##148##]. The FIR consensus sequence is D/E/S/T-D/E/S/T-F/W/Y/I/L/V-X-X-I/L/V [##REF##35133947##149##] that is very similar to the LIR consensus. The only important difference is that hydrophobic residues (I, L, V) are accepted in the aromatic Θ residue position, and there is also a stronger need for an acidic or phosphorylated residue in position -1 of a FIR. Because of this similarity, some LIR motifs (e. g. in p62 and OPTN) can function both as LIR and FIR. The dual LIR/FIR motif in OPTN (DS-FVEI) is activated by phosphorylation, and this strongly increases its binding efficiency for both Atg8s and RB1CC1/FIP200 [##REF##33692357##144##]. The FIR2 motif in CCPG1 is similarly activated by phosphorylation and while its sequence (S-D-I-V-T-L) suggests it is not a LIR motif, binding experiments show that it can bind to mammalian Atg8s and RB1CC1/FIP200 [##REF##33692357##144##]. The binding of LIR/FIR in p62 to RB1CC1/FIP200 may help in recruiting the core autophagy machinery, but the p62-RB1CC1/FIP200 interaction is probably excluded by the stronger interaction of polymeric p62 to lipidated Atg8s at the concave side of the phagophore [##UREF##10##143##]. There is an interesting similarity between the CLAW domain in RB1CC1/FIP200 and the domain in yeast Atg11 used for docking of SAR-cargo complexes to the PAS [##REF##35133947##149##].</p>", "<title>LIR-LDS interaction is evolutionary conserved in <italic toggle=\"yes\">C. elegans</italic> and <italic toggle=\"yes\">Drosophila</italic></title>", "<p id=\"P36\">Analysis of the LGG-1 and LGG-2 binding motifs in various interacting proteins in <italic toggle=\"yes\">C. elegans</italic> showed that LGG-1 and LGG-2, similar to Atg8 family members in other systems, interact with the [W/F/Y]-x-x-[I/L/V] motif (LIR) [##REF##26687600##74##]. Acidic residues (Glu or Asp) or Thr are preferred in the positions preceding the aromatic residue. The core sequence and surrounding residues of LIR confer binding specificity for LGG-1 or LGG-2. LGG-1 binds to “W”, “F” and “Y” type LIRs, while LGG-2 prefers Phe as the aromatic residue and Asp and Thr in the positions preceding the aromatic residue [##REF##26687600##74##]. LGG-1 and LGG-2 also bind to substrates independent of the canonical LIR motif.</p>", "<p id=\"P37\">Crystal structures of LGG-1 and LGG-2 reveal that they exhibit a typical Atg8-family fold, which contains two N-terminal α-helixes (α1, α2) and a ubiquitin fold consisting of a four-stranded β-sheet and two α-helixes [##REF##26687600##74##]. The N- and C-terminals of LGG-1 and LGG-2 display structural differences that result in differential binding to substrates. The C-terminal tails of Atg8 family members are flexible and divergent. The N-terminus of LGG-1, like GABARAP subfamily Atg8 proteins in mammals, exhibits a rigid closed conformation resulting from hydrophobic interactions between residues in the N-terminal arm and the ubiquitin fold (i.e. interactions formed by Met1 and Trp3 with Ala108 in LGG-1). The N-terminus of LGG-2, like the LC3 family and Atg8, is detached from the ubiquitin fold and thus adopts the open conformation. LGG-1 and LGG-2 possess the two hydrophobic pockets, HP1 and HP2, which recognize the aromatic [WFY]<sub>0</sub> and [I/L/V]<sub>+3</sub> core LIR residues, respectively, in their binding substrates. The HP1 and HP2 in LGG-1 and LGG-2 are structurally distinct. In the HP1 site in LGG-1, a key Phe residue is adjacent to a Gly residue, which allows rotation of the Phe benzene ring. Thus, HP1 shows plasticity in accommodating divergent aromatic residues. In the HP1 of LGG-2, the conformation of the corresponding Phe is restricted by juxtaposition with a bulky Val residue. The HP2 in LGG-1 and LGG-2, which differ in size and shape, show different preference for Leu and Val at the “+3” position in the LIR [##REF##26687600##74##]. The N-termini of LGG-1 and LGG-2 contribute to binding with the residues preceding the aromatic residue in LIR. For example, the Arg residues at the N-termini form electrostatic interactions with the acidic residues adjacent to LIR [##REF##26687600##74##]. Therefore, the structural differences in the HP1, the HP2, and the N-termini of LGG-1 and LGG-2 determine their binding specificity for interacting proteins.</p>", "<p id=\"P38\">In <italic toggle=\"yes\">Drosophila</italic>, a conservation of F3 and A108 residues in Atg8a suggests that this protein has a closed conformation typical for metazoan GABARAP subfamily. However, these residues are not conserved in Atg8b (##FIG##1##Figure 2##). A range of LIR-motif containing proteins found in mammals also have functional equivalents in <italic toggle=\"yes\">Drosophila</italic>, many of which interact with Atg8a via the aid of a LIR motif. As with many other lower complexity model organisms, often their relative ease of study allows for early and cost-effective investigations that can provide helpful insight to inform research into higher complexity models [##REF##32475477##150##]. Recently, a high-throughput yeast 2-hybrid (Y2H) screening identified several Atg8a-interacting proteins in <italic toggle=\"yes\">Drosophila</italic> [##REF##35081354##151##]. These proteins can be classified in 3 groups: 1) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, Ref(2)P and Kenny, 2) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4 and 3) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1 [##REF##35081354##151##]. Of note, upon sequence analysis using the LIR-prediction software iLIR, all proteins of this Y2H list are found to possess at least one, often several, candidate LIR motif(s) within positions that overlap with the predicted Atg8a-interaction region mapped by the Y2H screen for each hit [##REF##35081354##151##]. In accordance with the typical LIR peptide structure, most of the characterized LIR motifs for the known Atg8a-interactors in <italic toggle=\"yes\">Drosophila</italic>, are hexapeptide sequences that bear the typical the typical Θ<sub>0</sub>-X<sub>1</sub>-X<sub>2</sub>-Γ<sub>3</sub>pattern, (where Θ is an aromatic (W/F/Y) and Γ is a hydrophobic (L/I/V) residue) at their core [##REF##23908376##152##].</p>", "<title>The need for multivalent LIR-LDS interactions in docking of cargos to the phagophore in selective autophagy</title>", "<p id=\"P39\">Only a subset of Atg8 binding proteins work as SARs involved in selective autophagy, but these SARs are extremely important for this process since they are responsible for the docking of a selected cargo to the phagophore membrane. Presumably, a selected cargo must have a certain size to enable growth of a phagophore on its surface. In addition, the selectivity is driven by a tight binding between the SAR-cargo complex and Atg8 proteins lipidated to the phagophore membrane. Several studies have shown that autophagosome formation is possible without Atg8 proteins, but the efficiency is low and the GABARAP subfamily is required for the closure step [##REF##27864321##101##,##REF##29038162##102##]. In addition, SAR-cargo complexes are only randomly engulfed in the absence of Atg8 proteins and there is no selectivity in the process. Martens used the term exclusive autophagy to describe the growth of a phagophore on a selected cargo [##UREF##12##153##] emphasizing the need for multiple interactions between a SAR and Atg8 proteins in selective autophagy. Multivalency can be achieved in different ways, but we will here mention two different strategies. p62 is a polymeric protein that is presumably attached as a polymer to the phagophore membrane [##REF##16286508##154##–##REF##26413874##156##]. Atg19 is however monomeric, but it contains several cryptic LIR sequences that are activated if the main LIR in Atg19 is bound to an Atg8 [##UREF##12##153##].</p>", "<title>Non-canonical LIR motifs: Half-LIRs and α-helical LIRs</title>", "<p id=\"P40\">An increasing number of noncanonical LIR sequences either lack the aromatic Θ<sub>0</sub> residue or the hydrophobic Γ<sub>+3</sub> residue and can be seen as “half-LIRs”. The first non-canonical LIR to be described was the LIR of NDP52 (I<sup>333</sup>LVV) that lacks the aromatic Θ residue and therefore does not engage HP1 [##REF##23022382##129##]. The side chain of the Ile residue in Θ position is too short to occupy the aromatic pocket, but the LIR of NDP52 binds selectively and strongly to LC3C (therefore named CLIR). The LVV motif in CLIR forms compensating hydrophobic interactions with residues Lys32, Phe33, Leu64, Phe69 in LC3C, and these residues are misaligned or absent in other human Atg8s explaining the selectivity for LC3C [##REF##23022382##129##]. The importance of the missing aromatic Θ residue is illustrated by that mutation of the NDP52 core motif from ILVV to WLVV results in binding to all human Atg8s [##REF##23022382##129##]. Other reported non-canonical LIR sequences have no hydrophobic Γ residue and therefore do not engage HP2. Examples are the LIRs in TRIM5α (DW<sup>196</sup>E) [##REF##30282803##157##,##REF##25127057##158##] and BCL-2 (EW<sup>30</sup>D) [##REF##24240096##159##], which both have a Trp residue docked into HP1. Another non-canonical feature of the LIR in TRIM5α is that it is helical and located in a coiled-coil domain [##REF##30282803##157##]. In a canonical LIR-LDS interaction the LIR is kept within the structure of a β-sheet. Helical LIR motifs have higher structural flexibility, and the LIR consensus sequence is less rigid. This may result in a docking of non-consensus residues into pockets HP1 and HP2, and the distance separating the two residues may also vary. In the structure formed between the coiled-coil domain of TRIM5α and LC3B, no residue is docked into HP2, but Gln203 is located on the edge of the HP2 pocket and replacing it with a smaller hydrophobic residue gave increased binding affinity. The TRIM5α LIR has a rather weak binding affinity, but the affinity is strongly increased by dimerization of TRIM5β [##REF##30282803##157##]. Another type of non-canonical LIR motif is found in UBA5. This motif binds to the GABARAP subfamily, but also to the ubiquitin-like modifier UFM1 [##REF##26929408##160##]. The binding of this LIR motif results in the formation of an intermolecular β-sheet, but three hydrophobic pockets (not the usual two) are formed at the interphase on GABARAP. Rearrangements of GABARAP residues K46 and K/R47 results in the formation of an additional pocket termed HP0 (##FIG##2##Figure 3C##). The LIR in UBA5 (W<sup>341</sup>GIELV) has W<sup>341</sup> docked into HP0, while the canonical HP1 and HP2 sites are occupied by I<sup>343</sup> and L<sup>346</sup> [##REF##30990354##138##]. The GABARAP selectivity of this LIR motif strongly depends on the above-mentioned K/R47 residue unique for the GABARAP subfamily (D50 in LC3B). A few proteins, including the RavZ protein of the intracellular pathogen <italic toggle=\"yes\">Legionella pneumophila</italic> [##REF##28668392##161##], human FNIP1 [##REF##34597140##162##] and human ATG3 [##UREF##13##163##], have a LIR embedded in a β-sheet. The core LIR of human Atg3 (W<sup>107</sup>VDT) is unusual by having a Thr instead of the usual Ile, Leu or Val residues in position +3, but W<sup>107</sup> is docked into HP1 and T<sup>110</sup> into HP2 as in canonical interactions. However, while the interaction with GABARAP is completely blocked by a mutation of W <sup>107</sup>, mutation of T<sup>110</sup> does not affect the interaction [##UREF##13##163##].</p>", "<title>Regulation of LIR-LDS interactions by phosphorylation of LIR motifs</title>", "<p id=\"P41\">Phosphorylation of residues within the N-terminal flanking region of the core LIR (mostly preceding the aromatic residue at positions -1 or -2) may enhance the affinity of the SAR:Atg8/LC3/GABARAP binding [##REF##23805866##109##,##REF##28127051##132##,##REF##35417087##164##–##REF##23209295##170##], and serves as a key autophagy regulator in corresponding types of selective autophagy. LIR sequences usually contain residues that can be phosphorylated (Ser/Thr) and it is seen for a large number of investigated proteins that phosphomimetic mutations increase their affinity to Atg8/LC3/GABARAPs (##FIG##3##Figure 4##). The strongest effect is usually seen when a negatively charged or phosphorylated residue is located at positions -2 or -1 preceding Θ, but also more distant residues (positions -8 to -3) may affect the affinity of a LIR-LDS interaction [##REF##33845085##168##,##REF##28291748##171##]. For the optineurin (OPTN) LIR, phosphorylation of individual Ser residues up to position -8 still increases its affinity to LC3B [##REF##23805866##109##]. IKKα-mediated phosphorylation of AMBRA1 S1014 at position -6 promotes AMBRA1’s binding to LC3 and GABARAP (<italic toggle=\"yes\">in vitro</italic> and <italic toggle=\"yes\">in vivo)</italic> and serves as a positive regulator of AMBRA1-mediated mitophagy [##UREF##14##165##]. In some cases, like for FUNDC1, direct phosphorylation of the Tyr residue at the LIR Θ position leads to a weakening of the LIR:LC3/GABARAP binding affinity [##REF##27653272##166##].</p>", "<p id=\"P42\">Post-translational modification of a LIR motif is normally phosphorylation, and around 25% of LIR motifs has a Ser or Thr residue in the critical -1 position [##REF##23908376##152##]. It is convenient to distinguish between LIR motifs that depend on post-translational modification and those that do not. Known regulatory phosphorylation sites within LIR domains of mammalian proteins are shown in ##TAB##0##Table 1##. There is so far no direct evidence that LIR motifs in p62 or NBR1 are regulated by post-translational modification. This correlates with a constitutive degradation of these soluble SARs by selective autophagy [##REF##19250911##172##]. Post-translational activation of LIR motifs was first shown for the soluble SAR OPTN [##REF##21617041##167##]. The involvement of OPTN in selective autophagy (e. g. xenophagy, mitophagy, aggrephagy) depends on TBK1 mediated phosphorylation of S177 in its LIR motif (position -1) [##REF##21617041##167##]. Unlike other SARs, OPTN is degraded by the proteasome if not involved in selective autophagy [##REF##28334804##173##], and this correlates with the need for activation of its LIR motif. Organelle resident SARs sit on the surface of structures that are normally not degraded, and it is obvious that their functions in selective autophagy needs to be tightly regulated. Indeed, early studies on mitophagy receptors BNIP3L/NIX, BNIP3 and FUNDC1 revealed that activation by phosphorylation is a common mechanism for regulating LIR motifs in mitophagy receptors [##REF##26611876##174##]. The LIR in BNIP3 is activated by phosphorylation of residues Ser17 and Ser24 flanking the core motif in positions -1 and +6 [##REF##23209295##170##], LIR in NBIP3/NIX is activated by phosphorylation of Ser34 in position -1 [##REF##28127051##132##], while LIR in FUNDC1 is regulated both positively and negatively by phosphorylation. The function of FUNDC1 in mitophagy is induced by hypoxia, and its LIR motif (ESDDDSYEVL) is kept inactive under normoxia by the phosphorylation of two residues: i. e. Ser13 in position -5 [##REF##24746696##175##] and Tyr18 in position 0 [##REF##22267086##176##]. Hypoxia results in dephosphorylation of both these sites by PGAM5 and recruited ULK1 further activates the LIR motif by phosphorylating Ser17 in the -1 position [##REF##24746696##175##,##REF##24671035##177##]. Phosphorylation of Tyr18 is a good example on how Y-type LIR motifs can be inactivated by phosphorylation of the essential aromatic residue. The negative regulation seen for the Ser13 phosphorylation may reflect its location in the LIR motif that is more distant to the core motif (-5) than seen for most negatively charged residues or phosphorylation sites having a positive effect on the LIR-LDS interaction. Ser13 in FUNDC1 is phosphorylated by CK2 [##REF##24746696##175##]. The same kinase was recently shown to phosphorylate three Ser residues in positions -12, -11 and -7 in the LIR motif of reticulophagy receptor FAM134C, thereby inhibiting the binding of FAM134C to Atg8s under fed conditions [##REF##36044577##178##]. The authors explained the inhibitory effect by the notion that phosphorylated residues more distal to the core LIR interact with a region in LC3B with a neutral charge and are therefore disfavored.</p>", "<p id=\"P43\">A common way of testing the effect of phosphorylation on Atg8 binding is the use of phosphomimic mutations. However, this type of mutations may not always mimic the effect of phosphorylation. A recent study on the reticulophagy receptor TEX264 reported that phosphorylation of two Ser residues adjacent to the core LIR (<underline>SS</underline>FEEL), again by CK2, is essential for its interaction with Atg8s and induction of reticulophagy [##REF##35417087##164##]. In this case, phosphomimic mutations had no such effect, and crystal structures of GABARAP in complex with TEX264 LIR peptides revealed important structural differences in complexes formed with phosphorylated and phosphomimic mutated LIR peptides. The main difference was the presence of four specific hydrogen bonds with the phosphorylated peptide that were not formed with the mutated peptide that instead gave nonspecific salt bridges [##REF##35417087##164##].</p>", "<title>Interactions involving the UDS</title>", "<p id=\"P44\">A number of Atg8 binding proteins interact in a LIR-LDS independent manner [##REF##20562859##179##]. A search for other binding mechanisms led to the identification of ubiquitin interacting motif (UIM)-like sequences as candidates for a new type of Atg8 interacting motif. The UIM-like sequences were shown by yeast two hybrid assays and site directed mutagenesis to bind to a specific patch on the Atg8s which was termed the UIM-docking site (UDS) [##REF##30955882##122##]. The UDS was defined as a patch of four residues that are highly conserved in evolution and has the core consensus sequenceψ-F-ψ-ω/T [##REF##30955882##122##]. The UDS is localized on the opposite surface of the Atg8s relative to LDS and relatively close to the C-terminal Gly residue. This means that the UDS points towards the membrane when Atg8 is lipidated [##REF##34239122##37##], and it is therefore not fully understood how a UIM motif can interact with the lipidated form. Importantly, so far there are no structural data to support the notion of UIM-like sequences binding to the UDS. However, structural data exist for the interaction of ATG4B [##REF##19322194##93##] and Hfl1 [##UREF##4##42##] with the UDS region, but no UIM-like sequences has been identified in these proteins. A main question is if the UIM-UDS interaction is restricted to unlipidated Atg8s or if it is also possible for Atg8s that are lipidated to a membrane. Further studies and structural evidence is needed before a conclusion can be made. Interestingly, both the LDS and UDS regions were marked as important already in 2006, when it was shown that sites involving Y49 and L50 on one (LDS) side, and F77 and F79 on the opposite (UDS) side, of yeast Atg8 were essential sites for autophagy [##REF##16680092##180##].</p>", "<title>Composite interactions involving both LDS and UDS</title>", "<p id=\"P45\">The non-canonical CLIR motif of NDP52 (ILVV) is evolutionary conserved in the paralogs TAX1BP1 and CALCOCO1, but the selectivity for LC3C is not conserved and the non-canonical motif in CALCOCO1 (LLVV) has preference for the GABARAP subfamily [##REF##32525583##181##]. The sequence adjacent to core LIR differs in the three paralogs and the CLIR of NDP52 contains more negatively charged residues. The binding of the isolated LIR motif of CALCOCO1 is very weak even for the GABARAP subfamily. However, the interaction is strongly increased by dimerization of CALCOCO1 and further increased by an additional weak interaction with the hydrophobic UDS patch including the essential Phe residue (F<sup>77</sup> in GABARAP) [##REF##32525583##181##]. No UIM-like sequence was identified in CALCOCO1 and the UDS interacting motif mapped to residues 615-653 was therefore named UIR (UDS interacting region). Yeast vacuole membrane protein Hfl1 is another example of a protein where a weak non-canonical LIR motif is supported by an additional interaction with UDS [##UREF##4##42##]. Unlike UIR in CALCOCO1 that is located distal to LIR, the UDS interacting region in Hfl1 is located adjacent to LIR. The noncanonical LIR in Hfl1 is helical. The spacers separating the aromatic Θ and hydrophobic Γ residues are extended in the LIRs of ScHfl1 (W<sup>371</sup>xxxI) and SpHfl1 (F<sup>388</sup>xxxxxxxxxY), and the latter has a Tyr residue docked into HP2 [##UREF##4##42##]. In addition, a Tyr residue adjacent to the non-canonical LIR Y<sup>387</sup> in ScHfl1) interacts with a so-called Y-site in UDS of Atg8 and this increases the strength of the interaction (##FIG##2##Figure 3D##). These two examples demonstrate that simultaneous interaction with LDS and UDS is possible, but the frequency is not known since the weakest interaction is easily overlooked. It should be noted that yeast Hfl1 interacts with unlipidated Atg8 for its function, and it is not known if UDS is exposed and can facilitate binding if Atg8 is lipidated to a membrane. Another interesting example of a protein binding LDS and UDS is Atg4. There is no crystal structure of full-length Atg4, but structures of human LC3B bound to the catalytic domain of Atg4B revealed an interaction with UDS and residues surrounding UDS [##REF##19322194##93##]. In addition, the C-terminal tail of Atg4B contains an evolutionary conserved and canonical LIR motif that binds strongly to LDS, and functional studies of this LIR has been performed in yeast and mammals [##REF##28287329##94##,##REF##28330855##182##]. How the UDS and LDS interactions are utilized in Atg8 processing or delipidation is not clear. However, in mammals a highly selective role of Atg4B is stabilizing a free pool of unlipidated GABARAP and GABARAPL1 and this depends on a strong interaction enabled by using both interaction surfaces [##REF##28287329##94##].</p>", "<title>Interactions involving the N-terminal arm</title>", "<p id=\"P46\">The canonical LIR-LDS interaction is dependent on both the N-terminal arm domain (amino acids 1–28) and the ubiquitin core (amino acids 30–120) of LC3B [##REF##17580304##23##,##REF##18653543##140##,##REF##19250911##172##]. However, there are several reports of interactions with Atg8 family proteins that do not involve the LDS but rather the N-terminal arm. Hence, <italic toggle=\"yes\">Drosophila</italic> KEAP1 binds to the N-terminal 71 amino acids of <italic toggle=\"yes\">Drosophila</italic> Atg8a. The LDS mutant K48A/Y49A does not affect binding. However, the N-terminal arm alone is not sufficient as amino acids 1-26 does not bind to <italic toggle=\"yes\">Drosophila</italic> KEAP1, neither does a piece from amino acid 26 to 71 showing that the region 1-71 of Atg8 is required [##UREF##16##183##]. The same interaction pattern was seen between <italic toggle=\"yes\">Drosophila</italic> YL-1, a component of a nuclear acetyltransferase complex, and Atg8a [##REF##32460019##15##]. When oncogene induced senescence is triggered by expression of activated Ras, Lamin B1 interacts with nuclear LC3B and the complex is exported out of the nucleus to be degraded by autophagy in the cytoplasm. The N-terminal arm of LC3B with amino acids 1-28 is sufficient to bind to human Lamin B1 with R10 and R11 being essential for binding [##REF##26524528##13##]. Upon lysosomal damage, GABARAP binds to the core stress granule proteins NUFIP2 and G3BP1 and recruits them to damaged lysosomes where NUFIP2 helps to inactivate mTOR via the Ragulator-RagA/B complex. Both NUFIP2 and G3BP1 bind to the N-terminal arm of GABARAP and mutants of the LDS or UDS do not affect the binding [##UREF##2##12##]. Whereas NUFIP2 also interacts with the ubiquitin core of GABARAP, G3BP1 only binds to the N-terminal arm. Two regions of NUFIP2 bind to GABARAP whereas it is the N-terminal NTF2L domain of G3BP1 which binds to the N-terminal 1-26 amino acids of GABARAP. Recognition of damaged mitochondria is required for cellular health. Hence, redistribution of cardiolipin from the inner to the outer mitochondrial membrane acts as an “eat-me” signal for mitophagy in neuronal cells. Cardiolipin binds directly to LC3 with R10 and R11 in the N-terminal arm being essential for the binding and the biological response [##REF##24036476##119##]. We will surely see more examples in the future of interactions depending on the N-terminal arm of Atg8s. However, so far we sorely miss structural data on these type of interactions.</p>", "<title>Prediction of LIR motifs in proteins</title>", "<p id=\"P47\">Sequence motif-based prediction of LIR motifs in proteins employing regular expression pattern was pioneered by the iLIR software tool [##REF##24589857##184##]. Subsequently, the iLIR web resource for LIR-containing proteins in <italic toggle=\"yes\">Arabidopsis, C. elegans</italic>, chicken, human, mouse rat, zebrafish and <italic toggle=\"yes\">S. cerevisiae</italic> was established, followed by the iLIR@viral web resource for LIR-containing viral proteins [##REF##28806134##185##,##REF##27484196##186##]. The most recent development is the LIRcentral, a web accessible database (LIRcentral; <ext-link xlink:href=\"https://lircentral.eu\" ext-link-type=\"uri\">https://lircentral.eu</ext-link>) that contains information about literature-validated LIR-motifs and displays them along with features annotated in UniProt. By cross-referencing protein entities to UniProt entries, LIRcentral enables seamless data integration with other resources [##UREF##17##187##]. It is possible to improve on the iLIR and other regular expression based sequence prediction methods by manually curating the candidate hits by excluding motifs that contain residues within the core LIR that usually are inhibitory to binding such as glycine (G) and proline (P) that affect the secondary structure, and the basic lysine (K) and arginine (R) residues that can mediate charge repulsion, due to the basic residues surrounding the two hydrophobic pockets of the LDS [##REF##31310766##9##,##REF##23043107##95##].</p>", "<p id=\"P48\">However, iLIR, and other sequence motif-based prediction tools, cannot predict any of the non-canonical LIR sequences and it is challenging to predict half-LIRs as well, particularly in long sequences. An exciting new development that holds promise of more precise LIR predictions is the use of the AlphaFold2-multimer artificial intelligence-based protein modelling tool. Ibrahim et al. combined protein modelling using AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays [##UREF##18##188##]. Strikingly, the AF2-multimer enabled high accuracy prediction of canonical and also some non-canonical LIR motifs. When more non-canonical LIR/LDS structures are known the AF2 multimer predictions of such interactions will become more reliable.</p>", "<title>Binding motifs anticipated to be found in future investigations</title>", "<p id=\"P49\">The LC3/GABARAP interactome in human contains ~400 potential candidates under basal autophagy conditions [##REF##20562859##179##]. Only a small fraction of these proteins were validated and characterized as LC3/GABARAP binders, while validation of the rest and/or discovery of new candidates is complicated by the fact that researchers are looking mostly for the conventional and well-characterized canonical LIR motifs as the interaction determinant. This strategy, however, will not be sufficient in the light of the growing examples of unusual mechanism for interactions of Atg8/LC3/GABARAPs their partners. Below we summarize suggested (but not investigated so far) structural motifs which could be implicated in these interactions and may serve as a starting point for new investigations.</p>", "<title>Antiparallel LIR motifs</title>", "<p id=\"P50\">For all the canonical and non-canonical LIR sequences identified to date, the orientation of the extended β-stranded conformation of the LIR peptide is parallel to the β-strand β2 in Atg8/LC3/GABARAP. The only exception reported so far is the structure of an artificial peptide called K1 in complex with GABARAP [##REF##18638487##189##]. Therefore, one can predict that antiparallel β-stranded linear peptides with a reverse order of residues for Θ and Γ positions and with a corresponding C-terminal track of negatively charged residues after the core aromatic residue (N’-Γ-X-X-Θ-X<sup>–</sup>X<sup>–</sup>X<sup>–</sup> instead of N’-X<sup>–</sup>X<sup>–</sup>X<sup>–</sup>Θ-X-X-Γ) could efficiently bind Atg8/LC3/GABARAP proteins [##UREF##9##135##](##FIG##4##Figure 5A##).</p>", "<title>LIR motifs with non-canonical sequences</title>", "<p id=\"P51\">Considering other non-canonical linear LIR sequences (cLIR in NDP52 and LIR/UFIM in UBA5), one can predict existence of a high number of LIR-like sequences representing this category. The attempt to generate (by a phage display) high affinity and highly selective synthetic peptides capable of binding individual members of LC3 and GABARAP subfamilies in human cells led to generation of a number of sensor molecules; however, they all seem to contain canonical LIR motifs [##REF##28028054##190##]. The hypothetical motifs could be organized in various ways (displaying sequence complexity and using “through-space” binding modes) to facilitate effective and specific binding of Atg8-family proteins. For example, the evolutionary conserved ERphagy receptor C53 (called CDK5RAP3 in humans) binds plant and mammalian Atg8s via so-called shuffled AIM/LIR located within the intrinsic disordered central region of C53. These are versatile binding sites allowing both binding to Atg8s and to UFM1 and composed by shuffled AIM sequences (like IDWD, ##FIG##4##Figure 5B##). It is not clear, where and how exactly these shuffled AIMs interact with Atg8s. Hence, structural aspects of these interactions should be investigated in detail. But, as shown by NMR studies, the binding is different to Atg8s and UFM1. C53-mediated autophagy clears toxic incomplete polypeptides from translation generated under stress conditions. At normal conditions, C53 is inactive as an ERphagy receptor because it binds UFM1. Upon stress it is displaced by Atg8s activating ERphagy [##UREF##19##191##,##UREF##20##192##].</p>", "<title>Binding motifs that do not use the LDS/UDS sites on the Atg8/LC3/GABARAP surface</title>", "<p id=\"P52\">It was proposed [##UREF##9##135##], that the α-helical subdomain can be displaced from the ubiquitin core of Atg8/LC3/GABARAP proteins by another α-helical structure containing a combination of residues, which are more favorable for the binding of the ubiquitin core of a particular Atg8/LC3/GABARAP protein (##FIG##4##Figure 5C##). More aggressive conditions, which appear in close proximity to membranes or in cellular compartments with critical pH values, might facilitate the displacement. In this case, the amino acid content of the displacing α-helices could significantly differ from that for displaced helices α1 and α2, leading to the HP1 modulation in shape and dynamics.</p>", "<p id=\"P53\">The N-terminal α-helical subdomain in Atg8/LC3/GABARAP proteins is a key evolutionary addition to the core ubiquitin-like fold to separate structurally and functionally the autophagy modifiers from any other UBLs. The α-helices show a significant conformational exchange [##REF##23805866##109##,##REF##11779480##115##,##REF##31208283##125##,##REF##26284781##193##] and could potentially be separated from the ubiquitin core as the truncated LC3B and GABARAPL2 proteins were still able to perform some functions, like membrane fusion [##REF##21497758##118##]. The first α-helix in LC3B and GABARAPL2 was successfully swapped to emphasize their role in p62/SQSTM1 recognition [##REF##21460636##194##]. Moreover, it was shown that GABARAPL1 being truncated N-terminally for the α-helical subdomain and the β-strand β1 could still recognize and bind a number of cognate receptors and proteins (γ2 subunit of GABA<sub>A</sub> receptor [##REF##31857374##136##], human κ opioid receptor [##REF##16431922##195##], gephyrin [##UREF##21##196##]).</p>" ]
[ "<title>Acknowledgements</title>", "<p>We thank JC De La Concepcion for help with the phylogenetic tree presented in ##FIG##0##Figure 1##.</p>", "<title>Funding</title>", "<p>This work was supported by grants from Structural Genomics Consortium (SGC), a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute, EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA, Pfizer and Takeda to V.V.R., from the Norwegian Cancer Society (190214) and Research Council of Norway (249884) to T.J., from BBSRC (Biotechnology and Biological Sciences Research Council, UK) grants BB/L006324/1, BB/P007856/1 and BB/V014838/1 to I.P.N., from MEXT/JSPS KAKENHI (JP19H05707), JST CREST (JPMJCR20E3) to N.N.N., Austrian Science Fund (SFB-F-79) and European Research Council Grant (Project number: 101043370) to Y.D, from KAKENHI Grants-in-Aid for Scientific Research JP19H05708 from the Ministry of Education, Culture, Sports, Science and Technology of Japan AMED Grant Number JP21gm1410004 to H.N., from the National Natural Science Foundation of China (grants 31790403 and 82188101) to H.Z., and from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 259130777-SFB 1177 to C.B. and I.D., and the European Union (ERC, ER-REMODEL, 101055213) to I.D. M.W, S.M, and S.A.T. were supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001187, FC001999) and the Medical Research Council (FC001187, FC001999). This research was funded in whole, or in part, by the Wellcome Trust (FC001187, FC001999).</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><title>Structure and diversity of Atg8 family proteins.</title><p>(<bold>A</bold>) Structural organization of the Atg8/LC3/GABARAP proteins. (<bold>B</bold>) Neighbor-net analysis of 36 ATG8 nucleotide sequences from 14 eukaryotic species. The network was calculated with Splitstree4 applying the general time reversible (GTR) distance matrix using a conserved 332 nt position calculated with Gblocks from an original alignment of 666 nt positions in MUSCLE. Species labels are colored according to a major grouping into plant (green), fungal (orange), animal (black) or protozoan (blue) lineages. (Hs, <italic toggle=\"yes\">Homo sapiens</italic>; Sc, <italic toggle=\"yes\">Saccharomyces cerevisiae;</italic> D.m, <italic toggle=\"yes\">Drosophila melanogaster</italic>; C.e, <italic toggle=\"yes\">Caenorhabditis elegans</italic>; Dr, <italic toggle=\"yes\">Danio rerio</italic>; At, <italic toggle=\"yes\">Arabidopsis thaliana</italic>, Mp, <italic toggle=\"yes\">Marchantia polymorpha</italic>; Cr, <italic toggle=\"yes\">Chlamydomonas reinhardtii</italic>; Pt, <italic toggle=\"yes\">Paramecium tetraurelia</italic>; Dd, <italic toggle=\"yes\">Dictyostelium discoideum</italic>; Pf, <italic toggle=\"yes\">Plasmodium falciparum</italic>; Pt, <italic toggle=\"yes\">Phaeodactylum. tricornutum</italic>; Tv, <italic toggle=\"yes\">Trichomonas vaginalis</italic>)</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><title>Sequence alignment of Atg8/LC3/GABARAP proteins.</title><p>Sequence alignment of the Atg8-family members from 6 model species – yeast (<italic toggle=\"yes\">Saccharomyces cerevisiae</italic>), nematoda (<italic toggle=\"yes\">Caenorhabditis elegans</italic>), insects (<italic toggle=\"yes\">Drosophila melanogaster</italic>), bony fish (<italic toggle=\"yes\">Danio rerio</italic>), human (<italic toggle=\"yes\">Homo sapiens</italic>) and plant (<italic toggle=\"yes\">Arabidopsis thaliana</italic>). Secondary structure elements from the human LC3B (PDB ID 2ZJD) are shown at the top (H – α-helices, E – β-strands, L – long loops; rainbow color-code for α-helices – red, orange, yellow, green and cyan, all β-strands are in magenta). Every tenth residue in each sequence is marked bold/underlined, the catalytic Gly is marked green. The identity scores (* for identical residues,: for very similar residues, for analogous residues, space for residues without any similarity, - for gaps) are presented below each group of the Atg8/LC3/GABARAP. For the yeast Atg8 proteins, annotated UniProt entries for 11 yeast species Atg8 sequences were aligned to generate the identity score. The residues (or their absence) separating GABARAP/Atg8 and LC3 protein subtypes are marked red and blue, respectively. The consensus string for all 38 proteins aligned is presented at the bottom of alignment (named TOTAL SIMILARITY). The residues showed conservation are grouped within the following classes: residues participating in the protein folding (grey); residues forming HP1 (yellow); residues forming HP2 (light green); and residues forming UDS (cyan). The key residues indicating LC3 and Atg8/GABARAP subtypes difference are marked by green X.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><title>Atg8/LC3/GABARAP interactions with their partners.</title><p>(<bold>A</bold>). Structure of p62/SQSTM1-LIR:LC3B complex (PDB ID 2ZJD). Human LC3B is shown as a semi-transparent surface with the structural elements (α-helices and β-strands) visible. Murine p62/SQSTM1-LIR is shown as a main chain (orange) with sidechains of core LIR residues (W340 and L343, red) as sticks. Two hydrophobic pockets of LC3B, accommodating W340 and L343 sidechains, are shown on LC3B surface (HP1 – yellow, HP2 – light green). (<bold>B</bold>) Alignment of canonical (top) and non-canonical (bottom) LIR motifs with positions of residues indicated on top (from -6 to +8). Negatively charged residues (red), polar residues (magenta) and phosphorylatable residues (green) are indicated over the LIR sequences. The phosphorylatable residues confirmed to be phosphorylated are marked bold and underlined (see ##TAB##0##Table 1## for details). Residues at positions 0 and +3 within core LIR sequences whose sidechains are accommodated by HP1 and HP2 are boxed. Glutamate residues at position +7, forming additional intermolecular hydrogen bond to Arg/Lys at α-helix α3 in LC3/GABARAP proteins, are marked bold. Note that the enumeration in this work is according to N. Noda with the aromatic residue Θ as position 0, whereas this position is often numbered as +1 in many papers. (<bold>C</bold>) Interaction sites on Atg8/LC3/GABARAP surface. Left plot: surface representation of LC3B structure (the same orientation as in (A)), showing the main interacting sites - HP1 (yellow) and HP2 (light green), which form the LC3 docking site (LDS). Position of additional interacting site, HP0, is indicated by arrows. The alternative interacting area, the UIM docking site (UDS, including Y-site), is located on the opposite side of the LC3B molecule (right plot). The most relevant residues are colored dark red, additional hydrophobic residues around it are colored orange. (<bold>D</bold>) Structure of Hfl1 bound to the Y-site/UDS of Atg8 (PDB ID 6AAG). (<bold>E</bold>) Structural differences between LC3 and GABARAP proteins. (Right) Intramolecular contacts within LC3B (top) and GABARAP (bottom) proteins. Involved residues are presented as sticks, intramolecular hydrogen bonds between these residues are shown as black dashed lines. (Left) Orientation of H27 and K30 sidechains in LC3B (top) and corresponding Y25 and R28 sidechains in GABARAPs (bottom). Cation-π interactions (the non-covalent electrostatic interaction between an electron-rich face of aromatic rings and adjacent cations), stabilizing the specific orientation of Y25/R28 sidechains in GABARAPs are shown as blue dashed lines.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><title>Distribution of known phosphorylation sites on LIR motifs on some mammalian Atg8-interacting proteins.</title><p>Known positive (green circle with plus sign) or negative effects (red circle with minus sign) of phosphorylation (blue circle) of a particular residue relative to binding to Atg8s indicated. Yellow circle and half-filled green circle with plus sign indicate no regulation of ATG8 binding and positive effect specific to LC3 subfamily binding, respectively. The actual proteins are shown below each site. See also ##TAB##0##Table 1##.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><title>Emerging types of Atg8/LC3/GABARAP interacting motifs and elements</title><p>the antiparallel β-strand (<bold>A</bold>), the shuffled LIR motifs (<bold>B</bold>), and displacing α-helical structure (<bold>C</bold>). The interacting elements shown as blue arrows or cylinders on LC3B ribbon diagram (top) and on LC3B surface (bottom, with HP1 and HP2 indicated). The shuffled LIRs motifs are given as sequences in the blue boxes, their structural mechanism with Atg8/LC3/GABARAPs is not known, however, preliminary data indicate their LDS-guided binding.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Phosphorylation sites within LIR domains of mammalian proteins</title></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Protein</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">LIR domain</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Residue (kinase/phosphatase, if known)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AMBRA1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">EALN<bold><underline>S</underline></bold>GVEYY<bold>WDQL</bold>NETVFTVHSN</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S1014 (IKKβ)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##UREF##14##165##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ATG4B</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ERFFDSEDED<bold>FEIL<underline>S</underline></bold>L</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S249</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##28287329##94##,##REF##26378241##197##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Beclin 1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">RMM<bold><underline>S</underline></bold>TE<bold><underline>S</underline></bold>AN<bold><underline>S</underline>FTLI</bold>GEASDGGTME</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S90 (MAPKAPK2, MAPKAPK3, DAPK/PPP2); S93, S96 (AMPK)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##30767700##96##,##REF##26994142##198##–##UREF##22##200##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">BNIP3</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">GMQEE<bold><underline>S</underline></bold>LQG<bold><underline>S</underline>WVEL</bold>HF<bold><underline>S</underline></bold>NNGNGGS</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S13; S17 (ULK1), S24</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##23209295##170##,##REF##34654847##201##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">NIX/BNIP3L</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">LPPPAGLN<bold><underline>SS</underline>WVEL</bold>PMNSSNGNDN</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S34; S35 (ULK1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##28127051##132##,##REF##34654847##201##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">FAM134C</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold><underline>SS</underline></bold>DLD<bold><underline>T</underline></bold>DAEGDD<bold>FEEL</bold>DQSELSQLDP</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S435, S436, T440 (CK2)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##36044577##178##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">FUNDC1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">PQDYE<bold><underline>S</underline></bold>DDD<bold><underline>SY</underline>EVL</bold>DLTEYARRHQ</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S13 (CK2, PGAM5), S17 (ULK1), Y18 (PGAM5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##24746696##175##,##REF##22267086##176##,##REF##25126723##202##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">OPTN</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">LN<bold><underline>SS</underline></bold>G<bold><underline>SS</underline></bold>ED<bold><underline>S</underline>FVEI</bold>RMAEGEAEGS</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S170, S171, S173, S174; S177 (TBK1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##23805866##109##,##REF##21617041##167##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SCOC</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SRKEEEED<bold><underline>ST</underline>F<underline>T</underline>NI<underline>S</underline></bold>LADDIDHSS</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S12, T13, T15, S18</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##33845085##168##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">TEX264</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">YSE<bold><underline>S</underline></bold>GA<bold><underline>S</underline></bold>G<bold><underline>SS</underline>FEEL</bold>DLEGEGPLGE</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S266, S269, S271, S272 (CK2)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##REF##35417087##164##]</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">TNIP1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">KPPSSGT<bold><underline>SS</underline></bold>E<bold>FEVV</bold>TPEEQNSPES</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">S122, S123 (TBK1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">[##UREF##15##169##],[##REF##26118643##203##]</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P55\">\n<bold>Disclosure statement</bold>\n</p><p id=\"P56\">No potential conflict of interest was reported by the author(s).</p></fn></fn-group>" ]
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[{"label": ["2"], "person-group": ["\n"], "surname": ["Yamamoto", "Zhang", "Mizushima"], "given-names": ["H", "S", "N"], "article-title": ["Autophagy genes in biology and disease"], "source": ["Nat Rev Genet"], "year": ["2023"], "month": ["Jan"], "day": ["12"]}, {"label": ["10"], "person-group": ["\n"], "surname": ["Deretic", "Lazarou"], "given-names": ["V", "M"], "article-title": ["A guide to membrane atg8ylation and autophagy with reflections on immunity"], "source": ["J Cell Biol"], "year": ["2022"], "month": ["Jul"], "day": ["4"], "volume": ["221"], "issue": ["7"]}, {"label": ["12"], "person-group": ["\n"], "surname": ["Jia", "Wang", "Bhujabal", "Peters", "Mudd", "Duque", "Allers", "Javed", "Salemi", "Behrends", "Phinney"], "given-names": ["J", "F", "Z", "R", "M", "T", "L", "R", "M", "C", "B"], "article-title": ["Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage"], "source": ["J Cell Biol"], "year": ["2022"], "month": ["Nov"], "day": ["7"], "volume": ["221"], "issue": ["11"]}, {"label": ["20"], "person-group": ["\n"], "surname": ["Sanwald", "Dobner", "Simons", "Poschmann", "Stuhler", "Uffing", "Hoffmann", "Willbold"], "given-names": ["JL", "J", "IM", "G", "K", "A", "S", "D"], "article-title": ["Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition"], "source": ["Int J Mol Sci"], "year": ["2020"], "month": ["Dec"], "day": ["23"], "volume": ["22"], "issue": ["1"]}, {"label": ["42"], "person-group": ["\n"], "surname": ["Liu", "Yamasaki", "Du", "Coffman", "Ohsumi", "Nakatogawa", "Wu", "Noda", "Du"], "given-names": ["XM", "A", "XM", "VC", "Y", "H", "JQ", "NN", "LL"], "article-title": ["Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein"], "source": ["Elife"], "year": ["2018"], "month": ["Nov"], "day": ["19"], "volume": ["7"]}, {"label": ["58"], "person-group": ["\n"], "surname": ["Fukuda", "Ebi", "Saigusa", "Furukawa", "Yamashita", "Inoue", "Kobayashi", "Yoshida", "Kanki"], "given-names": ["T", "Y", "T", "K", "SI", "K", "D", "Y", "T"], "article-title": ["Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast"], "source": ["Elife"], "year": ["2020"], "month": ["Nov"], "day": ["3"], "volume": ["9"]}, {"label": ["62"], "person-group": ["\n"], "surname": ["Tomioka", "Kotani", "Kirisako", "Oikawa", "Kimura", "Hirano", "Ohsumi", "Nakatogawa"], "given-names": ["Y", "T", "H", "Y", "Y", "H", "Y", "H"], "article-title": ["TORC1 inactivation stimulates autophagy of nucleoporin and nuclear pore complexes"], "source": ["J Cell Biol"], "year": ["2020"], "month": ["Jul"], "day": ["6"], "volume": ["219"], "issue": ["7"]}, {"label": ["92"], "person-group": ["\n"], "surname": ["Jatana", "Ascher", "Pires", "Gokhale", "Thukral"], "given-names": ["N", "DB", "DEV", "RS", "L"], "article-title": ["Human LC3 and GABARAP subfamily members achieve functional specificity via specific stru ctural modulations"], "source": ["Autophagy"], "year": ["2019"], "month": ["Apr"], "day": ["14"], "fpage": ["1"], "lpage": ["17"]}, {"label": ["104"], "person-group": ["\n"], "surname": ["Tomczyk", "Suknovic", "Schenkelaars", "Wenger", "Ekundayo", "Buzgariu", "Bauer", "Fischer", "Austad", "Galliot"], "given-names": ["S", "N", "Q", "Y", "K", "W", "C", "K", "S", "B"], "article-title": ["Deficient autophagy in epithelial stem cells drives aging in the freshwater cnidarian Hydra"], "source": ["Development"], "year": ["2020"], "month": ["Jan"], "day": ["23"], "volume": ["147"], "issue": ["2"]}, {"label": ["135"], "person-group": ["\n"], "surname": ["Wesch", "Kirkin", "Rogov"], "given-names": ["N", "V", "VV"], "article-title": ["Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond"], "source": ["Cells"], "year": ["2020"], "month": ["Sep"], "day": ["1"], "volume": ["9"], "issue": ["9"]}, {"label": ["143"], "person-group": ["\n"], "surname": ["Turco", "Witt", "Abert", "Bock-Bierbaum", "Su", "Trapannone", "Sztacho", "Danieli", "Shi", "Zaffagnini", "Gamper"], "given-names": ["E", "M", "C", "T", "MY", "R", "M", "A", "X", "G", "A"], "article-title": ["FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates"], "source": ["Mol Cell"], "year": ["2019"], "month": ["Feb"], "day": ["26"]}, {"label": ["146"], "person-group": ["\n"], "surname": ["Ravenhill", "Boyle", "von Muhlinen", "Ellison", "Masson", "Otten", "Foeglein", "Williams", "Randow"], "given-names": ["BJ", "KB", "N", "CJ", "GR", "EG", "A", "R", "F"], "article-title": ["The Cargo Receptor NDP52 Initiates Selective Autophagy by Recruiting the ULK Complex to Cytosol-Invading Bacteria"], "source": ["Mol Cell"], "year": ["2019"], "month": ["Feb"], "day": ["28"]}, {"label": ["153"], "person-group": ["\n"], "surname": ["Sawa-Makarska", "Abert", "Romanov", "Zens", "Ibiricu", "Martens"], "given-names": ["J", "C", "J", "B", "I", "S"], "article-title": ["Cargo binding to Atg19 unmasks further Atg8-binding sites to mediate membrane-cargo apposition during selective autophagy"], "source": ["Nat Cell Biol"], "year": ["2014"], "volume": ["16"]}, {"label": ["163"], "person-group": ["\n"], "surname": ["Farnung", "Muhar", "Liang", "Tolmachova", "Benoit", "Corn", "Bode"], "given-names": ["J", "M", "JR", "KA", "RM", "JE", "JW"], "article-title": ["Human ATG3 contains a non-canonical LIR motif crucial for its enzymatic activity in autophagy"], "source": ["bioRxiv"], "year": ["2022"], "elocation-id": ["2022.08.02.502437"]}, {"label": ["165"], "person-group": ["\n"], "surname": ["Di Rita", "Peschiaroli", "Pasquale", "Strobbe", "Hu", "Gruber", "Nygaard", "Lambrughi", "Melino", "Papaleo"], "given-names": ["A", "A", "D", "D", "Z", "J", "M", "M", "G", "E"], "article-title": ["HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKK\u03b1"], "source": ["Nature communications"], "year": ["2018"], "volume": ["9"], "issue": ["1"], "fpage": ["1"], "lpage": ["18"]}, {"label": ["169"], "person-group": ["\n"], "surname": ["Zhou", "Rasmussen", "Olsvik", "Akimov", "Hu", "Evjen", "Kaeser-Pebernard", "Sankar", "Roubaty", "Verlhac", "van de Beck"], "given-names": ["J", "NL", "HL", "V", "Z", "G", "S", "DS", "C", "P", "N"], "article-title": ["TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1"], "source": ["J Cell Biol"], "year": ["2023"], "month": ["Feb"], "day": ["6"], "volume": ["222"], "issue": ["2"]}, {"label": ["183"], "person-group": ["\n"], "surname": ["Jain", "Rusten", "Katheder", "Elvenes", "Bruun", "Sjottem", "Lamark", "Johansen"], "given-names": ["A", "TE", "N", "J", "JA", "E", "T", "T"], "article-title": ["p62/sequestosome-1, autophagy-related gene 8 and autophagy in Drosophila are regulated by Nuclear factor erythroid 2-related factor 2 (NRF2), independent of transcription factor TFEB"], "source": ["J Biol Chem"], "year": ["2015"], "month": ["Apr"], "day": ["30"]}, {"label": ["187"], "person-group": ["\n"], "surname": ["Chatzichristofi", "Sagris", "Pallaris", "Eftychiou", "Kalvari", "Price", "Theodosiou", "Iliopoulos", "Nezis", "Promponas"], "given-names": ["A", "V", "A", "M", "I", "N", "T", "I", "IP", "VJ"], "article-title": ["LIRcentral: a manually curated online database of experimentally validated functional LIR-motifs"], "source": ["bioRxiv"], "year": ["2022"], "elocation-id": ["2022.06.21.496832"]}, {"label": ["188"], "person-group": ["\n"], "surname": ["Ibrahim", "Khandare", "Mirkin", "Tumtas", "Bubeck", "Bozkurt"], "given-names": ["T", "V", "FG", "Y", "D", "TO"], "article-title": ["AF2-multimer guided high accuracy prediction of typical and atypical ATG8 binding motifs"], "source": ["bioRxiv"], "year": ["2022"], "elocation-id": ["2022.09.25.509395"]}, {"label": ["191"], "person-group": ["\n"], "surname": ["Picchianti", "de Medina Hern\u00e1ndez", "Zhan", "Irwin", "Stephani", "Hornegger", "Beveridge", "Sawa-Makarska", "Lendl", "Grujic", "Martens"], "given-names": ["L", "VS", "N", "NAT", "M", "H", "R", "J", "T", "N", "S"], "article-title": ["Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy"], "source": ["bioRxiv"], "year": ["2022"], "elocation-id": ["2022.04.26.489478"]}, {"label": ["192"], "person-group": ["\n"], "surname": ["Stephani", "Picchianti", "Gajic", "Beveridge", "Skarwan", "Sanchez de Medina Hernandez", "Mohseni", "Clavel", "Zeng", "Naumann", "Matuszkiewicz"], "given-names": ["M", "L", "A", "R", "E", "V", "A", "M", "Y", "C", "M"], "article-title": ["A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress"], "source": ["Elife"], "year": ["2020"], "month": ["Aug"], "day": ["27"], "volume": ["9"]}, {"label": ["196"], "person-group": ["\n"], "surname": ["Kneussel", "Haverkamp", "Fuhrmann", "Wang", "W\u00e4ssle", "Olsen", "Betz"], "given-names": ["M", "S", "JC", "H", "H", "RW", "H"], "article-title": ["The \u03b3-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse"], "source": ["Proceedings of the National Academy of Sciences"], "year": ["2000"], "volume": ["97"], "issue": ["15"], "fpage": ["8594"], "lpage": ["8599"]}, {"label": ["200"], "person-group": ["\n"], "surname": ["Wei", "An", "Zou", "Sumpter", "Su", "Zang", "Sinha", "Gaestel", "Levine"], "given-names": ["Y", "Z", "Z", "R", "M", "X", "S", "M", "B"], "article-title": ["The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation"], "source": ["Elife"], "year": ["2015"], "month": ["Feb"], "day": ["18"], "volume": ["4"]}]
{ "acronym": [], "definition": [] }
203
CC BY
no
2024-01-13 00:12:45
Autophagy Rep. 2023 Mar 19; 2(1):27694127.2023.2188523
oa_package/cb/e3/PMC7615515.tar.gz
PMC7615516
38213473
[ "<title>Introduction</title>", "<p id=\"P5\">Obstructive sleep apnoea (OSA) is a multisystem, debilitating, chronic disorder of breathing during sleep, resulting in a relatively consistent pattern of cognitive deficits (##REF##25887982##Rosenzweig et al., 2015##; ##REF##28779504##Bucks et al., 2017##), particularly in attention, executive function and episodic memory (##REF##28779504##Bucks et al., 2017##). Moreover, there is a high prevalence of depression, anxiety and other psychiatric problems, which are only partially remediated by treatment (##REF##25887982##Rosenzweig et al., 2015##; ##REF##28779504##Bucks et al., 2017##).</p>", "<p id=\"P6\">Cognitive functions traditionally comprise broad domains of attention and memory, as well as those of higher order cognitive skills such as planning, problem-solving, and mental flexibility (grouped together as executive function), visuospatial abilities, processing speed, and both expressive and receptive language (##UREF##10##Esther Strauss, 2006##; ##REF##28779504##Bucks et al., 2017##; ##UREF##11##Rosenzweig et al., 2022##). Historically, a body of work has suggested significant impact of OSA on: attention and vigilance, long-term verbal and visual memory, expressive and receptive language (##REF##22913604##Bucks et al., 2013##; ##REF##23372268##Wallace and Bucks, 2013##), and visuo-spatial and constructional abilities (##REF##22913604##Bucks et al., 2013##). Similarly, deficits in the executive domain have also been demonstrated (##REF##23997362##Olaithe and Bucks, 2013##; ##REF##28779504##Bucks et al., 2017##), with somewhat less uniform evidence for short-term memory deficits (##UREF##11##Rosenzweig et al., 2022##). However, cognitive domains are not unitary constructs, and only judiciously deconstructed analysis of their different sub-capacities and their vulnerabilities to a range of risks and protective factors specific to OSA can provide a more accurate appraisal of a patient’s deficits (##UREF##12##Rosenzweig et al., 2017##, ##UREF##11##2022##).</p>", "<p id=\"P7\">Accordingly, OSA’s bidirectional link to neurodegenerative disorders, including Alzheimer’s disorder (AD), has similarly highlighted the importance of disentangling some of the major cognitive neuromechanisms at play (##REF##18795985##Ancoli-Israel et al., 2008##; ##REF##19968005##Cooke et al., 2009##; ##REF##25878183##Osorio et al., 2015##; ##REF##35550019##Bubu et al., 2022##). However, the links between severity of OSA, historically indexed by apnea-hypopnea index (AHI) or respiratory disturbance index (RDI), or by indices of hypoxia severity, sleep fragmentation, or sleepiness (##REF##15764750##Pépin et al., 2005##; ##REF##28779504##Bucks et al., 2017##), and the severity of the cognitive deficits observed, are far from being well understood (for more in-depth review please refer to ##REF##15327723##Aloia et al., 2004##; ##REF##23997362##Olaithe and Bucks, 2013##; ##REF##23372268##Wallace and Bucks, 2013##; ##REF##25070768##Gagnon et al., 2014##; ##REF##25887982##Rosenzweig et al., 2015##; ##REF##28779504##Bucks et al., 2017##). Similarly poorly understood, is the link between the timing of the hypoxia or arousal during each sleep cycle, and the severity or the cognitive phenotype (##REF##25887982##Rosenzweig et al., 2015##).</p>", "<p id=\"P8\">Moreover, the link between cognition, OSA, and aging, has proven equally difficult to fully discern (##UREF##11##Rosenzweig et al., 2022##). For instance, aging is known to be independently linked with physiological changes that may predispose to OSA (##UREF##11##Rosenzweig et al., 2022##). It has been proposed that this may be in part due to changes in upper airway morphology that can lead to a reduction in upper airway dilator muscle function at sleep onset (##REF##28779504##Bucks et al., 2017##), contributing to an age-related propensity for upper airway collapse in response to negative pressure (##REF##18420722##Kirkness et al., 2008##) independent of body mass index (##REF##17413053##Eikermann et al., 2007##; ##UREF##11##Rosenzweig et al., 2022##). Against this background, it has been argued that some of these aging-associated processes may underlie the failure to find a consistent relationship between the severity of OSA and the risk of cognitive impairment (##REF##28779504##Bucks et al., 2017##; ##UREF##11##Rosenzweig et al., 2022##).</p>", "<p id=\"P9\">This evident polymorphic picture is further compounded by the fact that not everyone with OSA is cognitively impaired (##REF##16815753##Quan et al., 2006##), with the individual’s cognitive reserve (##REF##15743336##Alchanatis et al., 2005##; ##REF##20394321##Sforza et al., 2010##; ##REF##22913604##Bucks et al., 2013##; ##REF##25325480##Martin et al., 2015##; ##REF##24839237##Olaithe et al., 2015##; ##UREF##14##Schembri et al., 2017##) and their genetic risk (##REF##18083630##Cosentino et al., 2008##; ##REF##23729930##Nikodemova et al., 2013##) for neurodegenerative decline (##REF##28779504##Bucks et al., 2017##) possibly also playing an important role (##UREF##11##Rosenzweig et al., 2022##).</p>", "<p id=\"P10\">Taken together, it has been proposed that only middle-aged patients with OSA may demonstrate a consistent pattern of cognitive deficits, otherwise lacking in older patients (##REF##31881487##Bubu et al., 2020##). Notably, it has also been argued that deficits are principally driven by common cardiovascular and metabolic comorbidities, rather than by distinct OSA-processes (##REF##31881487##Bubu et al., 2020##). In further support of this, patients with OSA invariably present with already established comorbidities, such as overweight or obesity, sleepiness in passive situations or while driving, and are often affected by systemic hypertension, type 2 diabetes, and dyslipidemia (##REF##27188535##Levy et al., 2015##), making it in most cases impossible to delineate the specific contribution of all associated risks to the resulting cognitive presentation (##REF##30809382##Bonsignore et al., 2019##; ##REF##31881487##Bubu et al., 2020##; ##UREF##11##Rosenzweig et al., 2022##).</p>", "<p id=\"P11\">Whether OSA itself, or these common comorbidities drive the cognitive effects has wide ranging clinical implications, and may impact future clinical guidelines with treatment of comorbidities taking precedence over treatment of the core determinants of neuropathological process in OSA, such as sleep fragmentation (##REF##23910433##Jordan et al., 2014##), sleep disruption and blood gas abnormalities (##REF##28760549##Olaithe et al., 2018##).</p>", "<p id=\"P12\">To this end, and in order to clarify whether, independent of concomitant metabolic or cardiovascular comorbidities, OSA-induced injury (##REF##25887982##Rosenzweig et al., 2015##) may present with abnormal functional outcomes (##REF##31881487##Bubu et al., 2020##; ##UREF##11##Rosenzweig et al., 2022##), we undertook a proof of the concept study and set to define the cognitive pattern in a (rare) group of male, middle-aged patients with untreated OSA who present without comorbidities, compared to matched controls.</p>" ]
[ "<title>Methods</title>", "<p id=\"P13\">Preliminary analysis of cognitive parameters in patients with different OSA severities was undertaken as a part of the multimodal clinical study InCOSA (<ext-link xlink:href=\"https://clinical.trials.gov/\" ext-link-type=\"uri\">Clinical.Trials.gov</ext-link>, identifier: NCT02967536). All experimental protocols were approved by the U.K. Research Ethics Committee [Integrated Research Application System (IRAS): IRAS-Project-ID-170912; REC-REF16/L0/0893] and informed consent for study participation was obtained from all participants. Due to the nature of several investigations in the overarching multimodal clinical study, some of which are known to have significant sex and gender differences, only male participants were included. Thus, 27 male adult (35–70 years-old), non-obese [body-mass-index (BMI) &lt; 30 kg/m<sup>2</sup>], mildly somnolent [Epworth Sleepiness Scale (ESS); 5 &gt; ESS &lt; 15] patients with no current or past co-morbidities, and no current or past alcohol or smoking history, with a <italic toggle=\"yes\">de novo</italic> diagnosis of OSA according to ICSD criteria (##UREF##0##American Academy of Sleep Medicine, 2014##), and a group of healthy sex- and education-matched individuals were identified, as previously described (##REF##33939202##Gnoni et al., 2021##) (##SUPPL##0##Supplementary material##). All methods were carried out in accordance with relevant UK and international guidelines and regulations.</p>", "<p id=\"P14\">All participants underwent a domiciliary respiratory testing <italic toggle=\"yes\">via</italic> WatchPAT system (<ext-link xlink:href=\"https://www.itamar-medical.com/\" ext-link-type=\"uri\">https://www.itamar-medical.com/</ext-link>), as previously described (##UREF##15##Walter et al., 2023##). Additionally, patients also underwent a video-polysomnography (vPSG) in the sleep center. Full night vPSG recordings were based on the international 10:20 system; for purposes of the PSG scoring, six EEG channels (i.e., F3, F4, C3, C4, O1, and O2) were referenced to the mastoid, and used along with electrooculography, submental-electromyography, respiratory inductance plethysmography, nasal pressure sensor, oronasal thermistor, pulse-oximeter, two-lead electrocardiogram, body position detector and synchronized audio-visual recording, as previously described (##REF##27322475##Rosenzweig et al., 2016##). The scoring was carried out according to AASM rules (##REF##28416048##Berry et al., 2017##), and as previously described (##REF##27322475##Rosenzweig et al., 2016##). Eleven cognitive domains were pre-selected (##REF##28779504##Bucks et al., 2017##), based on previous reports of OSA- and depression-related deficits, and assessed <italic toggle=\"yes\">via</italic> 23 automated Cambridge-Neuropsychological-Test-Automated-Battery (CANTAB) tests.</p>", "<title>Cambridge neuropsychological test automated battery</title>", "<p id=\"P15\">CANTAB is a highly sensitive, validated touchscreen-based cognitive assessment. In this study, 11 domains were tested with the following tests (for more in-depth explanations please refer to ##SUPPL##0##Supplementary material##). <italic toggle=\"yes\">Reaction Time Task (RTT)</italic> (##UREF##1##Cognition, n.d.a##) tests reaction time, movement time, and vigilance, which are associated with motor pathway and right anterior hemispheric functioning (##REF##9863686##Coull et al., 1998##). <italic toggle=\"yes\">Spatial Working Memory (SWM)</italic> tests (##UREF##2##Cognition, n.d.b##) the retention and manipulation of visuospatial data in non-verbal and visuospatial working memory (##UREF##2##Cognition, n.d.b##), which are associated with frontal lobe function. <italic toggle=\"yes\">Pattern Recognition Memory (PRM)</italic> tests (##UREF##1##Cognition, n.d.a##) short-term visual memory in a two choice forced discrimination paradigm in both immediate and delayed conditions, which are associated with frontoparietal and posterior parietal function (##REF##12177449##Pessoa et al., 2002##; ##REF##15085133##Todd and Marois, 2004##). <italic toggle=\"yes\">The Emotion Recognition Task (ERT)</italic> (##UREF##3##Cognition, n.d.c##) assesses social cognition and emotion recognition (##REF##30595441##Glenthøj et al., 2019##), which are associated with the limbic system, inferior frontal gyrus, parietal lobe, cingulate cortex and inferior and middle temporal lobe functioning (##REF##20194514##Keightley et al., 2011##). Participants are shown a computer-generated face for 200 ms, after which the emotion displayed by the face must be selected from six options, i.e., sadness, happiness, fear, anger, disgust, and surprise. The outcome measures are the median reaction time and the total number of correct answers (##UREF##3##Cognition, n.d.c##). People with depression are likely to provide more negative ratings of emotional expression, reflecting the well-known negative bias seen in depression (##UREF##3##Cognition, n.d.c##). During testing, brief presentation encourages implicit processing, as opposed to conscious appraisal of the faces (##UREF##3##Cognition, n.d.c##). Conversely, in individuals at ultra-high risk of developing psychosis, longer emotion recognition latency, rather than lower accuracy has been demonstrated (##REF##30595441##Glenthøj et al., 2019##). <italic toggle=\"yes\">The Attention Switching Task (AST)</italic> (##UREF##4##Cognition, n.d.d##) tests executive functioning and cued attentional set-shifting, which are functions of the medial frontal structures and the anterior right hemisphere (##REF##8232848##Bench et al., 1993##). <italic toggle=\"yes\">Spatial Span Memory (SSP)</italic> (##UREF##4##Cognition, n.d.d##) assesses visuospatial working memory capacity which is associated with frontoparietal function (##REF##22973241##Jones and Berryhill, 2012##; ##REF##26257053##Ester et al., 2015##). <italic toggle=\"yes\">The Paired Associates Learning (PAL)</italic> tests (##UREF##5##Cognition, n.d.e##) episodic visuospatial memory and associative learning, which are predominantly functions of the temporal lobe. One <italic toggle=\"yes\">Touch Stockings of Cambridge (OTS)</italic> (##UREF##6##Cognition, n.d.f##) tests spatial planning and working memory and it is a measure of dorsolateral prefrontal cortex function (##REF##7695894##Goldman-Rakic, 1995##). <italic toggle=\"yes\">Delayed Matching to Sample</italic> (DMS) (##UREF##7##Cognition, n.d.g##) assesses both simultaneous visual matching ability and short-term visual recognition memory, for non-verbalizable patterns, which is associated with medial temporal lobe function (##REF##11992524##Lavenex et al., 2002##; ##REF##16010661##Lee et al., 2005##). The <italic toggle=\"yes\">Rapid Visual Information Processing (RVP)</italic> (##UREF##7##Cognition, n.d.g##) is a measure of sustained attention, associated with frontoparietal function (##REF##11336780##Sarter et al., 2001##). <italic toggle=\"yes\">Stop Signal Task (SST)</italic> (##UREF##8##Cognition, n.d.h##) is a test of impulse control and response inhibition associated with prefrontal cortex function (##REF##11336780##Sarter et al., 2001##).</p>", "<title>Statistical analyses</title>", "<p id=\"P16\">Group differences were analyzed with 2-way ANCOVA, corrected for multiple comparisons using Bonferroni test with additional pairwise tests. Age and BMI are used as covariates in the ANCOVA model and are evaluated at the following values: Age = 41.59 years, BMI = 26.78 kg/m<sup>2</sup>. Differences in socio-demographic characteristics are evaluated with <italic toggle=\"yes\">t</italic>-test for independent samples (continuous variables) and Fisher’s exact test or Fisher-Freeman-Halton’s exact test (categorical variables). All <italic toggle=\"yes\">P</italic>-values below 0.05 were considered significant. MedCalc<sup>®</sup> Statistical Software version 20.216 (MedCalc Software Ltd., Ostend, Belgium; <ext-link xlink:href=\"https://www.medcalc.org\" ext-link-type=\"uri\">https://www.medcalc.org</ext-link>; 2023) was used in statistical analysis and graphical presentations.</p>" ]
[ "<title>Results</title>", "<p id=\"P17\">Twenty-seven OSA patients and seven healthy controls (see ##TAB##0##Table 1##) completed the CANTAB (##TAB##1##Table 2##). Of the 27 patients, 16 were diagnosed with mild OSA (AHI mean ± S.D.:11.7 ± 4.0 events/h; mean age 42.6 ± 8.2 years; mean BMI 26.7 ± 4.1 kg/m<sup>2</sup>), and 11 with severe OSA (AHI 41.8 ± 20.7 events/h; age: 46.9 ± 10.9 years, BMI: 28.0 ± 3.2 kg/m<sup>2</sup>), according to ICSD criteria (##UREF##0##American Academy of Sleep Medicine, 2014##).</p>", "<p id=\"P18\">The cognitive findings for the whole set of behavioral readouts for the three experimental groups (control, mild OSA, and severe OSA), controlled for age and BMI and grouped into CANTAB sub-tests are shown in ##TAB##2##Table 3##. Distinct deficits were observed in the tests investigating cognitive domains of vigilance, executive functioning, short-term visual recognition memory and social and emotion recognition, with the greatest number of differences between controls and those with severe OSA. Whilst subjects with mild OSA performed better than those with severe OSA on most of those same tasks, they were rarely worse than controls (see ##FIG##0##Figure 1##).</p>", "<p id=\"P19\">The most significant deficits, by comparison to the control group, were demonstrated in the tests that assess both simultaneous visual matching ability and short-term visual recognition memory for non-verbalizable patterns (##FIG##0##Figure 1##; DMS), tests of executive functioning and cued attentional set shifting (##FIG##0##Figure 1##; AST), in vigilance and psychomotor functioning (RTT), and lastly, in social cognition and emotion recognition (ERT).</p>", "<p id=\"P20\">For full details of the cognitive findings please refer to ##TAB##1##Tables 2##, ##TAB##2##3## and ##SUPPL##0##Supplementary material##.</p>" ]
[ "<title>Discussion</title>", "<p id=\"P21\">We report a distinct pattern of circumscribed cognitive deficits in middle-aged male patients with severe OSA, in the absence of any overt neuropsychiatric, cardiovascular or metabolic co-morbidities (##REF##31881487##Bubu et al., 2020##). The findings are largely in keeping with previous studies of OSA patients with associated multiple comorbidities that similarly showed aberrant executive-functioning, visuospatial short-term-memory, deficits in vigilance and psychomotor control (##REF##28779504##Bucks et al., 2017##; ##UREF##11##Rosenzweig et al., 2022##). Thus, arguably, our findings suggest that distinct OSA-driven processes, particularly when OSA is severe, may be sufficient for cognitive changes to occur as early as middle age, in otherwise healthy male individuals.</p>", "<p id=\"P22\">Remarkably, we also report, for the first time, diminished social cognition in this group of middle-aged severe OSA patients. Social and emotional cognition is an important ability to interpret and identify socially relevant information, known to be impaired in several psychiatric conditions, including major depressive disorder, and thought to be strongly associated with sleep physiology (##REF##20421251##Gujar et al., 2011##; ##REF##25566100##Weightman et al., 2014##). In past studies, sleep deprivation has been shown to selectively impair the accurate judgment of human facial emotions, especially threat relevant and reward relevant categories (##REF##20337191##van der Helm et al., 2010##). Significant deficits in emotional facial recognition have been previously also reported following a night of sleep fragmentation, without significant reduction of total sleep time (##REF##35181390##Lee et al., 2022##). In keeping, it has been suggested that the disruption of normal sleep process, and not the reduction of sleep time, may likewise play the role (##REF##35181390##Lee et al., 2022##). Moreover, there is evidence to suggest that emotional facial recognition can be sleep-stage dependent, with REM sleep known to play a critical role on both emotional and neutral face recognition (##UREF##9##Cunningham and Payne, 2017##; ##REF##35181390##Lee et al., 2022##). Thus, it is likely that sleep fragmentation and associated sleep loss in our OSA patients, particularly REM-related fragmentation (##UREF##9##Cunningham and Payne, 2017##), may act to impair discrete affective neural systems, disrupting the identification of salient affective social cues (##REF##20337191##van der Helm et al., 2010##).</p>", "<p id=\"P23\">More recently, in a thought provoking set of studies, sleep loss and sleep’s diminished quality and or quantity, which indeed present one of the important features of OSA, have also been linked to diminished altruism (##REF##35998121##Ben Simon et al., 2022##). Specifically, the authors argued that sleep loss represents one previously unrecognized factor that may dictate whether humans choose to help each other, which they based on their observations at three different scales, within individuals, across individuals, and across societies (##REF##35998121##Ben Simon et al., 2022##). For instance, in one of the studies, one night of sleep loss was shown to trigger the withdrawal of help from one individual to another, with the associated fMRI findings showing deactivation of key nodes within the social cognition brain network that facilitate prosociality (##REF##35998121##Ben Simon et al., 2022##).</p>", "<p id=\"P24\">Following this argument and our findings, as well as taking into account that currently around one-seventh of the world’s adult population, or approximately one billion people, are estimated to have OSA (##REF##32436658##Lyons et al., 2020##), the clinical and societal impact of OSA’s effects on cognition, even in the absence of any associated co-morbidities, dictates urgent attention and a joint multidisciplinary effort. It is increasingly evident that OSA’s functional neuropsychiatric impact may go well beyond OSA’s currently best recognized role in increasing driving and occupational accidents risks (##REF##28779504##Bucks et al., 2017##; ##UREF##11##Rosenzweig et al., 2022##).</p>", "<p id=\"P25\">We believe that our pilot study, despite limitations, including its size, a small control group, multiple comparisons, and a cross-sectional design, significantly contributes to understanding of the complex interplay between OSA-severity and cognitive problems. Critically, our data also reveal a threshold effect in the cognitive domain of executive functioning. Furthermore, it appears that cognitive deficits in this age group are greatest in male patients with severe OSA, likely suggestive of already existent widespread intricate physiologic central nervous changes, and in further support of early treatment for this patient group (##REF##25887982##Rosenzweig et al., 2015##; ##REF##33939202##Gnoni et al., 2021##).</p>", "<p id=\"P26\">Finally, another important limitation to any direct translational generalization of our findings lies in inclusion of male participants only. Whilst this enabled controlling for possible effects of the oestrous cycle, it also prevents us from generalizing to female patients. Moreover, over the last decade, pioneering new findings suggest a spectrum of changes in the brain metabolism during the pre-, peri-, and post-menopausal period (##REF##34108509##Mosconi et al., 2021##), all of which may arguably interplay with OSA pathomechanisms (##REF##16171289##Driver et al., 2005##; ##UREF##13##Saaresranta et al., 2015##), as well as underlie its links with neurodegenerative processes and cognitive deficits (##REF##29223769##Polsek et al., 2018##) in female patients with OSA.</p>", "<p id=\"P27\">In conclusion, future multi-center multi-modal longitudinal studies should confirm these findings, as well as decipher how these cognitive deficits may interplay in men and women with other comorbidity-driven impairments over time.</p>" ]
[]
[ "<title>Introduction</title>", "<p id=\"P1\">Obstructive sleep apnoea (OSA) is a multisystem, debilitating, chronic disorder of breathing during sleep, resulting in a relatively consistent pattern of cognitive deficits. More recently, it has been argued that those cognitive deficits, especially in middle-aged patients, may be driven by cardiovascular and metabolic comorbidities, rather than by distinct OSA-processes, such as are for example ensuing nocturnal intermittent hypoxaemia, oxidative stress, neuroinflammation, and sleep fragmentation.</p>", "<title>Methods</title>", "<p id=\"P2\">Thus, we undertook to define cognitive performance in a group of 27 middle-aged male patients with untreated OSA, who had no concomitant comorbidities, compared with seven matched controls (AHI mean ± S.D.: 1.9 ± 1.4 events/h; mean age 34.0 ± 9.3 years; mean BMI 23.8 ± 2.3 kg/m<sup>2</sup>). Of the 27 patients, 16 had mild OSA (AHI mean ± S.D.:11.7 ± 4.0 events/h; mean age 42.6 ± 8.2 years; mean BMI 26.7 ± 4.1 kg/m<sup>2</sup>), and 11 severe OSA (AHI 41.8 ± 20.7 events/h; age: 46.9 ± 10.9 years, BMI: 28.0 ± 3.2 kg/m<sup>2</sup>).</p>", "<title>Results</title>", "<p id=\"P3\">In our patient cohort, we demonstrate poorer executive-functioning, visuospatial memory, and deficits in vigilance sustained attention, psychomotor and impulse control. Remarkably, we also report, for the first time, effects on social cognition in this group of male, middle-aged OSA patients.</p>", "<title>Conclusion</title>", "<p id=\"P4\">Our findings suggest that distinct, OSA-driven processes may be sufficient for cognitive changes to occur as early as in middle age, in otherwise healthy individuals.</p>" ]
[ "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgments</title>", "<p>The authors are immensely grateful to all patients and colleagues at the Sleep Disorders Center, Guy’s and St. Thomas’ Hospital, as well as colleagues at the Sleep and Brain Plasticity Center, King’s College London for their input and help at various stages of this project. Part of these data were previously shown in the form of a poster presentation at the World Sleep Congress in Rome, 2022.</p>", "<title>Funding</title>", "<p>This research was funded in whole, or in part, by the Wellcome Trust (103952/Z/14/Z). For the purpose of open access, IR has applied a CC BY Public Copyright License to any Author Accepted Manuscript version arising from this submission. AY’s independent research was funded by the National Institute for Health Research (NIHR) Biomedical Research Center at South London and Maudsley NHS Foundation Trust and King’s College London.</p>", "<title>Data availability statement</title>", "<p id=\"P28\">The datasets presented in this article are not readily available because all data that support the findings of this study will be made available upon reasonable request from the corresponding author, once the appropriate ethics amendments are sought to accommodate this request. Requests to access the datasets should be directed to the U.K. Research Ethics Committee [Integrated Research Application System (IRAS)].</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Violin plots depict results of significant CANTAB tests’ findings for controls, mild OSA and severe OSA. When controlled for the influence of age and BMI, out of all CANTAB modalities (23 tests in 11 modalities), only Emotion Recognition Task, Attention Switching Task, Reaction Time and Delayed Matching to Sample showed significant differences between the groups, mainly between controls and severe OSA. Dots, squares and triangles show individual values (control, mild OSA and severe OSA, respectively). Thick dashed lines inside violins indicate group median, with thinner dashed lines indicating quartiles (*<italic toggle=\"yes\">P</italic> &lt; 0.05, **<italic toggle=\"yes\">P</italic> &lt; 0.01, ***<italic toggle=\"yes\">P</italic> &lt; 0.0001; two-way ANCOVA with Bonferroni’s correction for multiple comparisons controlled for age and BMI). RTIFDMRT, Median duration between stimulus onset and release of button; RTIFMMT, Mean time taken to touch stimulus after button release; ERTOMDRT, Median latency of response from stimulus onset to subject response; ERTTH, Number of correctly answered responses; ASTSWMD, Median latency of response in rule switching trials; ASTLCMD, Median latency of response on congruent trials; DMSPC, Percentage of assessment trials during which subject selected the correct box on their first box choice; DMSMDLAD, Median latency from the available choices being displayed to the subject choosing the correct choice.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Socio-demographic and clinical characteristics.</title></caption><table frame=\"box\" rules=\"all\"><thead style=\"background-color:#8F9496\"><tr><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">HC</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Mild OSA</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Severe OSA</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Control vs. Mild OSA</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Control vs. Severe OSA</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Mild OSA vs. Severe OSA</styled-content>\n</th></tr><tr><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">(<italic toggle=\"yes\">n</italic> = 7)</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">(<italic toggle=\"yes\">n</italic> = 16)</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">(<italic toggle=\"yes\">n</italic> = 11)</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">\n<italic toggle=\"yes\">P</italic>\n</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">\n<italic toggle=\"yes\">P</italic>\n</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">\n<italic toggle=\"yes\">P</italic>\n</styled-content>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Age (years)<sup><xref rid=\"TFN1\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean (SD)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">34.0 (9.3)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">42.6 (8.2)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">46.9 (10.9)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0380</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0199</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2484</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">BMI (kg/m<sup>2</sup>)<sup><xref rid=\"TFN1\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean (SD)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23.8 (2.3)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">26.7 (4.1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">28.0 (3.2)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0957</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0094</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.4137</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">AHI<sup><xref rid=\"TFN1\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean (SD)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.9 (1.4)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11.7 (4.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41.8 (20.7)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">&lt;0.0001</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0001</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">&lt;0.0001</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Ethnicity<sup><xref rid=\"TFN2\" ref-type=\"table-fn\">b</xref></sup></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.6971</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5769</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1986</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Caucasian</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5 (71.4)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13 (81.3)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7 (63.6)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Asian</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (28.6)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (12.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 (9.1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Indian</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 (6.3)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Afro-Caribbean</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (18.2)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Middle east</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 (9.1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Smoking<sup><xref rid=\"TFN2\" ref-type=\"table-fn\">b</xref></sup></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1243</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0535</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Ex-smoker</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6 (37.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Non-smoker</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7 (100.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10 (62.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11 (100.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Smoker</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Education<sup><xref rid=\"TFN2\" ref-type=\"table-fn\">b</xref></sup></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0735</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2450</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1614</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Undergrade</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (28.6)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11 (68.8)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4 (36.4)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Graduate</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5 (71.4)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3 (18.7)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6 (54.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">A level</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2 (12.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 (9.1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr style=\"background-color:#DEE1E1; border-top: hidden\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Age at leaving education<sup><xref rid=\"TFN1\" ref-type=\"table-fn\">a</xref></sup></td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mean (SD)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">25.6 (4.1)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.5 (3.2)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">26.3 (9.3)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0627</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.8535</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2184</td></tr><tr style=\"background-color:#DEE1E1; border-top: hidden\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Exercise regularly<sup><xref rid=\"TFN3\" ref-type=\"table-fn\">c</xref></sup></td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1243</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2450</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.6924</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6 (37.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3 (27.3)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">n</italic> (%)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7 (100.0)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10 (62.5)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8 (72.7)</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Estimated marginal means of analyzed clinical parameters controlled for the influence of age and BMI (two-way ANCOVA).</title></caption><table frame=\"box\" rules=\"all\"><thead style=\"background-color:#8F9496\"><tr><th align=\"center\" valign=\"top\" rowspan=\"4\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Dependent variable</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"12\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Group</styled-content>\n</th></tr><tr><th align=\"center\" valign=\"top\" colspan=\"4\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Control <italic toggle=\"yes\">n</italic> = 7</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"4\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Mild OSA <italic toggle=\"yes\">n</italic> = 16</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"4\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Severe OSA <italic toggle=\"yes\">n</italic> = 11</styled-content>\n</th></tr><tr><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Mean</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">SEM</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"2\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">95% CI</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Mean</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">SEM</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"2\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">95% CI</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Mean</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">SEM</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"2\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">95% CI</styled-content>\n</th></tr><tr><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Lower</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Upper</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Lower</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Upper</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Lower</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Upper</styled-content>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ASTLSWMD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">564.45</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">48.30</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">464.98</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">663.93</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">758.77</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31.92</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">693.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">824.50</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">789.18</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">704.30</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">874.07</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ASTLCMD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">702.94</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">36.67</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">627.42</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">778.46</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">626.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">24.23</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">576.97</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">676.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">520.67</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31.29</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">456.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">585.11</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ERTOMDRT</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,939.65</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">195.30</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,537.41</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,341.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,398.66</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">129.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,132.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,664.44</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,133.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">166.66</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">789.80</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,476.29</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ERTTH</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">48.92</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.96</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">40.76</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">57.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">57.49</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.62</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">52.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">62.89</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">64.89</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.38</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">57.92</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">71.85</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">PALTEA</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.76</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23.46</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.63</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.79</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20.37</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9.24</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">16.66</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">PALFAMS</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.45</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.49</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.38</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.52</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.81</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.98</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.84</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.35</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.27</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.73</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9.97</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">PRMPCI</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">99.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.15</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">93.30</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">106.25</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91.98</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.08</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">87.70</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">96.26</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">96.58</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.68</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">102.11</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">PRMPCD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78.29</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.58</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">66.79</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">89.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">88.51</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.69</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">80.91</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">96.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">88.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.76</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">78.36</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">97.98</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">RTIFDMRT</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">391.42</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">365.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">417.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">384.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.47</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">367.34</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">402.23</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">349.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10.94</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">326.65</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">371.70</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">RTIFMMT</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">266.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13.47</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">239.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">294.64</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">240.85</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">222.52</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">259.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">197.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11.49</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">173.92</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">221.25</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SWMBE</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23.81</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.33</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">33.28</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.79</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">25.31</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.58</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.93</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.49</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.66</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SWMS</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.25</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.03</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.16</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.47</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.77</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.16</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.51</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.70</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SSPSFSL</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.81</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.53</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.71</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.91</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.91</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.35</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.18</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.64</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.46</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.66</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.54</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SSPRSL</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.91</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.57</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.73</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.57</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.38</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.79</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.35</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.39</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.49</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.38</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.40</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">OTSPSFC</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10.28</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.55</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.73</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10.60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.33</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.94</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10.40</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.26</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DMSPC</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91.32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.62</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">85.93</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">96.70</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91.83</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.73</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">88.27</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">95.39</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">89.01</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.23</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">84.41</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">93.60</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DMSMDLAD</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4,835.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">332.58</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4,150.11</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5,520.01</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3,188.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">219.75</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,736.31</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3,641.50</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,466.32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">283.80</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,881.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3,050.82</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DMSPEGE</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-0.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-0.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">-0.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.20</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">RVPA</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.85</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.95</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.92</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.95</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.95</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.91</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.99</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">RVPMDL</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">657.81</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">68.07</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">517.61</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">798.01</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">494.93</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">44.98</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">402.29</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">587.57</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">421.81</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">58.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">302.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">541.45</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SSTSSRT</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">256.98</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.51</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">227.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">286.86</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">239.52</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">219.77</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">259.27</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">218.15</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12.38</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">192.64</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">243.65</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><title>Summary of statistical analysis (two-way ANCOVA with Bonferroni’s correction for multiple comparisons) for all CANTAB behavioral tasks and all pair-wise comparisons between groups (control, mild OSA, and severe OSA).</title></caption><table frame=\"box\" rules=\"all\"><thead style=\"background-color:#8F9496\"><tr><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Pairwise comparisons</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Predicted (LS) mean diff.</styled-content>\n</th><th align=\"center\" valign=\"top\" colspan=\"2\" rowspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">95% CI of diff.</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Below threshold</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Summary</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Adjusted <italic toggle=\"yes\">P</italic>-value</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Effect size</styled-content>\n</th></tr><tr><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Lower</styled-content>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<styled-content style-type=\"color\" style=\"#FFFFFF\">Upper</styled-content>\n</th></tr></thead><tbody><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>ASTLSWMD</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−194.32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−345.94</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−42.71</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN6\" ref-type=\"table-fn\">**</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0090</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.521</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−224.73</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−400.66</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−48.80</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN6\" ref-type=\"table-fn\">**</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0090</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.698</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−30.41</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−161.65</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">100.83</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.230</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>ASTLCMD</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">76.07</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−39.03</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">191.17</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.3070</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.784</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">182.28</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">48.71</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">315.84</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN6\" ref-type=\"table-fn\">**</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0050</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.815</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">182.28</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.57</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">205.84</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN5\" ref-type=\"table-fn\">*</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0340</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.058</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>ERTOMDRT</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">540.99</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−72.07</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,154.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0970</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.047</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">806.60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">95.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,517.98</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN5\" ref-type=\"table-fn\">*</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0220</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.508</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">265.61</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−265.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">796.29</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.6320</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.497</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>ERTTH</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−8.57</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−21.01</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.87</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2680</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.830</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−15.96</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−30.40</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.53</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN5\" ref-type=\"table-fn\">*</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0270</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.470</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−7.39</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−18.16</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.38</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2710</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.681</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>PALTEA</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−13.12</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">13.39</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.012</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.52</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−9.86</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.477</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.39</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−6.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">16.86</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.7190</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.466</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>PALFAMS</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−4.03</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.163</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.90</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−7.33</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.53</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.465</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.54</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−6.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.51</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.3590</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.623</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>PRMPCI</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.80</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.08</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">17.67</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1610</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.937</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.19</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−8.26</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14.65</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5380</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.371</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−4.60</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−13.15</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3.95</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5380</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.534</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>PRMPCD</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−10.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−27.74</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.30</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.4410</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.692</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−9.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−30.21</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10.44</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.6710</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.647</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.33</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−14.83</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">15.50</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.022</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>RTIFMDRT</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.64</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−33.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">46.87</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.196</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">42.25</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−4.44</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">88.94</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0860</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.203</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">35.61</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">70.44</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN5\" ref-type=\"table-fn\">*</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0440</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.015</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>RTIFMMT</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">26.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−16.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">68.32</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.3790</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.732</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">69.31</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20.26</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">118.36</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN6\" ref-type=\"table-fn\">**</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0040</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.879</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">43.26</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">6.67</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">79.85</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN5\" ref-type=\"table-fn\">*</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0170</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.173</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>SWMBE</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.76</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−9.68</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">19.20</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.443</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9.23</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−7.52</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">25.99</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.703</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.47</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−8.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">16.97</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.311</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>SWMS</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.13</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.79</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.797</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−4.46</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4.63</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.058</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−4.43</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.35</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.711</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>SSPFSL</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.78</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.58</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.3140</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.726</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.79</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−3.74</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0790</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.140</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.69</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.14</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.76</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.7030</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.439</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>SSPRSL</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.66</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.46</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.14</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.227</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.47</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−3.56</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.61</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2460</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.490</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.37</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.74</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5740</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.271</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>OTSPSFC</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−1.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−5.27</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.63</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5660</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.559</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−6.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.96</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.6050</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.615</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.23</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−3.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.76</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.125</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>DMSPC</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.52</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−8.72</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7.69</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.075</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.31</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−7.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11.83</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.322</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.82</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−4.28</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9.93</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.9530</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.395</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>DMSMDLAD</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,646.16</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">602.20</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,690.12</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN7\" ref-type=\"table-fn\">***</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0009</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.872</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2,368.74</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,157.36</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">3,580.13</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"TFN7\" ref-type=\"table-fn\">***</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0008</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2.600</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">722.59</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−181.08</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1,626.25</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1520</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.793</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>DMSPEGE</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.19</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.061</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.27</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.809</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.22</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−2.869</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>RVPA</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.02</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.260</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.05</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.14</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5920</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.693</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.03</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.7830</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−0.442</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>RVPMDL</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">162.88</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−50.81</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">376.56</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.1850</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.905</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">236.00</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−11.96</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">483.96</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0660</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.265</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">73.12</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−111.85</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">258.10</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.9600</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.392</td></tr><tr style=\"background-color:#DEE1E1\"><td align=\"left\" valign=\"middle\" colspan=\"8\" rowspan=\"1\">\n<bold>SSTSSRT</bold>\n</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. mild</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">17.46</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−28.09</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">63.01</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1.0000</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.455</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Control vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">38.83</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−14.03</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">91.69</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.2130</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.977</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Mild vs. severe</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">21.37</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">−18.06</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">60.80</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.5300</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.538</td></tr></tbody></table></table-wrap>" ]
[]
[]
[]
[]
[]
[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Supplement</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"con\"><p id=\"P29\">\n<bold>Author contributions</bold>\n</p><p id=\"P30\">VG and IR designed the study. VG, DO’R, MJ, RB, and MM analyzed the data. All authors contributed equally to the writing and revision of this manuscript.</p></fn><fn id=\"FN2\" fn-type=\"COI-statement\"><p id=\"P31\">\n<bold>Conflict of interest</bold>\n</p><p id=\"P32\">MM was employed by L&amp;M Data Science Ltd.</p><p id=\"P33\">The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></fn><fn id=\"FN3\"><p id=\"P34\">\n<bold>Ethics statement</bold>\n</p><p id=\"P35\">The studies involving human participants were reviewed and approved by the U.K. Research Ethics Committee [Integrated Research Application System (IRAS): IRAS-Project-ID-170912; REC-REF16/L0/0893]. The patients/participants provided their written informed consent to participate in this study.</p></fn><fn id=\"FN4\"><p id=\"P36\">\n<bold>Publisher’s note</bold>\n</p><p id=\"P37\">All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p></fn><fn id=\"FN5\"><p id=\"P38\">\n<bold>Author disclaimer</bold>\n</p><p id=\"P39\">The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><label>a</label><p id=\"P40\">T-test for independent samples.</p></fn><fn id=\"TFN2\"><label>b</label><p id=\"P41\">Fisher-Freeman-Halton’s exact test.</p></fn><fn id=\"TFN3\"><label>c</label><p id=\"P42\">Fisher’s exact test.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN4\"><p id=\"P43\">Covariates appearing in the model are evaluated at the following values: Age = 41.59 years, BMI = 26.78 kg/m<sup>2</sup>.</p><p id=\"P44\">RTIFDMRT, Median duration between stimulus onset and release of button; RTIFMMT, Mean time taken to touch stimulus after button release; SWMBE, Between errors—number of times that a box in which a token has been previously found is revisited; SWMS, Number of distinct boxes used for the subject to begin new search for a token; PRMPCI, Number of correct responses made in immediate condition; PRMPCD, Number of correct responses made in delayed condition; ERTOMDRT, Median latency of response from stimulus onset to subject response; ERTTH, Number of correctly answered responses; ASTSWMD, Median latency of response in rule switching trials; ASTLCMD, Median latency of response on congruent trials; SSPFSL, Longest sequence successfully recalled—forward variant; SSPRSL, Longest sequence successfully recalled—reversed variant; PALTEA, Number of times incorrect box is chosen + adjusted estimated number of errors that would have been made on any problems, attempts, and recalls that were not reached; PALFAMS, Number of correct box choices made on first attempt; DMSPC, Percentage of assessment trials during which subject selected the correct box on their first box choice; DMSMDLAD, Median latency from the available choices being displayed to the subject choosing the correct choice; DMSPEGE, Reports the probability of an error occurring when the previous trial was responded incorrectly; RVPMDL, Median response latency during assessment sequence blocks where the subject responded correctly; RVPA, Measure of how good the subject is at detecting target sequences; SSTSSRT, Length of time between the go stimulus and the stop stimulus at which the subject is able to successfully inhibit their response on 50% of the trials; OTSPSFC, Number of assessment problems on which the first box choice made was correct.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN5\"><label>*</label><p id=\"P45\"><italic toggle=\"yes\">P</italic> &lt; 0.05.</p></fn><fn id=\"TFN6\"><label>**</label><p id=\"P46\"><italic toggle=\"yes\">P</italic> &lt; 0.01.</p></fn><fn id=\"TFN7\"><label>***</label><p id=\"P47\"><italic toggle=\"yes\">P</italic> &lt; 0.001.</p></fn><fn id=\"TFN8\"><p id=\"P48\">All values are evaluated at the following values: Age = 41.59 years, BMI = 26.78 kg/m<sup>2</sup>.</p><p id=\"P49\">CANTAB tests (23 tests in 11 modalities) for controls, mild OSA and severe OSA. RTIFDMRT, Median duration between stimulus onset and release of button; RTIFMMT, Mean time taken to touch stimulus after button release; SWMBE, Between errors—number of times that a box in which a token has been previously found is revisited; SWMS, Number of distinct boxes used for the subject to begin new search for a token; PRMPCI, Number of correct responses made in immediate condition; PRMPCD, Number of correct responses made in delayed condition; ERTOMDRT, Median latency of response from stimulus onset to subject response; ERTTH, Number of correctly answered responses; ASTSWMD, Median latency of response in rule switching trials; ASTLCMD, Median latency of response on congruent trials; SSPFSL, Longest sequence successfully recalled—forward variant; SSPRSL, Longest sequence successfully recalled—reversed variant; PALTEA, Number of times incorrect box is chosen + adjusted estimated number of errors that would have been made on any problems, attempts, and recalls that were not reached; PALFAMS, Number of correct box choices made on first attempt; DMSPC, Percentage of assessment trials during which subject selected the correct box on their first box choice; DMSMDLAD, Median latency from the available choices being displayed to the subject choosing the correct choice; DMSPEGE, Reports the probability of an error occurring when the previous trial was responded incorrectly; RVPMDL, Median response latency during assessment sequence blocks where the subject responded correctly; RVPA, Measure of how good the subject is at detecting target sequences; SSTSSRT, Length of time between the go stimulus and the stop stimulus at which the subject is able to successfully inhibit their response on 50% of the trials; OTSPSFC, Number of assessment problems on which the first box choice made was correct; ns, non significant.</p></fn></table-wrap-foot>" ]
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[{"collab": ["American Academy of Sleep Medicine"], "article-title": ["The International Classification of Sleep Disorders - Third Edition (ICSD-3)"], "year": ["2014"]}, {"collab": ["Cambridge Cognition"], "source": ["Pattern Recognition Memory (PRM)"], "comment": ["(n.d.a) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/memory/pattern-recognition-memory-prm/"]}, {"collab": ["Cambridge Cognition"], "source": ["Spatial Working Memory (SWM)"], "comment": ["(n.d.b) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/executive-function/spatial-executive-function-swm/"]}, {"collab": ["Cambridge Cognition"], "source": ["Multitasking Test"], "comment": ["(n.d.c) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/executive-function/multitasking-test-mtt/"]}, {"collab": ["Cambridge Cognition"], "source": ["Spatial Span (SSP)"], "comment": ["(n.d.d) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/memory/spatial-span-ssp/"]}, {"collab": ["Cambridge Cognition"], "source": ["Paired Associates Learning (PAL)"], "comment": ["(n.d.e)"]}, {"collab": ["Cambridge Cognition"], "source": ["One Touch Stockings of Cambridge (OTS)"], "comment": ["(n.d.f) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/executive-function/one-touch-stockings-of-cambridge-ots/#:~:text=One%20Touch%20Stockings%20of%20Cambridge%20is%20a%20test%20of%20executive,and%20the%20working%20memory%20subdomains.&text=The%20participant%20is%20shown%20two%20displays%20containing%20three%20coloured%20balls"]}, {"collab": ["Cambridge Cognition"], "source": ["Delayed Matching to Sample (DMS)"], "comment": ["(n.d.g) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/memory/delayed-matching-to-sample-dms/#:~:text=Delayed%20Matching%20to%20Sample%20assesses,%2C%20for%20non%2Dverbalisable%20patterns.&text=The%20participant%20is%20shown%20a,patterns%2C%20after%20a%20brief%20delay"]}, {"collab": ["Cambridge Cognition"], "source": ["Stop Signal Task (SST)"], "comment": ["(n.d.h) Available online at: "], "ext-link": ["https://www.cambridgecognition.com/cantab/cognitive-tests/executive-function/stop-signal-task-sst"]}, {"person-group": ["\n", "\n"], "surname": ["Cunningham", "Payne", "Axmacher", "Rasch"], "given-names": ["TJ", "JD", "N", "B"], "part-title": ["Emotional memory consolidation during sleep"], "source": ["Cognitive Neuroscience of Memory Consolidation"], "publisher-name": ["Springer"], "publisher-loc": ["Cham"], "year": ["2017"], "fpage": ["133"], "lpage": ["159"], "pub-id": ["10.1007/978-3-319-45066-7_9"]}, {"person-group": ["\n"], "surname": ["Esther Strauss"], "given-names": ["EMSS"], "source": ["Otfried Spreen A Compendium of Neuropsychological Tests: Administration, Norms, and Commentary"], "edition": ["Third"], "publisher-name": ["Oxford University Press"], "publisher-loc": ["Oxford"], "year": ["2006"]}, {"person-group": ["\n"], "surname": ["Rosenzweig", "Gosselin", "Bucks"], "given-names": ["I", "N", "RS"], "part-title": ["Cognitive and neurologic aspects of obstructive sleep apnea"], "source": ["Encyclopedia of Respiratory Medicine"], "edition": ["2nd Edn"], "publisher-name": ["Elseivier"], "publisher-loc": ["Doyma"], "year": ["2022"], "volume": ["17"], "fpage": ["60"], "lpage": ["74"], "pub-id": ["10.1016/B978-0-08-102723-3.00127-X"]}, {"person-group": ["\n"], "surname": ["Rosenzweig", "Weaver", "Morrell"], "given-names": ["I", "TE", "MJ"], "part-title": ["Obstructive sleep apnea and the central nervous system"], "source": ["Principles and Practice of Sleep Medicine"], "publisher-name": ["Biashideng Publishing Group"], "publisher-loc": ["Pleasanton, CA"], "year": ["2017"], "fpage": ["1154"], "lpage": ["1166"], "elocation-id": ["e1155"], "pub-id": ["10.1016/b978-0-323-24288-2.00117-3"]}, {"person-group": ["\n"], "surname": ["Saaresranta", "Anttalainen", "Polo"], "given-names": ["T", "U", "O"], "article-title": ["Sleep disordered breathing: is it different for females?"], "source": ["ERJ Open Res"], "year": ["2015"], "volume": ["1"], "fpage": ["15"], "pub-id": ["10.1183/23120541.00063-2015"]}, {"person-group": ["\n"], "surname": ["Schembri", "Spong", "Graco", "Berlowitz"], "given-names": ["R", "J", "M", "DJ"], "collab": ["COSAQ study team"], "article-title": ["Neuropsychological function in patients with acute tetraplegia and sleep disordered breathing"], "source": ["Sleep"], "year": ["2017"], "volume": ["40"], "elocation-id": ["zsw037"], "pub-id": ["10.1093/sleep/zsw037"]}, {"person-group": ["\n"], "surname": ["Walter", "Lee", "Blake", "Kalluri", "Cziraky", "Stanek"], "given-names": ["J", "JY", "S", "L", "M", "E"], "article-title": ["A new wearable diagnostic home sleep testing platform: comparison with available systems and benefits of multi-night assessments"], "source": ["J Clin Sleep Med"], "year": ["2023"], "pub-id": ["10.5664/jcsm.10432"]}]
{ "acronym": [], "definition": [] }
66
CC BY
no
2024-01-13 00:12:45
Front Sleep. 2023 Apr 6; 2:1097946
oa_package/4c/ea/PMC7615516.tar.gz
PMC7615518
38096890
[ "<title>Introduction</title>", "<p id=\"P5\">Mortality from cancers in the UK and other industrialised countries has declined more slowly than from other major causes of death, and hence the share of deaths from cancers has steadily increased.<sup>##UREF##0##1##</sup> Cancers are now the leading cause of death in England, overtaking cardiovascular diseases.<sup>##UREF##1##2##</sup></p>", "<p id=\"P6\">Subnational data on trends in cancer mortality are currently limited to large areas.3,4 Small-area data can also guide primary prevention strategies to reduce incidence, and health care planning and delivery to improve survival. We report trends in cancer mortality for 314 districts in England from 2002 to 2019 and the association with poverty.</p>" ]
[ "<title>Methods</title>", "<title>Data</title>", "<p id=\"P7\">We used vital registration data for all deaths in England from 2002 to 2019 (8,648,191 death records). We used postcode of residence at death registration to assign each death to a local authority district (referred to as district hereafter). England had 314 districts in 2020. Records were categorised into the following age groups: 0, 1-4, 5-9, 10-14, …, 80-84 and ≥85 years. We did not use 130 death records (&lt;0.001%) for which sex was not recorded.</p>", "<p id=\"P8\">We used ICD-10 codes for underlying cause of death to assign each death to 136 cause groups of the WHO Global Health Estimates (GHE) study. We used the top ten leading cancer causes of death for each sex according to the total number of deaths from 2002 to 2019 (##FIG##0##Figure 1##) for cause-specific analysis, as well as a residual group comprising all other cancer deaths (##SUPPL##0##Appendix p6##).</p>", "<p id=\"P9\">Mid-year population data by age group, district, year, and sex were obtained from the ONS. We used data on income deprivation from the English Indices of Deprivation for 2019 (referred to as poverty hereafter), defined as the proportion of the district population claiming income-related benefits due to being out-of-work or having low earnings. The deprivation data are available at the Lower-layer Super Output Area (LSOA) level, which we aggregated to the district level by taking the population-weighted average. We did not use data on ethnicity because it is not recorded on death certificates and district-level data are only available for the 2011 census year.</p>", "<title>Statistical analysis</title>", "<p id=\"P10\">The number of deaths in each age group, district, year, and cancer cause group is small, which means that death rates calculated from observed data have an apparent variability from year to year, or from district to district, that is larger than the true differences in the risk of death. We adapted a Bayesian hierarchical model from previous studies<sup>##REF##34653419##5##,##REF##37069855##6##</sup> to obtain stable estimates of death rates by sharing information across age groups, districts and years. In our hierarchical model, death rates for each age group, district and year were informed by data in that district-age-year unit as well as by those in adjacent age groups, adjacent years, and nearby districts. We did not use district-level socioeconomic variables like poverty as a covariate in the model because such data are only available for some years, and the methodology for their calculation may vary from year to year. Detailed model specification is presented in the ##SUPPL##0##Appendix (pp2–4 and p7)##.</p>", "<p id=\"P11\">We conducted all analyses separately by sex and cancer cause of death. In addition, we ran a model for all cancer deaths combined. The results for total cancer mortality using the combined model and the sum of cause-specific models were nearly identical (correlation coefficient across all years 0.99 for both sexes; mean absolute difference in probability of death &lt;0.01 for both sexes).</p>", "<p id=\"P12\">Our primary reporting outcome is the unconditional probability of dying between birth and 80 years of age, which is the probability of death in the absence of competing causes of death, calculated by applying life tables to the posterior estimates of age-specific death rates (detailed calculation in the ##SUPPL##0##Appendix (p5)##). We used probability of death because it has an intuitive interpretation, and because, unlike age-standardised death rate, it does not depend on the choice of standard population. We used unconditional probability of death because removing competing causes of death enhances comparability and equity.<sup>##UREF##3##7##</sup> We limited the age range because the probability of death in the absence of competing causes equals 1.0 when the entire life course is considered. We selected 80 years of age as the upper bound because it covers a wide age range but does not include the very oldest ages where multimorbidity makes the assignment of cause of death increasingly difficult. We also calculated age-standardised death rates. The correlation coefficients between age-standardised death rates and the probability of dying between birth and 80 years of age ranged from 0.93 to &gt;0.99 across sex-year-cancer combinations for the years 2002 and 2019 (##SUPPL##0##Appendix pp8–10##).</p>", "<p id=\"P13\">The reported 95% credible intervals (CrI) represent the 2.5<sup>th</sup> to 97.5<sup>th</sup> percentiles of the posterior distribution of estimated probability of death. We also report the posterior probability (PP) that the estimated change over time in a district represents an increase versus a decrease in the probability of dying between birth and 80 years of age. If the estimated probability of dying is the same in 2002 and 2019 and an increase is statistically indistinguishable from a decrease, there is a 0.5 PP of an increase and a 0.5 PP of a decrease. PPs more distant from 0.5 indicate more certainty. We report Spearman rank correlation between the district (rank of) probability of dying between birth and 80 years and (rank of) poverty in 2019, together with its 95% CrI.</p>", "<title>Role of the funding source</title>", "<p id=\"P14\">The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.</p>" ]
[ "<title>Results</title>", "<p id=\"P15\">There were 2,453,173 deaths from cancers in England from 2002 to 2019; of these, 1,533,703 (62.5%) deaths occurred before 80 years of age. Of cancer deaths before 80 years of age, 697,953 (45.5%) were deaths in women and 835,750 (54.5%) in men. Nationally, the probability of dying from a cancer before 80 years of age declined for both sexes, from 0.16 to 0.13 for women and 0.22 to 0.17 for men from 2002-2019.</p>", "<p id=\"P16\">In 2019, the probability of dying from a cancer before 80 years of age ranged from one in ten (0.10 (95% CrI 0.10-0.11)) in Westminster to one in six (0.17 (0.16-0.18)) in Manchester for women, and from one in eight (0.12 (0.12-0.13)) in Harrow to one in five (0.22 (0.21-0.23)) in Manchester for men (##FIG##1##Figure 2##). The highest probabilities of dying were in northern cities such as Liverpool, Manchester, Hull and Newcastle, and in coastal areas to the east of London.</p>", "<p id=\"P17\">Among the ten leading cancer causes of death (##FIG##0##Figure 1##), for the mean age at death in 2019 among those who died before 80 years of age varied from 63.0 years for breast cancer to 69.0 years for lung cancer in women and from 66.1 years for the combined category of all other cancers to 72.2 for prostate cancer in men. The leading cancer cause of death for both sexes was lung cancer, with 218,561 deaths (18.7% of all cancer deaths; 148,551 before 80 years) in women and 282,422 deaths (22.0%; 201,862) in men from 2002-2019 (##FIG##0##Figure 1##). Lung cancer was one of the most unequal cancers, with 3.7-times (3.2-4.4) and 3.0-times (2.7-3.5) variation across districts in 2019 in the probability of death for women and men, respectively. The highest probabilities of dying were in the urban North West and North East (##SUPPL##0##Appendix pp11–33##), with the highest probability of death in 2019 being 0.06 (0.06-0.07) in Knowsley for women and 0.07 (0.07-0.08) in Manchester for men. The distribution of district-level probabilities of dying from lung cancer was nearly identical in 2002 and 2019 for women, whereas for men the probabilities declined everywhere (##FIG##2##Figure 3##).</p>", "<p id=\"P18\">Lung cancer was followed by major sex-specific cancers – breast cancer (177,528 (15.2%) deaths) for women and prostate cancer (164,871 (12.8%)) for men. In 2019, deaths from these cancers had less district-level inequality than from lung cancer (##FIG##2##Figure 3##), although the probability of dying from prostate cancer was noticeably lower in northwest London than elsewhere (##SUPPL##0##Appendix pp11–33##). The probability of dying from ovarian cancer was also lower in London, in contrast to corpus uteri cancer, where east London had the highest probabilities (##SUPPL##0##Appendix pp11–33##). High probabilities of death from liver cancer, one of the leading causes of cancer mortality among men, were very concentrated in the North West, the North East, and central London. Stomach cancer mortality showed vast inequality (3.3-times (2.4-4.6) for women and 3.2-times (2.6-4.1) for men), with the highest probabilities of dying in the urban North West. The cancers with the least geographical variability were lymphoma and multiple myeloma (1.2-times (1.1-1.4) for women and 1.2-times (1.0-1.4) for men) and leukaemia (1.1-times (1.0-1.4) for women and 1.2-times (1.0-1.5) for men).</p>", "<p id=\"P19\">For women, the district-level probabilities of dying from stomach, oesophageal, lung and the residual category of all other cancers were correlated, with pairwise correlation coefficients ranging 0.51-0.75 (##SUPPL##0##Appendix pp34–35##). For men, the probability of dying from lung cancer was correlated with those of stomach, liver, bladder, colorectal, oesophageal, and all other cancers. There were particularly high correlations between the probabilities of dying from bladder and oesophageal cancers (0.71), and from oesophageal and colorectal cancers (0.69). Probabilities of dying from leukaemia, lymphoma and multiple myeloma, and pancreatic and corpus uteri cancers were only weakly correlated with probabilities for other categories.</p>", "<p id=\"P20\">The probability of dying from a cancer was associated with poverty for both sexes, with Spearman correlation coefficients of 0.74 (0.72-0.76) for women and 0.79 (0.78-0.81) for men (##FIG##3##Figure 4##). In part, this was due to the probability of dying from the leading cancer for both sexes, lung cancer, exhibiting strong correlations with poverty (0.76 (0.74-0.78) for women and 0.85 (0.83-0.87) for men). There were also strong correlations between poverty and mortality from stomach cancer (0.58 (0.54-0.63) for women and 0.64 (0.60-0.67) for men), all other cancers (0.54 (0.50-0.59) and 0.56 (0.52-0.60)) and liver cancer (0.57 (0.51-0.62) for men for whom it was analysed). The probabilities of dying from breast cancer (0.02 (-0.06 to 0.12), prostate cancer (0.13 (0.05-0.21)), pancreatic cancer (0.02 (-0.09 to 0.13) and 0.01 (-0.11 to 0.14)), lymphoma and multiple myeloma (-0.02 (-0.14 to 0.10) and -0.05 (-0.16 to 0.07)), leukaemia (-0.02 (-0.17 to 0.12) and 0.09 (-0.04 to 0.22)), and colorectal cancer in women (0.06 (-0.03 to 0.16)) showed little or no association with poverty. Those in poor districts of London had lower probabilities of dying from lung, colorectal, oesophageal, bladder (men), and all other cancers than in comparably poor districts in the rest of the country (##FIG##3##Figure 4##).</p>", "<p id=\"P21\">The probability of dying from a cancer before 80 years of age declined from 2002-2019 in every district for both sexes with a PP &gt;0.97, with median reduction across districts of 18.7% for women and 23.4% for men. However, these reductions occurred at varying rates (##FIG##4##Figure 5##). For women, the greatest district-level reduction in the probability of dying from a cancer (30.1% (25.6-34.5%)) was nearly five times that of the smallest (6.6% (0.3-13.1%)). For men, the largest decrease was triple that of the smallest, 36.7% (32.2-41.2%) compared to 12.8% (7.1-18.8%). Districts in London achieved the largest declines.</p>", "<p id=\"P22\">Among cause categories, the largest reductions in mortality were for stomach cancer, with the declines from 2002-2019 in probability of death ranging 39.1-57.2% across districts for women, and 51.5-58.8% for men (PP &gt;0.99). The probability of dying from oesophageal cancer also decreased in every district for women, but varied for men from a 42.9% (27.7-56.0%) decrease in Plymouth to a 7.0% (-19.8% to 42.4%) increase in Gosport (##FIG##4##Figure 5##and ##SUPPL##0##Appendix pp11–33##).</p>", "<p id=\"P23\">Lung cancer mortality decreased everywhere for men with PP &gt;0.99, but for women, there were mixed trends. The largest declines were in London, (largest decrease: 29.5% (18.5-38.8%) in Newham), whereas the probability of dying increased in many districts in the East of England, (largest increase: 27.0% (6.8-49.7%) in Tendring) (##FIG##4##Figure 5##and ##SUPPL##0##Appendix pp11–33##). The PP that the observed decline for lung cancer in women was a true decline was &gt;0.80 in 197 (62.7%) districts. Women in 4 (1.3%) districts experienced an increase in lung cancer mortality with a PP &gt;0.80, and in the remaining 113 (36.0%) districts there was no detectable change at this PP.</p>", "<p id=\"P24\">The probability of dying from pancreatic cancer increased in all but one sex-district combination, but the PP of the observed increase was a true increase was &gt;0.80 in 259 (82.5%) districts for women and 302 (96.2%) districts for men. The median increase across districts was 8.6% for women and 6.7% for men, and the largest increases were in the South East (##FIG##4##Figure 5##). Liver cancer for men (median increase 78.3%) saw increases in mortality in all districts with PP &gt;0.98, and corpus uteri cancer for women (median increase 31.7%) saw increases in mortality in all but one district with PP&gt;0.80. There were increases in male liver cancer mortality in excess of 100% in 32 (10.2%) districts, mostly in northern England (##FIG##4##Figure 5##). All districts that experienced an increase in pancreatic cancer with a PP &gt;0.80 also experienced an increase in a second cancer at the same PP (corpus uteri for women, liver for men). Women in 3 (1.0%) districts experienced an increase in three cancers (lung, pancreatic and corpus uteri cancer) with a PP &gt;0.80.</p>" ]
[ "<title>Discussion</title>", "<p id=\"P25\">Although overall cancer mortality decreased everywhere, the gains were unequal, with the largest declines almost five times that of the smallest. Cancers with strong links to behavioural and environmental risk factors, and those with screening for pre-cancerous lesions<sup>##REF##22997325##8##</sup> (from the cancer groups in this study: lung, colorectal, breast, oesophageal, stomach, liver, and bladder cancers), saw some of the largest inequalities across districts and the widest variation in how much they changed. The cancers showing the least inequality, lymphoma and multiple myeloma and leukaemia, were those with weaker and more heterogeneous links to modifiable risk factors.</p>", "<p id=\"P26\">A strength of our study is the presentation of mortality from leading cancers, as well as all cancers together, for subnational spatial units in England over a period of substantial change in public health programmes and healthcare technology and provision. We used a Bayesian hierarchical model to robustly estimate death rates for different cancers, together with uncertainty in these areas, by sharing information over age, space, and time. A limitation of our work is that we only analysed deaths from the ten leading causes of death from cancer and did not split the residual group into more specific cancer groups. This was done so that there were sufficient deaths in each cause group for robust inference of age-specific death rates at the district level. Each cancer group was analysed separately rather than in a single model, which should be explored in methodological work. Our reporting metric, the probability of dying between birth and 80 years of age, intuitively presents mortality over the life-course, but does not include differences in age groups beyond its upper age limit. We found that this metric was highly correlated with age-standardised death rates. Although districts are valuable units for analysis as they are used for resource allocation and policy implementation, previous studies on total mortality have found heterogeneity among units smaller than districts.<sup>##REF##34653419##5##,##REF##37069855##6##</sup> Furthermore, we did not analyse or present data by healthcare geographies because these do not map to districts, and because regional cancer services vary within England. For example, there may be a single regional cancer centre in some areas, or a single specialist centre for a particular malignancy. There are also factors including patient choice (of where to receive care), age (younger patients may be treated at specialist children’s hospitals), cancer stage (late stage cancers are more aggressive and might be treated in a specialist centre), and year (health geographies are subject to change whenever the NHS undergoes a reorganisation) which mean people in the same district may not be treated at the same hospital for each cancer. The population in each district can change because of migration, both within the country and from overseas. Therefore, changes in cancer mortality should not be entirely attributed to changes in the health of the population, although studies from both the UK<sup>##REF##34653419##5##</sup> and USA<sup>##REF##18433290##9##</sup> have shown that migration alone is unlikely to fully explain trends in health outcomes. Moreover, the majority of moves in the UK are within the same district,<sup>##UREF##4##10##</sup> which does not affect the change in mortality in a given district. The correlations with poverty were reported at the district level, whereas there are variations in both mortality and poverty within each district.<sup>##REF##34653419##5##,##REF##37069855##6##</sup> The correlations between cancer risk and individual or household poverty may be even stronger because aggregation tends to attenuate correlation. To ensure the underlying cause of death data are comparable between places and times, the UK has national guidelines for death certification, as well as algorithms for the computer-based assignment of the underlying cause of death.<sup>##UREF##5##11##</sup> For example, a validation study found that that death certificates in the UK accurately identified deaths from prostate cancer.<sup>##REF##27253172##12##</sup> Despite this, there might be differences in the cancer cause of death assignment between districts.</p>", "<p id=\"P27\">Cancer mortality depends on both incidence and survival. Incidence is influenced by three factors: exposure to risk factors for cancer, including smoking, alcohol use, obesity, infections, and occupational and environmental exposures; the uptake of preventative treatments such as vaccinations; and the implementation of screening for pre-cancerous lesions. In England, smoking, alcohol use and obesity are higher where cancer mortality is highest, generally in the North.<sup>##UREF##6##13##–##UREF##8##15##</sup> The rise and fall in female smoking have lagged behind men by about 20-30 years,<sup>##REF##22345230##16##</sup> leading to the mix of increase and decrease in female lung cancer mortality while that of males declined everywhere. Similarly, after peaking in the mid-2000s, alcohol consumption has declined, and is lowest in London.<sup>##UREF##6##13##,##UREF##9##17##</sup> In contrast, obesity has risen,<sup>##UREF##8##15##</sup> mirrored by a rise in diabetes.<sup>##UREF##8##15##</sup> These trends could be partly responsible for cancers associated with diabetes and obesity increasingly co-occurring with those due to smoking and alcohol use, especially in men among whom smoking rates declined earlier. We observed substantial increases in liver cancer mortality for men, especially in northern districts, which mirror a long-run trend in the USA where it has increased both nationally and in nearly all counties.<sup>##REF##28118455##18##</sup>,<sup>##REF##12784323##19##</sup> The observed trends in liver cancer mortality could be linked to the heterogeneous trends in its major risk factors, namely infections, smoking, alcohol use and diabetes.<sup>##REF##22158327##20##–##UREF##11##23##</sup> Differences between men and women in the temporal and geographical distribution of tobacco smoking, alcohol use, poor diet, and obesity may potentially account for our observation that oesophageal cancer mortality decreased in all districts for women, but increased in some districts for men. Stomach cancer exhibited the largest decreases in mortality, as well as substantial inequality in the level across districts. In part this may be due to variable prevalence of, and heterogeneous decrease in, <italic toggle=\"yes\">H pylori</italic> infection.</p>", "<p id=\"P28\">Survival is influenced by awareness and utilisation of care, access and barriers to screening and early diagnosis, tumour factors (stage, topography, histology), comorbidities, and the quality of care. The UK has performed badly in terms of survival compared to other European countries.<sup>##REF##26241817##24##</sup> There were also subnational variations in survival for a number of cancers, both for cancers with relatively high average survival (e.g., colorectal and corpus uteri cancers) and those with relatively low average survival (e.g., pancreatic and liver cancers).<sup>##UREF##2##3##</sup> This survival variation, together with variations in incidence, would drive the observed variation in mortality.</p>", "<p id=\"P29\">Combinations of risk factors and healthcare can also explain London’s better performance for some cancers. Firstly, there can be differences in cancer risk factors between London’s ethnically diverse population and the rest of England. For example, smoking rates amongst Asian (8.3% of adults) and black (9.7%) groups are lower than the white ethnic group (14.4%).<sup>##UREF##12##25##</sup> Secondly, there could be differences in the quality of healthcare, either because advanced treatments such as immunotherapies are more available, or, because patients can travel to a specialist hospital rather than their local hospital thanks to the higher density of hospitals. However, districts in East London had the highest mortality for corpus uteri cancer, which has higher incidence rates in the black ethnic group in England.<sup>##REF##35233092##26##</sup></p>", "<p id=\"P30\">Since the turn of the century, two major national policy initiatives in the UK have aimed to improve cancer care and survival. Firstly, the NHS Cancer Plan of 2000 targeted socioeconomic inequalities in cancer survival through an increase in expenditure, a focus on centralisation and specialisation of cancer services, and a greater use of multidisciplinary teams. Secondly, the National Awareness and Early Diagnostic Initiative, launched in 2008, addressed both patient factors, such as reducing the stigma around cancer and the barriers to seeing a doctor, and tumour factors, such as improving access to screening and optimising referral pathways. However, no consistent evidence was found of a direct impact on one-year survival after these successive initiatives.<sup>##REF##20588275##27##</sup>,<sup>##REF##29540358##28##</sup> Recent NHS plans have renewed focus on survival targets and early diagnosis,<sup>##UREF##13##29##</sup> as well as the roll out of a national lung cancer screening programme targeted at those with a history of smoking.<sup>##UREF##14##30##</sup> Technological developments, including multicancer detection assays, cancer vaccines, new and emerging drugs, combination therapy, and a shift towards personalised cancer care as the cost of profiling tumours falls, all offer scope for advancement. However, equitable availability of novel diagnostics and treatments to all patients may lag behind successful development and approvals, and patients without access will be more likely to present with advanced cancers and receive less advanced treatment. Our results on heterogeneous trends in mortality, particularly for cancers with modifiable risk factors and potential for screening for pre-cancerous lesions, indicate that reducing these mortality inequalities requires addressing factors affecting both incidence and survival at the local level.</p>" ]
[]
[ "<title>Summary</title>", "<title>Background</title>", "<p id=\"P1\">Cancers are the leading cause of death in England. Our aim was to estimate trends from 2002-2019 in mortality from leading cancers for the 314 districts in England.</p>", "<title>Methods</title>", "<p id=\"P2\">We used vital registration data in England from 2002 to 2019 for ten leading cancers by sex according to the total number of deaths over the study period, and a residual group of all other cancers. We used a Bayesian hierarchical model to obtain robust estimates of age- and cause-specific death rates. We applied life tables to calculate the probability of dying between birth and 80 years of age by sex, cancer cause of death, district and year. We report Spearman rank correlation between the probability of dying from a cancer and district-level poverty in 2019.</p>", "<title>Findings</title>", "<p id=\"P3\">In 2019, the probability of dying from a cancer ranged from 0.10 (95% credible interval 0.10-0.11) to 0.17 (0.16-0.18) for women and from 0.12 (0.12-0.13) to 0.22 (0.21-0.23) for men. The most unequal cancers were lung cancer for women (3.7-times (3.2-4.4) variation between the districts with the highest and lowest probabilities of dying) and stomach cancer for men (3.2-times (2.6-4.1)). The cancers with the least geographical variability were lymphoma and multiple myeloma (1.2-times (1.1-1.4) for women and 1.2-times (1.0-1.4) for men), and leukaemia (1.1-times (1.0-1.4) for women and 1.2-times (1.0-1.5) for men). The correlation between probability of dying from a cancer and district poverty was 0.74 (0.72-0.76) for women and 0.79 (0.78-0.81) for men. The probability of dying declined in all districts from 2002 to 2019: the reductions ranged from 6.6% (0.3-13.1%) to 30.1% (25.6-34.5%) for women and 12.8% (7.1-18.8%) to 36.7% (32.2-41.2%) for men.</p>", "<title>Interpretation</title>", "<p id=\"P4\">Cancers with modifiable risk factors and potential for screening for pre-cancerous lesions had heterogeneous trends and the greatest inequality. Reducing these inequalities requires addressing factors affecting both incidence and survival at the local level.</p>" ]
[ "<title>Supplementary Material</title>" ]
[ "<title>Acknowledgements</title>", "<p>The statistical methods and their implementation have benefited from discussions with Christopher Paciorek. This work was supported by the Pathways to Equitable Healthy Cities grant from the Wellcome Trust (209376/Z/17/Z) and by a grant from the UK Medical Research Council (grant number MR/V034057/1). TR is supported by an Imperial College President’s PhD scholarship. DCM received funding from Cancer Research UK, The National Cancer Institute, and the European Commission. The mortality data used for this study were supplied by the Office for National Statistics and held by the UK Small Area Health Statistics Unit (SAHSU). The work of the SAHSU is overseen by UK Health Security Agency (UK HSA) and funded by the National Institute for Health Research (NIHR) through its Health Protection Units (HPRUs) at Imperial College London in Environmental Exposures and Health and in Chemical and Radiation Threats and Hazards, and through Health Data Research UK (HDR UK). This paper does not necessarily reflect the views of ONS, UKHSA, the NIHR or the Department of Health and Social Care. Authors acknowledge infrastructure support for the Department of Epidemiology and Biostatistics provided by the NIHR Imperial Biomedical Research Centre (BRC). SAHSU holds approval from the Health Research Authority Confidentiality Advisory Group under regulation 5 of the health service (Control of Patient Information) regulations 2002 (section 251; reference 20/CAG/0028), and the National Research Ethics Service: London-South East Research Ethics Committee (reference 22/LO/0256). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any author accepted manuscript version arising from this submission.</p>", "<title>Funding</title>", "<p>Wellcome Trust, Imperial College London, UKRI (MRC), National Institute of Health Research</p>", "<title>Data sharing</title>", "<p id=\"P31\">The Small Area Health Statistics Unit does not release data to third parties except in the form of non-disclosive statistical tables or conclusions suitable for publication. Individual-record mortality data can be requested through the Office for National Statistics (ONS) (<ext-link xlink:href=\"https://www.ons.gov.uk\" ext-link-type=\"uri\">https://www.ons.gov.uk</ext-link>). Mid-year population estimates (<ext-link xlink:href=\"https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationestimates/datasets/middlesuperoutputareamidyearpopulationestimates\" ext-link-type=\"uri\">https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationestimates/datasets/middlesuperoutputareamidyearpopulationestimates</ext-link>) and the English Indices of Deprivation 2019 (<ext-link xlink:href=\"https://www.gov.uk/government/statistics/english-indices-of-deprivation-2019\" ext-link-type=\"uri\">https://www.gov.uk/government/statistics/english-indices-of-deprivation-2019</ext-link>) are available online. The code for the model (<ext-link xlink:href=\"https://globalenvhealth.org/code-data-download/\" ext-link-type=\"uri\">https://globalenvhealth.org/code-data-download/</ext-link>) is also available online.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><title>Total number of deaths for the ten leading cancers in England from 2002 to 2019 by sex. See the ##SUPPL##0##Appendix (p6)##for ICD-10 codes in each category.</title></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Probability of dying from a cancer between birth and 80 years of age in 314 local authority districts in England in 2019 and change from 2002 to 2019. See the ##SUPPL##0##Appendix (pp11–33)##for results for specific cancers.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><title>Ranked probability of dying between birth and 80 years of age in 314 local authority districts in England in 2002 and 2019 for the ten leading cancers.</title><p>Each point shows one district and the vertical line going through the point its 95% credible interval. The districts were ranked by the posterior median estimate for the probability of dying.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Local authority district probability of death in 2019 in relation to poverty for each of the leading cancers. The points are coloured by the regions in England. The median Spearman rank correlations (ρ) between the probability of dying from the cancer and poverty ranking and the corresponding 95% credible intervals, calculated at the posterior sample-level, are in the top left of each panel.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><title>Change in probability of dying between birth and 80 years of age in 314 local authority districts in England in 2002 and 2019 for the ten leading cancers.</title><p>Each point shows one district and the vertical line going through the point its 95% credible interval. The districts were first ordered by region and then ranked by the posterior median estimate for change in the probability of dying. The points are coloured by the regions in England.</p></caption></fig>" ]
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[ "<boxed-text id=\"BX1\" position=\"float\"><caption><title>Research in context</title></caption><sec id=\"S15\"><title>Evidence before the study</title><p>We searched PubMed for articles published from database inception up to July 5, 2023, using search terms “mortality” AND “cancer” AND (“sub-national” OR “small area” OR “local”) AND (“trend” OR “time”) AND (“England” OR “United Kingdom”) for papers that had analysed trends in cancer mortality in England, with no language restrictions. We also searched for relevant reports through the websites of the Office for National Statistics and Public Health England. We found estimates of cancer mortality for health boards, which are much larger than districts. One study reported risk relative to the national population for multiple cancers in census wards, which is a finer resolution than district level, by aggregating data over a 25-year period but did not analyse change over time. We found two studies which investigated trends in cancer mortality: one looked at upper-tier local authorities from 1990 to 2016, of which there are fewer than half the number of districts in this study. The other aggregated communities into deciles of deprivation within each region and only focussed on four leading cancers from 2001 to 2016. None of the studies estimated cancer mortality for areas as small as districts nor included all leading cancer causes of death.</p><p>Elsewhere, cancer atlases have reported cancer mortality or incidence for coarse geographical units and aggregated multiple years of data rather than presenting information on trends. One study in the USA looked at trends in cancer mortality at the county level.</p></sec><sec id=\"S16\"><title>Added value of this study</title><p>To our knowledge, we present estimates for trends in cancer mortality for districts in England at a higher resolution than previous studies. By using a Bayesian spatiotemporal model based on patterns of mortality over age, space and time, we obtained robust yearly estimates of cancer mortality for small geographies, together with uncertainty in these estimates.</p></sec><sec id=\"S17\"><title>Implications of all the available evidence</title><p>Declines in overall cancer mortality have been unequal both geographically and among groups of cancers. The greatest geographical inequality was seen for cancers with modifiable risk factors and potential for screening for pre-cancerous lesions. Addressing risk factors like smoking and alcohol use, expanding access to and utilisation of screening for prevention and early detection, and improving the quality of care should all be used to reduce deaths where they remain highest. High-resolution spatiotemporal data can help identify where intervention is required and track progress.</p></sec></boxed-text>" ]
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[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Appendix</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\" fn-type=\"con\"><p id=\"P32\">\n<bold>Contributors</bold>\n</p><p id=\"P33\">TR, JEB, HID and BD obtained or managed data. TR, JEB and ME developed analytical methods. TR implemented methods in consultation with JEB and ME. TR, JEB and ME designed presentation of results and wrote the first draft of the paper. All other authors contributed to revision and finalisation of the paper. The corresponding author was responsible for submitting the article for publication. TR, HID and JEB had full access to all mortality data. TR, JEB and HID checked and verified the mortality data. Due to data permission restrictions, not all authors were able to access the raw data used in the study.</p></fn><fn id=\"FN2\" fn-type=\"COI-statement\"><p id=\"P34\">\n<bold>Declaration of interests</bold>\n</p><p id=\"P35\">JP-S is chair of the Royal Society for Public Health and a partner at Lane Clark &amp; Peacock, and reports personal fees from Novo Nordisk A/S and Pfizer Ltd, all outside the submitted work. All other authors declare no competing interests.</p></fn></fn-group>" ]
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[{"label": ["1"], "collab": ["Office for National Statistics"], "source": ["Leading causes of death, UK: 2001 to 2018"], "year": ["2020"], "date-in-citation": ["accessed July 31,2023"], "publisher-name": ["Office for National Statistics"], "comment": ["published online March 27. "], "ext-link": ["https://www.ons.gov.uk/peoplepopulationandcommunity/healthandsocialcare/causesofdeath/articles/leadingcausesofdeathuk/2001to2018"]}, {"label": ["2"], "person-group": ["\n"], "surname": ["Vos", "Lim", "Abbafati"], "given-names": ["T", "SS", "C"], "article-title": ["Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019"], "source": ["The Lancet"], "year": ["2020"], "volume": ["396"], "fpage": ["1204"], "lpage": ["22"]}, {"label": ["3"], "collab": ["NHS Digital. CancerData. CancerData"], "year": ["2022"], "date-in-citation": ["accessed May 11,2023"], "comment": ["published online Oct 20. "], "ext-link": ["https://www.cancerdata.nhs.uk/incidence_and_mortality"]}, {"label": ["7"], "person-group": ["\n"], "surname": ["Bennett", "Stevens", "Mathers"], "given-names": ["JE", "GA", "CD"], "article-title": ["NCD Countdown 2030: worldwide trends in non-communicable disease mortality and progress towards Sustainable Development Goal target 3.4"], "source": ["The Lancet"], "year": ["2018"], "volume": ["392"], "fpage": ["1072"], "lpage": ["88"]}, {"label": ["10"], "person-group": ["\n"], "surname": ["van Dijk", "Lansley", "Longley"], "given-names": ["JT", "G", "PA"], "article-title": ["Using linked consumer registers to estimate residential moves in the United Kingdom"], "source": ["Journal of the Royal Statistical Society: Series A (Statistics in Society)"], "year": ["2021"], "volume": ["184"], "fpage": ["1452"], "lpage": ["74"]}, {"label": ["11"], "collab": ["Office for National Statistics"], "source": ["User guide to mortality statistics"], "year": ["2022"], "date-in-citation": ["accessed March 2, 2023"], "publisher-name": ["Office for National Statistics"], "comment": ["published online July 1. "], "ext-link": ["https://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/methodologies/userguidetomortalitystatisticsjuly2017"]}, {"label": ["13"], "collab": ["Public Health England"], "source": ["Local Alcohol Profiles for England"], "year": ["2021"], "date-in-citation": ["accessed April 14, 2023"], "publisher-name": ["Public Health England"], "comment": ["\n"], "ext-link": ["https://fingertips.phe.org.uk/profile/local-alcohol-profiles"]}, {"label": ["14"], "collab": ["Public Health England"], "source": ["Local Tobacco Control Profiles"], "year": ["2023"], "date-in-citation": ["accessed May 17, 2023"], "publisher-name": ["Public Health England"], "comment": ["\n"], "ext-link": ["https://fingertips.phe.org.uk/profile/tobacco-control/"]}, {"label": ["15"], "collab": ["NHS Digital"], "source": ["Health Survey for England"], "year": ["2023"], "date-in-citation": ["accessed May 18, 2023"], "publisher-name": ["NHS Digital"], "comment": ["published online May 16. "], "ext-link": ["https://digital.nhs.uk/data-and-information/publications/statistical/health-survey-for-england"]}, {"label": ["17"], "collab": ["Office for National Statistics"], "source": ["Adult drinking habits in Great Britain"], "year": ["2018"], "date-in-citation": ["accessed May 17, 2023"], "publisher-name": ["Office for National Statistics"], "comment": ["publishedonlineMay1. "], "ext-link": ["https://www.ons.gov.uk/peoplepopulationandcommunity/healthandsocialcare/drugusealcoholandsmoking/bulletins/opinionsandlifestylesurveyadultdrinkinghabitsingreatbritain/2017"]}, {"label": ["21"], "person-group": ["\n"], "surname": ["Pearson-Stuttard", "Zhou", "Kontis", "Bentham", "Gunter", "Ezzati"], "given-names": ["J", "B", "V", "J", "MJ", "M"], "article-title": ["Worldwide burden of cancer attributable to diabetes and high body-mass index: a comparative risk assessment"], "source": ["The Lancet Diabetes & Endocrinology"], "year": ["2018"], "volume": ["6"], "fpage": ["e6"], "lpage": ["15"]}, {"label": ["23"], "person-group": ["\n"], "surname": ["Danaei", "Hoorn", "Lopez", "Murray", "Ezzati"], "given-names": ["G", "SV", "AD", "CJ", "M"], "article-title": ["Causes of cancer in the world: comparative risk assessment of nine behavioural and environmental risk factors"], "source": ["The Lancet"], "year": ["2005"], "volume": ["366"], "fpage": ["1784"], "lpage": ["93"]}, {"label": ["25"], "collab": ["Office for National Statistics"], "source": ["Cigarette smoking among adults"], "year": ["2021"], "date-in-citation": ["accessed June 15, 2023"], "publisher-name": ["GOV.UK"], "comment": ["published online Feb 2. "], "ext-link": ["https://www.ethnicity-facts-figures.service.gov.uk/health/alcohol-smoking-and-drug-use/adult-smokers/latest"]}, {"label": ["29"], "collab": ["NHS"], "source": ["The NHS Long Term Plan"], "year": ["2019"], "date-in-citation": ["accessed May 17, 2023"], "publisher-name": ["NHS"], "ext-link": ["https://www.longtermplan.nhs.uk/publication/nhs-long-term-plan/"]}, {"label": ["30"], "collab": ["Department of Health and Social Care"], "source": ["New lung cancer screening roll out to detect cancer sooner"], "year": ["2023"], "date-in-citation": ["accessed July 6, 2023"], "publisher-name": ["GOV.UK"], "comment": ["published online June 26. "], "ext-link": ["https://www.gov.uk/government/news/new-lung-cancer-screening-roll-out-to-detect-cancer-sooner"]}]
{ "acronym": [], "definition": [] }
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2024-01-13 23:49:37
Lancet Oncol. 2024 Jan 11; 25(1):86-98
oa_package/61/bb/PMC7615518.tar.gz
PMC7615519
0
[ "<title>Introduction</title>", "<p id=\"P2\">Large-scale hypothesis testing is now ubiquitous in a variety of biomedical and technological applications. For example, many major technology companies perform tens of thousands of randomised controlled experiments (known as A/B tests) each year to make data-driven decisions about how to improve products (##REF##31898511##Kohavi et al., 2020##). Meanwhile, in genomics it is now routine to test hundreds of thousands of genetic variants for an association with particular phenotypic trait(s). Even in the setting of randomised controlled trials (RCTs) in medicine, there is a growing push towards the use of “overarching” trial frameworks to allow the efficient testing of multiple experimental drugs for multiple patient subpopulations.</p>", "<p id=\"P3\">Performing a large number of hypothesis tests naturally gives rise to the problem of multiple comparisons (##UREF##32##Tukey, 1953##): given a collection of multiple hypotheses to be tested, the goal is to distinguish which hypotheses are null and non-null, while controlling a suitable error rate (see <xref rid=\"S7\" ref-type=\"sec\">Section 1.2</xref>). This error rate is generally formed around the probability of incorrectly classifying a null hypothesis as non-null. Typically, a p-value is calculated for each hypothesis and is then used to decide whether to reject the null hypothesis. Multiple hypothesis testing is one of the core problems in statistical inference, and has led to a wide range of procedures that can be used to correct for multiplicity and ensure that a suitable error rate is controlled. In contrast, uncorrected hypothesis testing contributes to serious concerns over reproducibility, publication bias and ‘p-hacking’ in scientific research (##REF##16060722##Ioannidis, 2005##; ##REF##25768323##Head et al., 2015##).</p>", "<p id=\"P4\">Multiplicity, as broadly understood, is naturally linked to scientific reproducibility. ##UREF##14##Goodman, Fanelli and Ioannidis (2016)## state that “Multiplicity, combined with incomplete reporting, might be the single largest contributor to the phenomenon of nonreproducibility, or falsity, of published claims” and go on to say that “Scientific fields that routinely work with multiple hypotheses without correcting for or reporting the occurrence of multiplicity run a higher risk of nonreproducibility of results or inferences”. As an example of this, ##UREF##37##Zeevi, Astashenko and Benjamini (2020)## recently showed that adjusting for multiplicity greatly enhances the reproducibility of results from psychology experiments. Similarly, in the context of drug development, ##UREF##3##Bretz and Westfall (2014)## in a paper titled “Multiplicity and replicability: two sides of the same coin” showed that there is a close link between between multiplicity and replicability in terms of the observed effect sizes of selected subgroups, with further examples given in ##UREF##2##Bretz, Maurer and Xi (2019)##.</p>", "<p id=\"P5\">Traditionally, multiple hypothesis testing is <italic toggle=\"yes\">offline</italic> in nature, in the sense that a procedure for testing <italic toggle=\"yes\">N</italic> hypotheses will receive all of the corresponding <italic toggle=\"yes\">p</italic>-values (<italic toggle=\"yes\">P</italic><sub>1</sub>, …, <italic toggle=\"yes\">P<sub>N</sub></italic>) at once. Step-up and step-down multiple testing procedures (for example) require knowledge about all <italic toggle=\"yes\">p</italic>-values in advance. In the offline setting, the seminal Benjamini-Hochberg (BH) procedure is the dominant method used for FDR control. However, this paradigm is often incompatible with modern data-driven decision-making processes, as demonstrated by our motivating examples in <xref rid=\"S3\" ref-type=\"sec\">Section 1.1</xref>. Once the data is available to make a decision about a particular hypothesis, it can be desirable to take a corresponding action (e.g. to update a tech product) relatively quickly, and not to wait for the results of ongoing or future hypothesis tests. Linked with this, in many application areas it may not even be possible to know in advance how many tests in total will be performed. Moreover, the repeated application of traditional offline multiple testing procedures as the family of hypotheses grows can lead to repeatedly changing past decisions, which may be undesirable in some contexts.</p>", "<p id=\"P6\">What is needed therefore are procedures for <italic toggle=\"yes\">online</italic> multiple hypothesis testing, which better take into account the nature of modern data analysis. This is defined as follows: A stream of hypotheses arrives online. At each step, the analyst must decide whether to reject the current null hypothesis without having access to the number of hypotheses (potentially infinite) or any future data, but solely based on the previous decisions and evidence against the current hypothesis.</p>" ]
[ "<title>Online Error Rate Control Methodology</title>", "<title>Generalised alpha-investing (GAI)</title>", "<p id=\"P33\">The first proposals for online error rate control were based on “alpha-investing” by ##UREF##13##Foster and Stine (2008)## and its generalisation (GAI) (##UREF##0##Aharoni and Rosset, 2014##). (An alternative early line of work instead focused on extensions of gatekeeping procedures that allow for online control of the FWER or FDR for ordered hypotheses (##REF##20528862##Finos and Farcomeni, 2011##; ##REF##23844579##Farcomeni and Finos, 2013##) but these turn out to be far less powerful in practice, so we do not discuss them further.) Any GAI rule begins with an error budget, or <italic toggle=\"yes\">alpha-wealth</italic>, which is allocated to the different hypothesis tests over time. That is, there is a price to be paid each time a hypothesis is tested, which can be viewed as making an investment in the hypothesis in question. If the hypothesis is rejected, alpha-wealth is earned back, which can be viewed as a return or payout on the alpha-investment. Since the alpha-wealth can increase in this way, as long as discoveries continue to be made, hypotheses can be tested indefinitely without the test levels tending towards zero. The intuition behind the alphawealth increasing after a rejection is that the denominator in the FDP increases, therefore allowing the numerator (i.e. the number of false rejections) to also increase for future hypothesis tests while still controlling the FDR.</p>", "<p id=\"P34\">Formally, a GAI rule produces a series of test levels (<italic toggle=\"yes\">α</italic><sub>1</sub>, <italic toggle=\"yes\">α</italic><sub>2</sub>, <italic toggle=\"yes\">α</italic><sub>3</sub>, …) based on which it uses (1) to produce the corresponding decisions (<italic toggle=\"yes\">R</italic><sub>1</sub>, <italic toggle=\"yes\">R</italic><sub>2</sub>, <italic toggle=\"yes\">R</italic><sub>3</sub>, …). Of course, <italic toggle=\"yes\">α<sub>t</sub></italic> must be based only on <italic toggle=\"yes\">R</italic><sub>1</sub>, …, <italic toggle=\"yes\">R</italic><sub><italic toggle=\"yes\">t</italic>−1</sub>. At each time point <italic toggle=\"yes\">t</italic>, the alpha-wealth <italic toggle=\"yes\">W</italic>(<italic toggle=\"yes\">t</italic>) decreases by an amount <italic toggle=\"yes\">ϕ<sub>t</sub></italic>. If the hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic> is rejected (<italic toggle=\"yes\">R<sub>t</sub></italic> = 1), then the alpha-wealth is increased by <italic toggle=\"yes\">φ<sub>t</sub></italic>. In other words, the price <italic toggle=\"yes\">ϕ<sub>t</sub></italic> is the amount paid for testing (i.e., investing in) a new hypothesis, and the payout (or return on the investment) <italic toggle=\"yes\">φ<sub>t</sub></italic> is the amount earned if a discovery is made at that time. Hence the initial wealth is <italic toggle=\"yes\">W</italic>(0) = <italic toggle=\"yes\">w</italic><sub>0</sub> and it is updated via: </p>", "<p id=\"P35\">##FIG##1##Figure 2## give a diagrammatic summary of how GAI works. The total wealth <italic toggle=\"yes\">W</italic>(<italic toggle=\"yes\">t</italic>) must always be nonnegative, and hence <italic toggle=\"yes\">ϕ<sub>t</sub></italic> ≤ <italic toggle=\"yes\">W</italic>(<italic toggle=\"yes\">t</italic> − 1). Additionally, there are restrictions on <italic toggle=\"yes\">α<sub>t</sub></italic>, <italic toggle=\"yes\">ϕ<sub>t</sub></italic>, <italic toggle=\"yes\">φ<sub>t</sub></italic>, namely that when a rejection is made, the payout <italic toggle=\"yes\">φ<sub>t</sub></italic> is capped. This upper bound is there to ensure control of the FDR (and its variants).</p>", "<p id=\"P36\">Given these constraints, the user is free to choose the sequences <italic toggle=\"yes\">α<sub>t</sub></italic>, <italic toggle=\"yes\">ϕ<sub>t</sub></italic> and <italic toggle=\"yes\">φ<sub>t</sub></italic>. As an example, the alpha-investing rule explored in ##UREF##13##Foster and Stine (2008)## chooses and </p>", "<p id=\"P37\">The choice of <italic toggle=\"yes\">α<sub>t</sub></italic>, <italic toggle=\"yes\">ϕ<sub>t</sub></italic> and <italic toggle=\"yes\">φ<sub>t</sub></italic> was explored in terms of the trade-off between the sequences <italic toggle=\"yes\">α<sub>t</sub></italic> and <italic toggle=\"yes\">φ<sub>t</sub></italic> in ##UREF##0##Aharoni and Rosset (2014)##. However, in this paper, we focus on the new ‘statistical’ perspective for constructing online algorithms that control the FDR (see the start of <xref rid=\"S10\" ref-type=\"sec\">Section 2.2</xref>), which <italic toggle=\"yes\">implicitly</italic> give choices for <italic toggle=\"yes\">ϕ<sub>t</sub></italic> and <italic toggle=\"yes\">φ<sub>t</sub></italic>. As is pre-dominately the case in offline multiple testing, we often use <italic toggle=\"yes\">monotone</italic> decision rules for <italic toggle=\"yes\">α<sub>t</sub></italic> considered as a function of (<italic toggle=\"yes\">R</italic><sub>1</sub>, …, <italic toggle=\"yes\">R<sub>t</sub></italic>): if <italic toggle=\"yes\">R̃<sub>t</sub></italic> ≥ <italic toggle=\"yes\">R<sub>i</sub></italic> for all <italic toggle=\"yes\">i</italic> ≤ <italic toggle=\"yes\">t</italic> − 1, then we have <italic toggle=\"yes\">α<sub>t</sub></italic> (<italic toggle=\"yes\">R̃</italic><sub>1</sub>, …, <italic toggle=\"yes\">R̃</italic><sub><italic toggle=\"yes\">t</italic>−1</sub>) ≥ <italic toggle=\"yes\">α<sub>t</sub></italic> (<italic toggle=\"yes\">R</italic><sub>1</sub>, …, <italic toggle=\"yes\">R<sub>t</sub></italic>).</p>", "<p id=\"P38\">##UREF##25##Ramdas et al. (2017)## defined a class of improved GAI algorithms, called GAI++, as follows. Set <italic toggle=\"yes\">w</italic><sub>0</sub> so that 0 ≤ <italic toggle=\"yes\">w</italic><sub>0</sub> ≤ <italic toggle=\"yes\">α</italic> and choose the payout <italic toggle=\"yes\">φ<sub>t</sub></italic> to satisfy where <italic toggle=\"yes\">b<sub>t</sub></italic> = <italic toggle=\"yes\">α</italic> − <italic toggle=\"yes\">w</italic><sub>0</sub>1{<italic toggle=\"yes\">R</italic>(<italic toggle=\"yes\">t</italic> − 1) = 0}. This upper bound on the payout is different from the original GAI algorithms in order to guarantee FDR control while giving the largest possible payout for rejecting a hypothesis, with the choice of <italic toggle=\"yes\">w</italic><sub>0</sub> determining the payout received for the very first rejection (see e.g. the LORD++ algorithm in <xref rid=\"S10\" ref-type=\"sec\">Section 2.2</xref>). ##UREF##25##Ramdas et al. (2017)## show that any monotone GAI++ rule comes with the following guarantee:</p>", "<p id=\"P39\">T<sc>heorem</sc> 2.1. <italic toggle=\"yes\">If the null <italic toggle=\"yes\">p</italic>-values (i.e., the subsequence of <italic toggle=\"yes\">p</italic>-values where the null hypothesis is true) are independent of all other <italic toggle=\"yes\">p</italic>-values, any monotone GAI++ rule satisfies the bound</italic>\n\n<italic toggle=\"yes\">for all t</italic> ≥ 1. <italic toggle=\"yes\">Since W</italic>(<italic toggle=\"yes\">t</italic>) ≥ 0, <italic toggle=\"yes\">the FDR is controlled at level α</italic>.</p>", "<p id=\"P40\">This is in contrast to the GAI rules (including alphainvesting as proposed by ##UREF##13##Foster and Stine (2008)##), which only control the mFDR.</p>", "<p id=\"P41\">We note that the independence assumption refers to independence between <italic toggle=\"yes\">different</italic> hypotheses. The important case of sequential testing of any <italic toggle=\"yes\">single</italic> hypothesis can be seamlessly incorporated through the use of anytime-valid <italic toggle=\"yes\">p</italic>-values as described in the Introduction. A related framework is to use ‘asynchronous’ online testing, as discussed in <xref rid=\"S22\" ref-type=\"sec\">Section 5</xref>, which gives the added flexibility of allowing hypothesis tests to overlap in time.</p>", "<title>Algorithms for online FDR control: LORD, SAFFRON and ADDIS</title>", "<p id=\"P42\">Although an “algorithmic perspective” led to the GAI procedures initially used in the online testing literature, ##UREF##25##Ramdas et al. (2017)## posited a “statistical perspective” to construct procedures, which is to keep an estimate of the FDP less than <italic toggle=\"yes\">α</italic>. First, the oracle FDP is defined as where 𝓗<sub>0</sub> denotes the set of true null hypotheses. If we can keep FDP*(<italic toggle=\"yes\">t</italic>) ≤ <italic toggle=\"yes\">α</italic> at all times <italic toggle=\"yes\">t</italic>, then (depending on dependence assumption on the <italic toggle=\"yes\">p</italic>-values) we can prove that mFDR(<italic toggle=\"yes\">t</italic>) ≤ <italic toggle=\"yes\">α</italic> or FDR(<italic toggle=\"yes\">t</italic>) ≤ <italic toggle=\"yes\">α</italic>. This technique has been used to derive the LORD, SAFFRON and ADDIS algorithms (see below), by designing different estimates , , for FDP*(<italic toggle=\"yes\">t</italic>).</p>", "<title>LORD</title>", "<p id=\"P43\">The LORD algorithm was conceptualised by ##UREF##18##Javanmard and Montanari (2018)##, and is an instance of a monotone GAI rule. More precisely, given an infinite non-increasing sequence of positive constants that sums to one, the test levels <italic toggle=\"yes\">α<sub>t</sub></italic> for LORD are chosen as follows: where <italic toggle=\"yes\">τ<sub>j</sub></italic> denotes the time of the <italic toggle=\"yes\">j</italic>-th rejection and we must have <italic toggle=\"yes\">w</italic><sub>0</sub> + <italic toggle=\"yes\">b</italic><sub>0</sub> ≤ <italic toggle=\"yes\">α</italic> for FDR control to hold.</p>", "<p id=\"P44\">Following this, ##UREF##25##Ramdas et al. (2017)## defined a simple upper bound of FDP*(<italic toggle=\"yes\">t</italic>): and showed that LORD can be viewed as an algorithm that keeps . Here corresponds to the alpha-wealth used for testing while <italic toggle=\"yes\">αR</italic>(<italic toggle=\"yes\">t</italic>) corresponds to the total <italic toggle=\"yes\">earned</italic> alpha-wealth that can be used for subsequent tests. Exploiting this view, they derived a uniform improvement of LORD, termed LORD++, (presented below). In brief, LORD++ is able to replace <italic toggle=\"yes\">b</italic><sub>0</sub> = <italic toggle=\"yes\">α</italic> − <italic toggle=\"yes\">w</italic><sub>0</sub> with the choice <italic toggle=\"yes\">b</italic><sub>0</sub> = <italic toggle=\"yes\">α</italic> while still maintaining FDR control, with the catch that for the very first rejection only <italic toggle=\"yes\">b</italic><sub>0</sub> = <italic toggle=\"yes\">α</italic> − <italic toggle=\"yes\">w</italic><sub>0</sub> (see below).</p>", "<p id=\"P45\">Given an infinite non-increasing sequence of positive constants that sums to one, the test levels <italic toggle=\"yes\">α<sub>t</sub></italic> for LORD++ are chosen as follows: </p>", "<p id=\"P46\">The above formula may look daunting but it is interpretable. The first term is the fraction of the initial wealth <italic toggle=\"yes\">w</italic><sub>0</sub> that is used by the <italic toggle=\"yes\">t</italic>-th test. The other terms are the fractions of the earnings from rejections before <italic toggle=\"yes\">t</italic> that are spent in the <italic toggle=\"yes\">t</italic>-th round: LORD++ awards <italic toggle=\"yes\">α</italic> − <italic toggle=\"yes\">w</italic><sub>0</sub> for the first rejection and <italic toggle=\"yes\">α</italic> for every subsequent rejection, and on receiving this reward, the method immediately allocates that reward to future rounds according to the same schedule of constants {<italic toggle=\"yes\">γ<sub>t</sub></italic>}, shifted to start at the next instant. This rule ensures that LORD++ never spends more than it has earned, thus keeping .</p>", "<p id=\"P47\">The intuitive reason why LORD++ cannot award <italic toggle=\"yes\">α</italic> for the very first rejection can be seen in the definition . The denominator <italic toggle=\"yes\">R</italic>(<italic toggle=\"yes\">T</italic>) ⋁ 1 = 1 when the number of rejections equals zero or one, and hence only starts increasing at the second rejection. This means that the sum of <italic toggle=\"yes\">w</italic><sub>0</sub> and the first reward must be at most <italic toggle=\"yes\">α</italic>, following which <italic toggle=\"yes\">α</italic> may be rewarded at every rejection. As for the choice of the sequence <italic toggle=\"yes\">γ<sub>t</sub></italic>, this depends on the data application at hand, with a reasonable default choice given by , which has been shown to maximise power in the Gaussian setting (i.e. where the test statistics follow a normal distribution) (##UREF##18##Javanmard and Montanari, 2018##).</p>", "<p id=\"P48\">The manner in which is a simple upper bound on FDP*(<italic toggle=\"yes\">t</italic>) is reminiscent of the BH procedure for offline testing, which can be derived in a similar fashion. More precisely, suppose that one rejects all <italic toggle=\"yes\">p</italic>-values below some fixed threshold <italic toggle=\"yes\">s</italic> ∈ (0, 1). The BH procedure overestimates the FDP using the quantity , where 𝓡(<italic toggle=\"yes\">s</italic>) denotes the set of rejected <italic toggle=\"yes\">p</italic>-values using the fixed threshold <italic toggle=\"yes\">s</italic>. The BH procedure then rejects the set 𝓡(<italic toggle=\"yes\">ŝ</italic><sub>BH</sub>) where . This leads us to view LORD++ as the online analog of the BH procedure.</p>", "<p id=\"P49\">Guarantees for LORD++ hold under different <italic toggle=\"yes\">p</italic>-value dependencies, which we now formalise. Define the filtration at time <italic toggle=\"yes\">t</italic> as 𝓕<sub><italic toggle=\"yes\">t</italic></sub> = <italic toggle=\"yes\">σ</italic>(<italic toggle=\"yes\">R</italic><sub>1</sub>, …, <italic toggle=\"yes\">R<sub>t</sub></italic>) (representing the collection of the observed rejections up to time <italic toggle=\"yes\">t</italic>) and let <italic toggle=\"yes\">α<sub>t</sub></italic> = <italic toggle=\"yes\">f<sub>t</sub></italic>(<italic toggle=\"yes\">R</italic><sub>1</sub>, …, <italic toggle=\"yes\">R</italic><sub><italic toggle=\"yes\">t</italic>−1</sub>) where <italic toggle=\"yes\">f<sub>t</sub></italic> is a [0, 1]-valued function. The null <italic toggle=\"yes\">p</italic>-values are said to be conditionally super-uniform if Pr{<italic toggle=\"yes\">P<sub>t</sub></italic> ≤ <italic toggle=\"yes\">α<sub>t</sub></italic>|𝓕<sup><italic toggle=\"yes\">t</italic>−1</sup>} ≤ <italic toggle=\"yes\">α<sub>t</sub></italic> for any 𝓕<sup><italic toggle=\"yes\">t</italic>−1</sup>-measurable <italic toggle=\"yes\">α<sub>t</sub></italic>. Armed with this definition, we have the following theorem from ##UREF##25##Ramdas et al. (2017)##:</p>", "<p id=\"P50\">T<sc>heorem</sc> 2.2. (<italic toggle=\"yes\">a</italic>) <italic toggle=\"yes\">If the null <italic toggle=\"yes\">p</italic>-values are conditionally super-uniform, then the condition</italic>\n\n<italic toggle=\"yes\">for all t</italic> ≥ 1 <italic toggle=\"yes\">implies that mFDR</italic>(<italic toggle=\"yes\">t</italic>) ≤ <italic toggle=\"yes\">α</italic> for all <italic toggle=\"yes\">t</italic> ≥ 1.</p>", "<p id=\"P51\"><italic toggle=\"yes\">(b) If the null p</italic>-<italic toggle=\"yes\">values are independent of each other and of the p</italic>-<italic toggle=\"yes\">values corresponding to the non-null hypotheses, and</italic> {<italic toggle=\"yes\">α<sub>t</sub></italic>} <italic toggle=\"yes\">is chosen to be a monotone function of past rejections, then the condition</italic>\n\n<italic toggle=\"yes\">for all t</italic> ≥ 1 <italic toggle=\"yes\">implies that FDR</italic>(<italic toggle=\"yes\">t</italic>) ≤ <italic toggle=\"yes\">α for all t</italic> ≥ 1.</p>", "<p id=\"P52\">Finally, in terms of theoretical power guarantees for LORD, ##UREF##5##Chen and Arias-Castro (2021)## considered the setting of a (generalised) Gaussian model (see reference for further details) and showed that LORD is asymptotically optimal, in particular by being as powerful as BH to first asymptotic order.</p>", "<title>SAFFRON</title>", "<p id=\"P53\">##UREF##26##Ramdas et al. (2018)## derived an adaptive version of LORD++ called SAFFRON, which is based on an estimate of the proportion of true null hypotheses. By not wasting its earnings on attempting to reject weaker signals (i.e. larger <italic toggle=\"yes\">p</italic>-values), SAFFRON preserves alpha-wealth and hence can have a higher power than LORD++. To this end, we choose λ ∈ (0, 1) and define the candidate <italic toggle=\"yes\">p</italic>-values as those that satisfy <italic toggle=\"yes\">P<sub>t</sub></italic> ≤ λ, since SAFFRON will never reject a <italic toggle=\"yes\">p</italic>-value larger than λ. We also choose an infinite nonincreasing sequence of positive constants that sums to one. Reasonable default choices for these hyper-parameters are λ = 0.5 and <italic toggle=\"yes\">γ<sub>t</sub></italic> ∝ <italic toggle=\"yes\">t</italic><sup>−1.6</sup> (##UREF##26##Ramdas et al., 2018##). The formulae for the test levels <italic toggle=\"yes\">α<sub>t</sub></italic> for SAFFRON are given in ##SUPPL##0##Appendix A##.</p>", "<p id=\"P54\">SAFFRON starts off with alpha-wealth (1 − λ)<italic toggle=\"yes\">w</italic><sub>0</sub> and does not lose any of this wealth when testing candidate <italic toggle=\"yes\">p</italic>-values. Of course, this has to be done in a principled way and is accounted for in the formulation of the test levels <italic toggle=\"yes\">α<sub>t</sub></italic>, which intuitively helps explain the (1 − λ) multiplicative factor (see ##SUPPL##0##Appendix A##). It gains an alpha-wealth of (1 − λ)<italic toggle=\"yes\">α</italic> for each discovery after the first. SAFFRON can make more rejections than LORD++ if there is a significant fraction of non-nulls and the signals are strong.</p>", "<p id=\"P55\">Similar to LORD++, SAFFRON provably controls the mFDR at all times if the null <italic toggle=\"yes\">p</italic>-values are conditionally super-uniform. Also, SAFFRON controls the FDR at all times if the null <italic toggle=\"yes\">p</italic>-values are independent of each other and of the non-nulls, and {<italic toggle=\"yes\">α<sub>t</sub></italic>} is chosen to be a monotone function of (<italic toggle=\"yes\">R</italic><sub>1</sub>, …, <italic toggle=\"yes\">R</italic><sub><italic toggle=\"yes\">t</italic>−1</sub>, <italic toggle=\"yes\">C</italic><sub>1</sub>, …, <italic toggle=\"yes\">C</italic><sub><italic toggle=\"yes\">t</italic>−1</sub>), where <italic toggle=\"yes\">C<sub>t</sub></italic> = 𝟙{<italic toggle=\"yes\">P<sub>t</sub></italic> ≤ λ}; see ##UREF##26##Ramdas et al. (2018)## for details.</p>", "<title>ADDIS</title>", "<p id=\"P56\">stands for an ADaptive algorithm that DIScards conservative nulls, and was proposed by ##UREF##31##Tian and Ramdas (2019)##. ADDIS can invest alpha-wealth more effectively than LORD++ or SAFFRON by explicitly discarding the weakest signals (i.e. the largest <italic toggle=\"yes\">p</italic>-values) in a principled way, which can lead to a higher power. More formally, in practice it is common to encounter <italic toggle=\"yes\">conservative</italic> nulls, where a null <italic toggle=\"yes\">p</italic>-value <italic toggle=\"yes\">P</italic> is conservative if Pr{<italic toggle=\"yes\">P</italic> ≤ <italic toggle=\"yes\">x</italic>} &lt; <italic toggle=\"yes\">x</italic> for all <italic toggle=\"yes\">x</italic> ∈ [0, 1]. Often nulls are <italic toggle=\"yes\">uniformly</italic> conservative, which means that under the null, </p>", "<p id=\"P57\">For example, for a one-dimensional exponential family with parameter <italic toggle=\"yes\">θ</italic>, when the true parameter <italic toggle=\"yes\">θ</italic> is strictly smaller than <italic toggle=\"yes\">θ</italic><sub>0</sub>, the uniformly most powerful test of <italic toggle=\"yes\">H</italic><sub>0</sub> : <italic toggle=\"yes\">θ</italic> ≤ <italic toggle=\"yes\">θ</italic><sub>0</sub> versus <italic toggle=\"yes\">H</italic><sub>1</sub> : <italic toggle=\"yes\">θ</italic> &gt; <italic toggle=\"yes\">θ</italic><sub>0</sub> will give uniformly conservative nulls (##UREF##39##Zhao, Small and Su, 2019##). Another setting is using always-valid <italic toggle=\"yes\">p</italic>-values (##UREF##19##Johari et al., 2021##) in the context of continuous monitoring for A/B testing, which will always be conservative.</p>", "<p id=\"P58\">In general, adaptivity (used by both SAFFRON and ADDIS) helps when there is a significant fraction of non-nulls (like 10% or 20%). Discarding (used only by ADDIS) helps when the nulls are conservative, meaning that instead of being exactly uniform, they are stochastically larger than uniform. Discarding helps even without adaptivity, and adaptivity helps without discarding. The key idea behind discarding is intuitive: if you see a <italic toggle=\"yes\">p</italic>-value larger than (say) 0.5, throw it away, but if you see a <italic toggle=\"yes\">p</italic>-value smaller than 0.5, then double it (to condition on selection) and pass it onto the multiple testing procedure. Roughly, if there are mostly nulls and these are uniformly distributed, this doesn’t do much at all – the tested <italic toggle=\"yes\">p</italic>-values are doubled, but only about half the <italic toggle=\"yes\">p</italic>-values are tested so the multiplicity correction is halved, cancelling the effects. However, if the nulls are stochastically much larger than uniform, then we may throw away most of the nulls in this step, eventually testing only a much smaller number of <italic toggle=\"yes\">p</italic>-values (which have been doubled).</p>", "<p id=\"P59\">In terms of formal definitions, with λ and the corresponding candidate <italic toggle=\"yes\">p</italic>-values defined as for SAFFRON, we let <italic toggle=\"yes\">S<sub>t</sub></italic> = 𝟙 {<italic toggle=\"yes\">P<sub>t</sub></italic> ≤ <italic toggle=\"yes\">η</italic>} be the indicator of <italic toggle=\"yes\">H<sub>t</sub></italic> being selected for testing (i.e. not discarded). Hence <italic toggle=\"yes\">η</italic> is the discarding threshold and must be greater than λ. We also choose an infinite non-increasing sequence of positive constants that sums to one. Reasonable default choices for these hyper-parameters are λ = 0.25, <italic toggle=\"yes\">η</italic> = 0.5 and <italic toggle=\"yes\">γ<sub>t</sub></italic> ∝ (<italic toggle=\"yes\">t</italic> + 1)<sup>−1.6</sup>, as justified empirically in ##UREF##31##Tian and Ramdas (2019)##. The formulae for the test levels <italic toggle=\"yes\">α<sub>t</sub></italic> for ADDIS are given in ##SUPPL##0##Appendix B##. As can be seen, ADDIS starts off with an alpha-wealth of (<italic toggle=\"yes\">η</italic> – λ)<italic toggle=\"yes\">w</italic><sub>0</sub> and (like SAFFRON) does not lose any of this wealth when testing candidate <italic toggle=\"yes\">p</italic>-values. The <italic toggle=\"yes\">p</italic>-values that are greater than <italic toggle=\"yes\">η</italic> do not affect the test levels for ADDIS at all, i.e. as if they did not exist in the sequence of <italic toggle=\"yes\">p</italic>-values of all (reflecting the term ‘discarding’). It gains an alpha-wealth of (<italic toggle=\"yes\">η</italic> – λ)<italic toggle=\"yes\">α</italic> for each rejection after the first.</p>", "<p id=\"P60\">Like for LORD++ and SAFFRON, ADDIS provably controls the mFDR at all times if the null <italic toggle=\"yes\">p</italic>-values are conditionally uniformly conservative. ADDIS provably controls the FDR at all times if the null <italic toggle=\"yes\">p</italic>-values are independent of each other and of the non-nulls, and is a monotone function of the past; see ##UREF##31##Tian and Ramdas (2019)## for full details.</p>", "<title>Monotone AI</title>", "<p id=\"P61\">As a comparator to the above algorithms, we also consider a version of the original AI algorithm of ##UREF##13##Foster and Stine (2008)##, as modified by ##UREF##25##Ramdas et al. (2017)## to ensure it is a monotone rule and hence that FDR control holds. We will refer to this rule as ‘monotone AI’.</p>" ]
[]
[ "<title>Discussion</title>", "<p id=\"P112\">Online error rate control methodology provides a powerful and flexible framework for large-scale hypothesis testing that takes into account the temporal nature of modern data analysis. Over the past 15 years since this framework was first proposed, there have been many proposed improvements and extensions, which better reflect the nature of real-world data and expand the scope of potential applications. In particular, continuous progress has been made towards increasing the statistical power of online testing algorithms, so that they can match (and in some cases even exceed) the power of traditional offline algorithms. The issue of accounting for dependent <italic toggle=\"yes\">p</italic>-values remains open, although progress has been made here too. As the methodology becomes increasingly mature, the next natural step is to see application of online testing algorithms in practice. To this end, and as seen in <xref rid=\"S19\" ref-type=\"sec\">Section 4</xref>, there have been a number of papers that are specifically focused on application examples, including in the context of growing data repositories (##REF##30873526##Robertson et al., 2019##), anomaly detection in time series (##UREF##27##Rebjock et al., 2021##), platform trials (##REF##37005003##Robertson et al., 2023##) and RNAseq data (##UREF##22##Liou, Hornburg and Robertson, 2023##). Further work may be required to explore and solve practical challenges that may arise in different application settings. Finally, the provision of software and training will also be key to promoting the use of online error rate control in practice. The <monospace>onlineFDR</monospace> package we described earlier is a key step in that regard, but software tuned to specific applications may also be desirable.</p>" ]
[]
[ "<p id=\"P1\">Modern data analysis frequently involves large-scale hypothesis testing, which naturally gives rise to the problem of maintaining control of a suitable type I error rate, such as the false discovery rate (FDR). In many biomedical and technological applications, an additional complexity is that hypotheses are tested in an online manner, one-by-one over time. However, traditional procedures that control the FDR, such as the Benjamini-Hochberg procedure, assume that all <italic toggle=\"yes\">p</italic>-values are available to be tested at a single time point. To address these challenges, a new field of methodology has developed over the past 15 years showing how to control error rates for online multiple hypothesis testing. In this framework, hypotheses arrive in a stream, and at each time point the analyst decides whether to reject the current hypothesis based both on the evidence against it, and on the previous rejection decisions. In this paper, we present a comprehensive exposition of the literature on online error rate control, with a review of key theory as well as a focus on applied examples. We also provide simulation results comparing different online testing algorithms and an up-to-date overview of the many methodological extensions that have been proposed.</p>", "<title>Key words and phrases</title>" ]
[ "<title>Online and sequential testing</title>", "<p id=\"P7\">Online hypothesis testing has a sequential nature, in the sense that individual hypotheses (or batches of hypotheses) are tested one after the other over time. However, this is distinct from the more traditional concept of <italic toggle=\"yes\">sequential testing</italic>, which refers to the testing of a <italic toggle=\"yes\">single</italic> hypothesis in a sequential manner with data accumulating over time. In sequential testing, the sample size for the experiment is not fixed in advance, and the accumulating data is evaluated as they are collected to allow the experiment to be stopped adaptively, such as when statistical significance is achieved. The framework of online multiple testing can be expanded to be “doubly sequential”, where the inner sequential process is a single sequential test, and the outer sequential process refers to the multiple experiments that are performed to test different hypotheses.</p>", "<p id=\"P8\">For each null hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic>, an anytime-valid <italic toggle=\"yes\">p</italic>-value is a sequence of <italic toggle=\"yes\">p</italic>-values (<italic toggle=\"yes\">P<sub>t,n</sub></italic>)<sub><italic toggle=\"yes\">n</italic>≥1</sub> where <italic toggle=\"yes\">n</italic> indexes the sample size in the experiment corresponding to hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic>, such that Pr(<italic toggle=\"yes\">P<sub>t,N</sub></italic> ≤ <italic toggle=\"yes\">x</italic>) ≤ <italic toggle=\"yes\">x</italic>, for all <italic toggle=\"yes\">x</italic> ∈ [0, 1] and <italic toggle=\"yes\">any</italic> data-dependent stopping time <italic toggle=\"yes\">N</italic>. In other words, the stopped anytime <italic toggle=\"yes\">p</italic>-value is a valid <italic toggle=\"yes\">p</italic>-value in the classical sense, no matter how the experiment was stopped. In online multiple testing, we typically drop the second index and focus on the “outer sequential process” (across experiments/hypotheses), which means that we assume that for each hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic>, we have a valid <italic toggle=\"yes\">p</italic>-value <italic toggle=\"yes\">P<sub>t</sub></italic>, but we keep in mind that this could have been achieved by stopping an anytime-valid <italic toggle=\"yes\">p</italic>-value (the “inner sequential process”, corresponding to the evidence within a single experiment).</p>", "<p id=\"P9\">We also wish to draw a distinction between online hypothesis testing and <italic toggle=\"yes\">multi-armed bandit (MAB)</italic> testing. While both frameworks allow the comparison of multiple experimental arms over time, an MAB can be considered as a <italic toggle=\"yes\">single</italic> experiment in which resources are iteratively allocated to the different arms in order to adaptively trade off certain costs and benefits, and this allocation depends on the previously observed outcomes on each arm. Again, the two testing frameworks can be combined within a doubly sequential framework where there is a sequence of MAB problems over time, see ##UREF##36##Yang et al. (2017)##.</p>", "<p id=\"P10\">##FIG##0##Figure 1## gives a diagrammatic representation of online multiple testing, where different hypotheses (corresponding to experiments) are tested over time (corresponding to the collection of data samples). As discussed above, each experiment could itself be a sequential experiment or take the form of an MAB.</p>", "<p id=\"P11\">Since the framework was first proposed by ##UREF##13##Foster and Stine (2008)##, a variety of procedures that control error rates for online hypothesis testing have been developed (##UREF##0##Aharoni and Rosset, 2014##; ##UREF##17##Javanmard and Montanari, 2015##; ##UREF##26##Ramdas et al., 2018##). Our aim in this paper is to provide an expository overview of this literature on online error rate control, with a review of the underlying theory, key methods and applied examples.</p>", "<p id=\"P12\">The bulk of the literature has focused on the setting of independent hypothesis tests for provable FDR control (with slightly weaker conditions allowing for control of variants of the FDR). Another important feature of many of the algorithms presented here is that they are <italic toggle=\"yes\">adaptive</italic>: when some fraction of the tests actually have the alternative hypothesis true (as evidenced by <italic toggle=\"yes\">p</italic>-values), they adapt and use less conservative tests.</p>", "<p id=\"P13\">In the rest of this section, we give motivating examples for online multiple testing and present formal definitions of error rate control. <xref rid=\"S8\" ref-type=\"sec\">Section 2</xref> describes the key procedures for online error rate control in detail. <xref rid=\"S15\" ref-type=\"sec\">Section 3</xref> presents a simulation study of the procedures, while <xref rid=\"S19\" ref-type=\"sec\">Section 4</xref> presents two case studies of applying online error rate control. We describe further methodological extensions as well as future directions in <xref rid=\"S22\" ref-type=\"sec\">Section 5</xref>. In <xref rid=\"S40\" ref-type=\"sec\">Section 6</xref> we provide a summary and some practical guidance, and conclude with a discussion in <xref rid=\"S41\" ref-type=\"sec\">Section 7</xref>.</p>", "<title>Motivating examples</title>", "<p id=\"P14\">We now present three motivating examples from a spectrum of ‘easier’ to ‘harder’ settings for currently available online testing algorithms, in terms of the statistical dependency between hypotheses.</p>", "<title>A/B testing in tech companies (independent hypotheses)</title>", "<p id=\"P15\">The development of web applications and services in the tech industry increasingly relies on the use of randomised controlled experiments known as A/B tests. There are a number of widely used platforms now available that streamline and handle the implementation of A/B tests. A typical application is in the development of different versions of webpages. As described in ##UREF##1##Berman and Van den Bulte (2021)##, in this context there are two webpage variations (A and B). When an online user visits, the platform randomly assigns the visitor to one of the variations for the duration of the experiment. The platform records the actions that the visitor takes, where the monitored actions reflect the experimenter’s goal(s), such as increasing visitor engagement (defined appropriately) or increasing revenue. One of the variations is designated as the baseline, and the performance of the other variation is compared to the baseline using suitable test statistics. If run correctly using anytime-valid <italic toggle=\"yes\">p</italic>-values (##UREF##19##Johari et al., 2021##) and/or confidence sequences (##UREF##15##Howard et al., 2021##), the data can be continuously monitored by the experimenter, and a decision can be made at any stopping time.</p>", "<p id=\"P16\">Many tech companies run tens of thousands of A/B tests per year, as part of a continuous process of designing, delivering, monitoring and improving webpages and other web services. However, there is reason to reduce the number of false alarms which result in making changes to web products that do no better (or even perform worse) than the current iteration, corresponding to an incorrect rejection of the null, particularly when such changes are potentially costly or disruptive to users. Hence, the framework of online error rate control provides a framework to do so while still allowing a large number of A/B tests to be performed in a flexible manner.</p>", "<title>Platform trials (known positive dependence)</title>", "<p id=\"P17\">A platform trial has a single master protocol that evaluates multiple treatments across one or more patient types, and allows a potentially large number of treatments to be added during the course of the trial (##REF##26908536##Saville and Berry, 2016##). A new treatment can be added to the trial (corresponding to testing a new hypothesis) when a new experimental therapy becomes available, such as when a safe drug candidate for the disease in question is identified from a successful phase I clinical trial. Treatments are dropped from the trial after they have been formally tested for effectiveness. Such a trial could (in theory) be ‘perpetual’ in that new treatments can continue to enter into the trial and be tested. ##FIG##4##Figure 5## in <xref rid=\"S21\" ref-type=\"sec\">Section 4.2</xref> gives a diagram of an example platform trial showing what this looks like.</p>", "<p id=\"P18\">In a platform trial, treatments are introduced at different time points by design. However, the trial investigators will wish to make a decision on whether a treatment is beneficial as soon as the data are ready, without waiting for results from the other treatment arms. Hence, the treatment effects are tested sequentially in an online manner, where the number of treatments to be tested in the future may be unknown. More formally, a platform trial generates a sequence of null hypotheses (<italic toggle=\"yes\">H</italic><sub>1</sub>, <italic toggle=\"yes\">H</italic><sub>2</sub>, <italic toggle=\"yes\">H</italic><sub>3</sub>, …) which are tested sequentially. Hypothesis <italic toggle=\"yes\">H<sub>i</sub></italic> tests the value of some parameter <italic toggle=\"yes\">θ<sub>i</sub></italic>, such as an estimate of the treatment difference compared to a control arm.</p>", "<p id=\"P19\">The <italic toggle=\"yes\">p</italic>-values generated from the platform trial described above will not be independent in general. Dependencies will primarily arise due to the shared control data that is re-used to test multiple hypotheses. A current example of a long-running platform trial is the STAMPEDE trial (##REF##18760574##James et al., 2008##) for patients with locally advanced or metastatic prostate cancer, which we return to as a case study in <xref rid=\"S21\" ref-type=\"sec\">Section 4.2</xref>.</p>", "<title>Data repositories (unknown arbitrary dependence)</title>", "<p id=\"P20\">Public databases and shared data resources are becoming increasingly pervasive and important in modern biomedical research, particularly in the fields of genetics, molecular biology and routinely collected healthcare records. Some well-known examples include the 1000 Genomes Project (##REF##26432245##1000 Genomes Project Consortium et al., 2015##) and the Wellcome Trust Case Control Consortium (##REF##17554300##Wellcome Trust Case Control Consortium et al., 2007##). Another example is the International Mouse Phenotyping Consortium database (##REF##24194600##Koscielny et al., 2013##; ##REF##27626380##Dickinson et al., 2016##), which we describe as one of our case studies in <xref rid=\"S19\" ref-type=\"sec\">Section 4</xref>. Meanwhile, the increase in routinely collected healthcare data allows evaluation of different healthcare technologies used in practice through emulation of target trials (##UREF##8##Dickerman et al., 2019##).</p>", "<p id=\"P21\">Multiple testing naturally occurs in this setting in two ways. Firstly, such databases can be accessed by multiple independent researchers at different times. When a researcher or research group comes up with a new hypothesis, they can fetch the relevant data from a database and perform a statistical test. Secondly, in some databases the family of hypotheses to be tested grows over time as new new hypotheses are tested (e.g., corresponding to new experiments being performed that measure phenotype expression for a previously untested gene knockout. In both of these scenarios, the number of hypotheses being tested will be unknown and potentially very large, and lead to concern about overlapping data allowing for arbitrary correlation patterns between hypothesis tests. The issues such dependence causes will be considered throughout the rest of this paper.</p>", "<p id=\"P22\">In order to control the number or proportion of false discoveries in this context, new procedures are required that allow a researcher to decide whether to reject a current hypothesis with minimal information about previous hypotheses, and without prior knowledge of even the number of hypotheses that are going to be tested in the future. This is precisely the online multiple testing framework described earlier.</p>", "<title>Error rates</title>", "<p id=\"P23\">We now formally define some error rates of interest. The basic problem setup is as follows. At each time step <italic toggle=\"yes\">t</italic> = 1, 2, … the experimenter observes a <italic toggle=\"yes\">p</italic>-value <italic toggle=\"yes\">P<sub>t</sub></italic> corresponding to testing a null hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic>, and must make a decision whether to reject <italic toggle=\"yes\">H<sub>t</sub></italic> before the next time step. We assume that all <italic toggle=\"yes\">p</italic>-values are valid, i.e. if the null hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic> is true, then Pr{<italic toggle=\"yes\">P<sub>t</sub></italic> ≤ <italic toggle=\"yes\">x</italic>} ≤ <italic toggle=\"yes\">x</italic> for all <italic toggle=\"yes\">x</italic> ∈ [0, 1].<sup><xref rid=\"FN1\" ref-type=\"fn\">1</xref></sup> At time <italic toggle=\"yes\">t</italic> = 0, the experimenter fixes the level <italic toggle=\"yes\">α</italic> at which a suitable error rate is meant to be controlled at all times.</p>", "<p id=\"P24\">A general testing procedure provides a sequence of test levels α<sub>t</sub> with decision rule </p>", "<p id=\"P25\">At any time <italic toggle=\"yes\">T</italic>, let denote the number of rejections (also known as discoveries) made so far and <italic toggle=\"yes\">V</italic> (<italic toggle=\"yes\">T</italic>) denote the total number of falsely rejected hypotheses (also known as false discoveries).</p>", "<p id=\"P26\">The <italic toggle=\"yes\">false discovery proportion</italic> (FDP) up to time <italic toggle=\"yes\">T</italic> is defined as where <italic toggle=\"yes\">α</italic> ⋁ <italic toggle=\"yes\">b</italic> = max(<italic toggle=\"yes\">a</italic>, <italic toggle=\"yes\">b</italic>). The <italic toggle=\"yes\">false discovery rate</italic> (FDR) is then the expectation of the FDP: </p>", "<p id=\"P27\">A commonly studied variant is the <italic toggle=\"yes\">marginal</italic> FDR (mFDR): </p>", "<p id=\"P28\">Another related error rate is the <italic toggle=\"yes\">false discovery exceedance</italic> (FDX), which is the probability the supremum of the FDP exceeds a predefined threshold <italic toggle=\"yes\">ϵ</italic>: </p>", "<p id=\"P29\">We view the FDR as the central metric of interest, given its long history, widespread use in applied fields such as genetics, and intuitive interpretation. The mFDR can be a convenient theoretically tractable proxy for the FDR when it is not possible to prove FDR control for a particular algorithm and data application, as we highlight in the rest of the paper. In some settings previously explored in the literature, it has been shown empirically that the realised FDR and mFDR of online hypothesis testing algorithms are very similar (see e.g. ##SUPPL##0##Appendix F## of ##UREF##40##Zrnic, Ramdas and Jordan (2021)##), although this is not true in general (see e.g. the Supplementary material in ##UREF##18##Javanmard and Montanari (2018)##). Hence, the FDR would be the default choice for most users, with the mFDR then being the pragmatic alternative error rate choice if a suitable algorithm for FDR control is not available for the particular data application in mind.</p>", "<p id=\"P30\">In contrast, the FDX gives a stricter guarantee about the distribution of the FDP: whereas the FDR controls the expectation of the FDP, the FDX controls the tail probability of the FDP (i.e., controlling the (1 − <italic toggle=\"yes\">ϵ</italic>)-quantile of the FDP distribution). Control of the FDX makes most sense in settings where the FDP can deviate significantly from its expectation, such as when the number of hypotheses to be tested is not very large, or there is significant correlation (##UREF##18##Javanmard and Montanari, 2018##). As for the choice of <italic toggle=\"yes\">ϵ</italic> for the FDX, a default choice of <italic toggle=\"yes\">ϵ</italic> = 0.05 or 0.10 is one option, but a pre-hoc choice of <italic toggle=\"yes\">ϵ</italic> may also be motivated on practical grounds, such as choosing <italic toggle=\"yes\">ϵ</italic> based on the required sample size to achieve a desired power given control of the FDX at level <italic toggle=\"yes\">α</italic>. Another approach is to use recently proven post-hoc bounds of the FDX<sub><italic toggle=\"yes\">ϵ</italic></sub> for online testing algorithms under independence, which allows the user to choose <italic toggle=\"yes\">ϵ</italic> (and <italic toggle=\"yes\">α</italic>) freely by examining the corresponding rejections and seeing what makes most sense (##UREF##21##Katsevich and Ramdas, 2020##).</p>", "<p id=\"P31\">An alternative error rate to those based on the FDP is the <italic toggle=\"yes\">familywise error rate</italic> (FWER), which is more commonly considered in clinical trial contexts due to the relatively small number of hypotheses and regulatory requirements. The FWER is the probability of falsely rejecting any null hypothesis: </p>", "<p id=\"P32\">The FWER and hence the FDR can be controlled at level α in a simple manner by using a Bonferonni-type correction, also known as <italic toggle=\"yes\">alpha-spending</italic>. More precisely, we can choose significance levels <italic toggle=\"yes\">α<sub>t</sub></italic> for <italic toggle=\"yes\">H<sub>t</sub></italic>, such that . We reiterate that this corresponds to the setting where each nominal critical value <italic toggle=\"yes\">α<sub>t</sub></italic> corresponds to testing a single hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic>, with the possibility of repeated testing of the same hypothesis (as in the sequential testing literature) included implicitly, see our remark in the Introduction. However, alpha-spending suffers from a low statistical power, with the probability of the null hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic> being rejected tending to zero as <italic toggle=\"yes\">t</italic> increases. This motivates the development of more sophisticated algorithms for online error rate control.</p>", "<title>Simulation Studies</title>", "<p id=\"P62\">In this section we compare the performance of the LORD++, SAFFRON and ADDIS algorithms in terms of the FDR and statistical power. We do not aim to present an exhaustive simulation of all the algorithms currently available in the literature, but rather select a representative set of algorithms to demonstrate some key general features for the core problem of online FDR control. To this end, we use LORD++ as a representative ‘basic’ online algorithm, given that it is the natural online analog of the BH procedure. We then use SAFFRON as a representative of an adaptive online algorithm, while ADDIS is a representative of an adaptive online algorithm that also incorporates discarding. As an additional comparison, we also include the ‘monotone AI’ rule. In <xref rid=\"S18\" ref-type=\"sec\">Section 3.3</xref> we refer the reader to further simulation studies that have been published in the literature. First though, we briefly describe software implementation of algorithms for online error rate control.</p>", "<title>Software: onlineFDR package</title>", "<p id=\"P63\">The <monospace>onlineFDR</monospace> package is an open-source R package that aims to provide a comprehensive and up-to-date implementation of algorithms for online error rate control. It is freely available via <italic toggle=\"yes\">Bioconductor</italic> (##UREF##29##Robertson et al., 2021##). The package implements the LORD++, SAFFRON and ADDIS algorithms, as well as almost all of the algorithms corresponding to the further extensions of online error rate control methodology (see <xref rid=\"S23\" ref-type=\"sec\">Section 5.1</xref>). In particular, it also provides functions for algorithms for online FWER and online FDX control. The package documentation provides a user-friendly introduction to the use of the package, and there is also a Shiny app available (##UREF##23##Liou and Robertson, 2021##) to allow users to explore algorithms for online FDR control in an interactive way without having to program. All results for the simulation and case studies in this paper were calculated using the package.</p>", "<title>Testing with Gaussian observations</title>", "<p id=\"P64\">In order to examine the relative performance of the online FDR algorithms, we use a simple experimental setup of testing Gaussian means, with a total of <italic toggle=\"yes\">T</italic> hypotheses. Note that although <italic toggle=\"yes\">T</italic> is fixed in the simulations, all the methods do not use this knowledge of <italic toggle=\"yes\">T</italic> to normalise the sequence <italic toggle=\"yes\">γ<sub>t</sub></italic>. The null hypotheses take the form <italic toggle=\"yes\">H<sub>t</sub></italic> : <italic toggle=\"yes\">μ<sub>t</sub></italic> ≤ 0 which are tested against the alternative for <italic toggle=\"yes\">t</italic> = 1, …, <italic toggle=\"yes\">T</italic>. We observe independent observations <italic toggle=\"yes\">Z<sub>t</sub></italic> ~ <italic toggle=\"yes\">N</italic>(<italic toggle=\"yes\">μ<sub>t</sub></italic>, 1) which are transformed to one-sided <italic toggle=\"yes\">p</italic>-values <italic toggle=\"yes\">P<sub>t</sub></italic> = Φ(−<italic toggle=\"yes\">Z<sub>t</sub></italic>), where Φ denotes the standard Gaussian CDF. The motivation for using one-sided <italic toggle=\"yes\">p</italic>-values is from A/B testing, where one wishes to detect larger effects, not smaller. The means <italic toggle=\"yes\">μ<sub>t</sub></italic> are set according to the following mixture model: where <italic toggle=\"yes\">F</italic><sub>1</sub> ~ <italic toggle=\"yes\">N</italic>(3, 1) and <italic toggle=\"yes\">F</italic><sub>0</sub> is defined as below.</p>", "<p id=\"P65\">We use the default settings for LORD++, SAFFRON and ADDIS that are implemented in the <monospace>onlineFDR</monospace> package (following suggestions in the literature). For LORD++, we use the default choice of . We also use this choice of <italic toggle=\"yes\">γ<sub>t</sub></italic> for alpha-spending, where is simply given by <italic toggle=\"yes\">α<sub>t</sub></italic> = <italic toggle=\"yes\">αγ<sub>t</sub></italic>. For SAFFRON we set λ = 0.5 and . Finally, for ADDIS we set λ = 0.25, <italic toggle=\"yes\">η</italic> = 0.5 and . We use the exponent 1.6 in the denominator for <italic toggle=\"yes\">γ<sub>t</sub></italic> because this was found empirically to work well in a range of different simulation studies in the original papers. More precisely, the sequence <italic toggle=\"yes\">γ<sub>t</sub></italic> satisfies .</p>", "<p id=\"P66\">##FIG##2##Figure 3## shows how the test levels (displayed on the log<sub>10</sub> scale) evolve over time for LORD++, SAFFRON, ADDIS and monotone AI compared with uncorrected testing (where <italic toggle=\"yes\">α<sub>t</sub></italic> ≡ <italic toggle=\"yes\">α</italic>) and alpha-spending. Here, <italic toggle=\"yes\">T</italic> = 300, <italic toggle=\"yes\">α</italic> = 0.05, <italic toggle=\"yes\">π</italic><sub>1</sub> = 0.5 and we choose <italic toggle=\"yes\">F</italic><sub>0</sub> ≡ 0.</p>", "<p id=\"P67\">All of the online FDR algorithms have higher test levels than alpha-spending (apart from LORD++ briefly early on in this particular experiment). The relative difference increases with <italic toggle=\"yes\">t</italic> as the online algorithms ‘earn back’ wealth over time, which alpha-spending cannot do. SAFFRON, ADDIS and monotone AI have higher test levels than LORD++, reflecting how they can more efficiently invest the alpha-wealth. In this setting, since <italic toggle=\"yes\">μ<sub>t</sub></italic> = 0 under the null, the nulls are exactly uniform and so ADDIS cannot take advantage of conservative nulls. Hence the testing levels of ADDIS are similar or slightly lower than those for SAFFRON and monotone AI. Finally, we see that SAFFRON has similar test levels as uncorrected testing, and SAFFRON, ADDIS and monotone AI can even have test levels above the nominal <italic toggle=\"yes\">α</italic>.</p>", "<p id=\"P68\">##FIG##3##Figure 4## compares the statistical power of LORD++, SAFFRON, ADDIS and monotone AI compared with uncorrected testing and alpha-spending, as <italic toggle=\"yes\">π</italic><sub>1</sub> varies from 0.01 to 0.9. Here, we define power as where 𝓗<sub>1</sub> denotes the index set of the non-null hypotheses. We also include the standard Benjamini-Hochberg (BH) procedure as an additional comparison. We stress that BH is an offline procedure and so could not be used for online testing in practice. In our simulation, we set <italic toggle=\"yes\">T</italic> = 1000, <italic toggle=\"yes\">α</italic> = 0.05 and <italic toggle=\"yes\">F</italic><sub>0</sub> ~ <italic toggle=\"yes\">N</italic>(−0.5, 0.1). Results are based on averaging 10<sup>4</sup> simulation replicates (which implies a Monte Carol standard error when power = 0.5 of 0.005).</p>", "<p id=\"P69\">Starting with alpha-spending, as expected the power is very low (&lt; 0.2) for all <italic toggle=\"yes\">π</italic><sub>1</sub>. LORD++ has substantial power gains compared with alpha-spending (as long as <italic toggle=\"yes\">π</italic><sub>1</sub> is not close to zero) and this advantage increases with <italic toggle=\"yes\">π</italic><sub>1</sub>. However, LORD++ has substantially lower power than BH for all values of <italic toggle=\"yes\">π</italic><sub>1</sub>. As expected, SAFFRON performs better as the fraction of non-nulls <italic toggle=\"yes\">π</italic><sub>1</sub> increases, with a higher power than LORD++ for <italic toggle=\"yes\">π</italic><sub>1</sub> &gt; 0.05, BH for <italic toggle=\"yes\">π</italic><sub>1</sub> &gt; 0.5 and even uncorrected testing for <italic toggle=\"yes\">π</italic><sub>1</sub> &gt; 0.7. Since <italic toggle=\"yes\">F</italic><sub>0</sub> ~ <italic toggle=\"yes\">N</italic>(−0.5, 0.1), almost all the means for the null hypotheses will be negative, i.e. we are in a setting with conservative nulls. Hence, as expected, ADDIS outperforms SAFFRON in terms of power (except for very high values of <italic toggle=\"yes\">π</italic><sub>1</sub>). ADDIS also has a higher power than BH for <italic toggle=\"yes\">π</italic><sub>1</sub> &gt; 0.2 and uncorrected testing for <italic toggle=\"yes\">π</italic><sub>1</sub> &gt; 0.6. Finally, in this setting, SAFFRON performs very similarly to the monotone AI algorithm in terms of power.</p>", "<p id=\"P70\">In ##SUPPL##0##Appendix C## (##FIG##5##Figure 6##) we show the corresponding FDR for all of the algorithms considered. We see that uncorrected testing can have substantial inflation of the FDR, with the FDR inflated above the nominal α = 0.05 level for <italic toggle=\"yes\">π</italic><sub>1</sub> &lt; 0.3. The FDR reaches as high as 0.65 when <italic toggle=\"yes\">π</italic><sub>1</sub> =0.01. All other algorithms control the FDR below the nominal 0.05 level, as expected.</p>", "<title>Observations from other simulations</title>", "<p id=\"P71\">Here, we summarise a few take-home messages for LORD, SAFFRON and ADDIS from simulation results already found in the literature. ##UREF##18##Javanmard and Montanari (2018)## investigated the effect of the ordering of the hypotheses for online testing rules, including LORD. In some applications, hypotheses can be ordered using side information, such that those that are most likely to be rejected come first. With this favourable ordering, the statistical power of LORD can substantially increase as long as <italic toggle=\"yes\">π</italic><sub>1</sub> is not too large (since ordering is less relevant in that case). Similar findings for LORD++, SAFFRON and ADDIS in the context of platform trials can be found in ##REF##37005003##Robertson et al. (2023)##, which also looked at the adversarial setting where hypotheses happen to be ordered so that those most likely to be rejected come last, resulting in lower power.</p>", "<p id=\"P72\">##UREF##26##Ramdas et al. (2018)## considered the impact on LORD++ and SAFFRON of choosing sequences of the form <italic toggle=\"yes\">γ<sub>t</sub></italic> ∝ <italic toggle=\"yes\">t</italic><sup>−<italic toggle=\"yes\">s</italic></sup>, where the parameter <italic toggle=\"yes\">s</italic> &gt; 1 controls the ‘aggressiveness’ of the procedure (since the larger the value of <italic toggle=\"yes\">s</italic>, the more the alpha-wealth is concentrated at small values of <italic toggle=\"yes\">t</italic>). For Gaussian alternatives, the simulation results suggested that less aggressive sequences are to be preferred in terms of increased power for SAFFRON and LORD++. Meanwhile, ##UREF##31##Tian and Ramdas (2019)## showed that ADDIS can match the power of SAFFRON when the nulls are not conservative (i.e. uniform nulls). The power advantage of ADDIS over LORD++ and SAFFRON increases the more conservative the nulls are, i.e. the more negative the means for the null hypotheses are (in the Gaussian setting).</p>", "<p id=\"P73\">The theory presented in <xref rid=\"S8\" ref-type=\"sec\">Section 2</xref> for provable FDR control requires null <italic toggle=\"yes\">p</italic>-values to be independent of one another (with a weaker condition sufficing for mFDR control). ##REF##37005003##Robertson et al. (2023)## explored the performance of online testing rules, including LORD++, SAFFRON and ADDIS, in the setting of platform trials with a common control, which induces positive correlations between the <italic toggle=\"yes\">p</italic>-values for testing concurrent arms. There was no evidence of FDR inflation for these algorithms under a range of assumed treatment effects and overlap of control data. ##UREF##28##Robertson and Wason (2018)## considered the setting where the test statistics are assumed to come from a multivariate normal distribution where the covariance matrix has ones along the diagonals and off-diagonal entries equal to ±0.5. There was no evidence of FDR inflation when using LORD++ under a range of non-null distributions. However, with a two-sided test under a Gaussian alternative, the SAFFRON procedure had an inflated FDR for smaller values of <italic toggle=\"yes\">π</italic><sub>1</sub>. This inflation persisted and even increased as <italic toggle=\"yes\">T</italic> increased from 100 to 1000. For further discussion handling dependent <italic toggle=\"yes\">p</italic>-values, we refer the reader to the end of <xref rid=\"S34\" ref-type=\"sec\">Section 5.2</xref>.</p>", "<p id=\"P74\">The simulation studies in ##UREF##28##Robertson and Wason (2018)## also highlighted the value of using ‘bounded’ versions of online testing algorithms. This requires setting an <italic toggle=\"yes\">a</italic>-<italic toggle=\"yes\">priori</italic> upper bound <italic toggle=\"yes\">M</italic> on the number of hypotheses to be tested, so that the <italic toggle=\"yes\">γ<sub>t</sub></italic> ≡ 0 for <italic toggle=\"yes\">t</italic> &gt; <italic toggle=\"yes\">M</italic>, which allows setting <italic toggle=\"yes\">γ<sub>t</sub></italic> ≡ 1/<italic toggle=\"yes\">M</italic> for <italic toggle=\"yes\">t</italic> ≤ <italic toggle=\"yes\">M</italic> (for example). The bounded versions have a uniformly higher power than the versions presented in <xref rid=\"S8\" ref-type=\"sec\">Sections 2</xref> (with the default choices of <italic toggle=\"yes\">γ<sub>t</sub></italic> given in <xref rid=\"S17\" ref-type=\"sec\">Section 3.2</xref>) which assume no upper bound on <italic toggle=\"yes\">T</italic>, and empirically a substantial gain can be observed for small <italic toggle=\"yes\">T</italic> (i.e. <italic toggle=\"yes\">T</italic> &lt; 100). Finally, another general observation is that the power advantages of online testing algorithms compared with alpha-spending increase as <italic toggle=\"yes\">T</italic> increases. Indeed, when <italic toggle=\"yes\">T</italic> is small and <italic toggle=\"yes\">π</italic><sub>1</sub> is low, online testing algorithms may no longer be competitive in terms of power. We return to this issue in <xref rid=\"S34\" ref-type=\"sec\">Section 5.2</xref>.</p>", "<title>Case Studies</title>", "<title>IMPC dataset</title>", "<p id=\"P75\">Our first case study uses high-throughput phenotypic data from the International Mouse Phenotyping Consortium (IMPC) data repository, which aims to generate and phenotypically characterize knockout mutant strains for every protein-coding gene in the mouse (##REF##24194600##Koscielny et al., 2013##). The IMPC database is an example of a growing dataset mentioned in <xref rid=\"S3\" ref-type=\"sec\">Section 1.1</xref>, since the family of hypotheses is constantly growing as new knockout mice lines are generated and phenotyping data is uploaded to the data repository.</p>", "<p id=\"P76\">We focus on the analysis of IMPC data performed by ##UREF##20##Karp et al. (2017)##, who looked at the influence of sex in mammalian phenotypic traits in both wildtype and mutants. As part of their analysis, Karp et al. analysed the role of sex as a modifier of the genotype effect (for continuous traits) using a two stage pipeline. Stage 1 tested the role of genotype using a likelihood ratio test comparing models (a) and (c). Similarly, stage 2 tested the role of sex using a likelihood ratio test comparing models (a) and (b).</p>", "<p id=\"P77\"><italic toggle=\"yes\">Y</italic> ~ Genotype + Sex + Genotype * Sex + Weight + (1|Batch)</p>", "<p id=\"P78\"><italic toggle=\"yes\">Y</italic> ~ Genotype + Sex + Weight + (1|Batch)</p>", "<p id=\"P79\"><italic toggle=\"yes\">Y</italic> ~ Sex + Weight + (1|Batch)</p>", "<p id=\"P80\">The above procedure resulted in two sets of <italic toggle=\"yes\">N</italic> = 172 328 distinct <italic toggle=\"yes\">p</italic>-values, ordered by the date of the corresponding genomic assay. Note that these <italic toggle=\"yes\">p</italic>-values will not be independent, due to positive and negative associations between different genes (caused for instance by linkage disequilibrium). In addition, multiple variables are being measured for the same gene, and these can be aspects of the same phenotype or be biologically correlated.</p>", "<p id=\"P81\">##TAB##0##Table 1## below shows the number of traits that had a statistically significant genotype effect or were classed as having a statistically significant sexual dimorphism (SD) using LORD++, SAFFRON, ADDIS and monotone AI. As a comparison, we include the results from alpha-spending, BH and uncorrected testing. The ‘Fixed Threshold’ procedure is the fixed significance threshold of 0.0001 used in practice for the IMPC pipeline.</p>", "<p id=\"P82\">Starting first with the results for the genotype data, the online testing algorithms make two to three times as many rejections as fixed testing. ADDIS and SAFFRON in turn make substantially more rejections than LORD++, with an increase of almost 50% and 70%, respectively. ADDIS makes a similar number of rejections to BH, but SAFFRON makes noticeably more rejections than both BH and ADDIS for these data. For the SD data, again the online testing algorithms make substantially more rejections than fixed testing, but the relative increase is much less. ADDIS and SAFFRON make a very similar number of rejections, about 50% more than the number of rejections for LORD++. Finally, for these data we see that monotone AI makes substantially fewer rejections then either ADDIS or SAFFRON.</p>", "<title>Platform trial: STAMPEDE</title>", "<p id=\"P83\">Our second case study is the ongoing STAMPEDE (Systemic Therapy for Advancing or Metastatic Prostate Cancer) platform trial, which evaluates the effect of systemic therapies for prostate cancer on overall survival (##REF##18760574##James et al., 2008##). The trial started with 5 experimental treatment arms (B-–F), and compared these with the control arm A, which was standard-of-care (SOC) hormone therapy. ##FIG##4##Figure 5## shows a schematic of the treatment comparisons that have already been reported from STAMPEDE. Two additional experimental arms (G and H) were added to the trial in 2011 and 2013, respectively.</p>", "<p id=\"P84\">##TAB##1##Table 2## shows the reported <italic toggle=\"yes\">p</italic>-values (unadjusted for multiplicity) when comparing the experimental arms with the control (arm A), as given in ##UREF##16##James et al. (2016##, ##REF##28578639##2017)##; ##REF##28300506##Mason et al. (2017)##; ##UREF##24##Parker et al. (2018)##. The dashed lines denote the four ‘batches’ present in the trial, where a batch corresponds to multiple hypotheses being available to be tested at the same time, as reflected in ##FIG##4##Figure 5##.</p>", "<p id=\"P85\">Following ##REF##37005003##Robertson et al. (2023)##, we apply the online testing algorithms to these observed <italic toggle=\"yes\">p</italic>-values, keeping the alphabetical ordering of <italic toggle=\"yes\">p</italic>-values within the batches. We set the upper bound on the number of treatments <italic toggle=\"yes\">M</italic> = 20 (i.e. twice as many arms that have already entered the STAMPEDE trial as of the end of 2021), and use the bounded versions of alpha-spending (i.e. a Bonferroni correction at level <italic toggle=\"yes\">α</italic>/<italic toggle=\"yes\">M</italic>), LORD++, SAFFRON and ADDIS. ##TAB##2##Table 3## shows which of the hypotheses corresponding to each experimental arm can be rejected at level <italic toggle=\"yes\">α</italic> = 0.05, as well as the current significance level <italic toggle=\"yes\">α</italic><sub>8</sub> that would be used to test the next experimental treatment after the 7 already evaluated in the trial.</p>", "<p id=\"P86\">Uncorrected testing rejects the hypotheses corresponding to three experimental arms (C, E, G), and has by far the highest value of <italic toggle=\"yes\">α</italic><sub>8</sub>. Both SAFFRON and the BH procedure reject hypotheses C and G, and SAFFRON has a substantially higher value of <italic toggle=\"yes\">α</italic><sub>8</sub> than for the other online testing algorithms. ADDIS and alpha-spending only reject hypothesis G, and have similar <italic toggle=\"yes\">α</italic><sub>8</sub>. Finally, LORD++ does not reject any hypotheses and the value of a8 is substantially lower than any of the other algorithms. For further discussion and results, see ##REF##37005003##Robertson et al. (2023)##.</p>", "<title>Extensions and Future Directions</title>", "<title>Further extensions</title>", "<title>Prior weights, penalty weights and decaying memory</title>", "<p id=\"P87\">##UREF##25##Ramdas et al. (2017)## proposed a number of extensions that apply to the class of GAI++ algorithms, including LORD++. Firstly, they showed how to incorporate certain types of prior information about the different hypotheses as expressed through <italic toggle=\"yes\">prior weights w<sub>t</sub></italic> and <italic toggle=\"yes\">penalty weights u<sub>t</sub></italic>. Prior weights allow the experimenter to exploit domain knowledge about which hypotheses are more likely to be non-null. By assigning a higher prior weight <italic toggle=\"yes\">w<sub>t</sub></italic> &gt; 1 to a hypothesis, the algorithm will have a higher chance of rejecting <italic toggle=\"yes\">H<sub>t</sub></italic>. Meanwhile, penalty weights express the different importance attached to the hypotheses being tested, with <italic toggle=\"yes\">u<sub>t</sub></italic> &gt; 1 indicating a more impactful or important test. Importantly, both <italic toggle=\"yes\">w<sub>t</sub></italic> and <italic toggle=\"yes\">u<sub>t</sub></italic> are allowed to depend on past rejections in this framework. ##UREF##25##Ramdas et al. (2017)## proposed doubly-weighted GAI++ rules that provably control the <italic toggle=\"yes\">penalty-weighted</italic> FDR when using both prior and penalty weights under independence. Recently, ##UREF##6##Chen and Kasiviswanathan (2020)## showed how to exploit contextual information associated with each hypothesis to re-weight the testing levels in an online manner, leading to increased power while controlling the FDR.</p>", "<p id=\"P88\">The second proposal of ##UREF##25##Ramdas et al. (2017)## dealt with problems of ‘<italic toggle=\"yes\">piggybacking</italic>’ and ‘<italic toggle=\"yes\">alpha-death</italic>’. Piggybacking happens when a substantial number of rejections are made so that the online testing algorithms earn and accumulate enough alpha-wealth to reject later hypotheses at much less stringent thresholds (hence the later tests ‘piggyback’ on the success of earlier tests). This can lead to a spike in the FDR <italic toggle=\"yes\">locally in time</italic>, even though the FDR over all time is controlled. Meanwhile, alpha-death occurs when there is a long stretch of null hypotheses, so that online testing algorithms make (almost) no rejections and lose nearly all of their alpha-wealth. Subsequently, the algorithm may have essentially no power, unless a non-null hypotheses with extremely strong signal (small <italic toggle=\"yes\">p</italic>-value) is observed. ##UREF##25##Ramdas et al. (2017)## proposed the <italic toggle=\"yes\">decaying memory</italic> FDR (mem-FDR), which pays more attention to recent discoveries through a user-defined discount factor <italic toggle=\"yes\">δ</italic> ∈ (0, 1] and thus smoothly forgets the past. They then proposed GAI++ rules that control the mem-FDR (which can also include penalty weights) under independence. In addition, they showed how to allow the algorithm to <italic toggle=\"yes\">abstain</italic> from testing in order to recover from alpha-death.</p>", "<title>Local dependence – asynchronous and batched testing</title>", "<p id=\"P89\">In most of the literature mentioned so far, an implicit assumption is that each hypothesis test can only start when the previous test has finished (the <italic toggle=\"yes\">synchronous</italic> setting, where synchronous refers to synchronising the start and end time of hypothesis tests). In reality, experimentation is “doubly sequential” like in ##FIG##0##Figure 1##, where it is common to have hypothesis tests that overlap in time, where each test may itself be run sequentially (the <italic toggle=\"yes\">asynchronous</italic> setting). One natural adjustment for asynchronous testing is to use an online FDR algorithm whenever each test finishes (that is, whichever test is the <italic toggle=\"yes\">t</italic>-th one to finish, test it at level <italic toggle=\"yes\">α<sub>t</sub></italic>). However, this would only assign <italic toggle=\"yes\">α<sub>t</sub></italic> at the end of a hypothesis test, which would not be appropriate for sequential hypothesis testing and multiarm bandit approaches that typically require specification of the target type I error level in advance because it is an important component of their stopping rule. Hence, the testing levels must be specified at the start of a hypothesis test. The asynchronous setting also means that potentially arbitrary dependence between some <italic toggle=\"yes\">p</italic>-values must be considered. Indeed, hypothesis tests that are being conducted concurrently are often likely to be dependent, since they may use the same or highly correlated data during their overlap.</p>", "<p id=\"P90\">To address these challenges of asynchronous testing, ##UREF##40##Zrnic, Ramdas and Jordan (2021)## derived asynchronous versions of LORD++ and SAFFRON that output test levels <italic toggle=\"yes\">α<sub>t</sub></italic> dynamically at the beginning of the <italic toggle=\"yes\">t</italic>-th test, such that, despite arbitrary local dependence and regardless of the decision times for each hypothesis, the mFDR is controlled at level <italic toggle=\"yes\">α</italic>. These procedures achieve this goal both at all fixed times <italic toggle=\"yes\">t</italic>, as well as certain adaptively chosen stopping times. ##UREF##31##Tian and Ramdas (2019)## also showed how to derive asynchronous versions of ADDIS. Note that in order to account for the uncertainty about the tests in progress, the test levels assigned by asynchronous online procedures will often be more conservative. Thus, there is a trade-off in that although asynchronous procedures take less time to perform a given number of tests, they can be less powerful than their synchronous counterparts.</p>", "<p id=\"P91\">##UREF##41##Zrnic et al. (2020)## considered the related setting of online <italic toggle=\"yes\">batched</italic> testing, where a potentially infinite number of batches of hypotheses are tested over time (see <xref rid=\"S21\" ref-type=\"sec\">Section 4.2</xref> for an example). To this end, they introduced online, FDR-preserving versions of the most widely used offline algorithms, namely the BH procedure and Storey’s improvement of the BH method (##UREF##30##Storey, 2002##). These online “mini-batched” testing algorithms interpolate between online and offline methodology, thus trading off the best of both worlds. When there is only one batch, the algorithms recover the BH (or Storey-BH) procedure. On the other hand, when all batches are of size one, the algorithms recover the LORD++ (or SAFFRON) procedure. These algorithms control the FDR under independence (an algorithm valid under positive dependence was also derived), and have a higher power than the fully online testing algorithms. Further, since they consist of compositions of offline FDR algorithms, they imply FDR control over each constituent batch, and not just over the whole sequence of tests.</p>", "<title>A Bayesian approach</title>", "<p id=\"P92\">##REF##35757777##Gang, Sun and Wang (2021)## developed a new class of structure-adaptive sequential testing (SAST) rules for online FDR control, which instead of being based on <italic toggle=\"yes\">p</italic>-values, are based upon estimates of the conditional local FDR (Clfdr; ##UREF##4##Cai and Sun (2009)##), which can optimally adapt to important local structures in the data stream. This results in a novel alpha-investing framework that more precisely characterises the effects of rejection of hypotheses: rather than viewing each rejection as a gain of alpha-wealth, the Clfdr characterisation does not view all rejections as equal. Rejections with small Clfdr will lead to increased alphawealth whereas rejections with large Clfdr will lead to decreased alpha-wealth. SAST learns the optimal rejection thresholds adaptively and optimises the alpha-wealth allocation across different time periods. ##REF##35757777##Gang, Sun and Wang (2021)## showed that SAST can achieve substantial power gain over existing methods, but it comes at the cost of only asymptotically controlling the FDR and requiring the underlying Bayesian model to be well-specified.</p>", "<title>Online FWER control</title>", "<p id=\"P93\">##REF##33413033##Tian and Ramdas (2021)## focused on developing methods for online control of the FWER (see <xref rid=\"S7\" ref-type=\"sec\">Section 1.2</xref>). Starting with the observation that only alpha-spending had previously been proposed for online FWER control, the authors first extended existing offline algorithms for FWER control, namely the Sidak method (##UREF##33##Šidák, 1967##) and the fallback procedure (##REF##19142850##Burman, Sonesson and Guilbaud, 2009##). Given <italic toggle=\"yes\">T</italic> hypotheses and FWER level <italic toggle=\"yes\">α</italic>, the offline Sidak method uses the testing level 1 − (1 − <italic toggle=\"yes\">α</italic>)<sup>1/<italic toggle=\"yes\">T</italic></sup> for each hypothesis. The online Sidak method analogously tests hypothesis <italic toggle=\"yes\">H<sub>t</sub></italic> at level <italic toggle=\"yes\">α<sub>t</sub></italic> = 1 − (1 − <italic toggle=\"yes\">α</italic>)<sup><italic toggle=\"yes\">γ<sub>t</sub></italic></sup>. Meanwhile, the offline fallback procedure partitions the overall <italic toggle=\"yes\">α</italic> between the hypotheses and allows the significance levels to be ‘recycled’ from rejected hypotheses.</p>", "<p id=\"P94\">The online Sidak and online fallback procedures control the FWER at all times <italic toggle=\"yes\">t</italic> under independence and arbitrary dependence, respectively. However, although these online FWER control methods are guaranteed to be uniformly more powerful than alpha-spending, the improvements are usually minor in practice, except for extreme cases. Hence, ##REF##33413033##Tian and Ramdas (2021)## proposed the ADDIS-spending algorithm for online FWER control, which (like ADDIS does for online FDR control) benefits from adaptivity to the fraction of nulls, but also gains power by discarding conservative nulls (if they exist). ADDIS-spending controls the FWER when null <italic toggle=\"yes\">p</italic>-values are uniformly conservative, and independent of each other and of the non-nulls. Finally, ##UREF##10##Fischer, Roig and Brannath (2023)## showed how to extend the ADDIS-spending procedure to the setting of ‘graphical’ testing procedures while maintaining control of the FWER. In graphical testing procedures, vertices represent the null hypotheses and weights represent the local significance levels, which are ‘recycled’ through weighted, directed edges. They also showed how to improve the power of the ADDIS-spending procedure under local dependence.</p>", "<title>Online FDX control and FDR at stopping times</title>", "<p id=\"P95\">##UREF##35##Xu and Ramdas (2022)## focused on online control of the FDX (see <xref rid=\"S7\" ref-type=\"sec\">Section 1.2</xref>). Prior to this work, the only online procedure that controlled the FDX was proposed by ##UREF##18##Javanmard and Montanari (2018)##, but this had very low power (no better than alpha-spending). ##UREF##35##Xu and Ramdas (2022)## proposed the supLORD algorithm, which has a higher power and provably controls the FDX when the null <italic toggle=\"yes\">p</italic>-values are conditionally superuniform. One feature of this algorithm is that it allows the user to choose the number of rejections after which FDX control begins, in exchange for more power. supLORD is based on the GAI framework, and the authors also show how to dynamically choose larger test levels <italic toggle=\"yes\">α<sub>t</sub></italic> when the wealth is large, allowing the algorithm to fully utilise its wealth and increase its power as a result. Finally, the authors show that supLORD also controls the mFDR and FDR at both fixed times and stopping times. Hence, supLORD provides the first guarantee for online FDR control at stopping times (LORD++, SAFFRON and ADDIS only control the mFDR at stopping times and not the FDR).</p>", "<title>Retesting of hypotheses</title>", "<p id=\"P96\">One feature of online testing algorithms that has not been explicitly pointed out in the literature is the option of <italic toggle=\"yes\">retesting</italic> hypotheses (i.e. using the same <italic toggle=\"yes\">p</italic>-value again later in the testing sequence, when the alpha-wealth may be higher). Crucially however, the choice of whether to retest must be made without using knowledge of the <italic toggle=\"yes\">p</italic>-value itself, but only that it was not rejected (e.g. that it is greater 0.01). In this example, under the null the <italic toggle=\"yes\">p</italic>-value will still be conditionally uniform in [0.01, 1]. Since this implies that the assumption of conditional super-uniformity under the null still holds, the same <italic toggle=\"yes\">p</italic>-value can be used for retesting. In practice, retesting could happen within an automated testing setting for example, perhaps with additional prior information. Meanwhile, ##UREF##12##Fisher (2022)## proposed a framework for online testing where each hypothesis requires an immediate <italic toggle=\"yes\">preliminary</italic> decision, which allows the analyst to update that decision until a preset deadline while controlling the FDR.</p>", "<title>Discrete test statistics</title>", "<p id=\"P97\">##UREF##9##Döhler, Meah and Roquain (2021)## focused on the setting where the null <italic toggle=\"yes\">p</italic>-values are conservative due to the discreteness of the test statistics, i.e. where the individual tests are based on counts or contingency tables. The authors proposed uniform improvements of LORD++, SAFFRON and ADDIS-spending, and showed that the power gains can be substantial when the discreteness is high (e.g. the counts in the contingency are moderate).</p>", "<title>Incorporating experimental costs</title>", "<p id=\"P98\">##UREF##7##Cook et al. (2022)## considered the setting of online multiple hypothesis testing where the cost of data collection (i.e. the cost of conducting an experiment) is not negligible. They proposed an extension of the GAI framework to take into account the cost of data collection, the choice of sample size for each experiment, as well as prior beliefs about the probability of rejection. The proposed methods ensure control of the mFDR and performs particularly well in settings where the aim is to maximise a limited budget of tests to achieve the highest possible power.</p>", "<title>Post-hoc FDP bounds</title>", "<p id=\"P99\">##UREF##21##Katsevich and Ramdas (2020)## proposed a class of simultaneous FDP bounds that apply to a variety of settings, including online testing. These bounds are finite-sample have a simple closed form. The results can be used as a diagnostic tool for FDR procedures: after running an FDR procedure, one can obtain a valid 1 − <italic toggle=\"yes\">α</italic> confidence bound on the FDP of the resulting rejection set. Since the guarantees are post hoc, they apply to any sequence of rejections produced by any online algorithm, that may or may not have been designed for FDR or FDP control.</p>", "<title>Online control of the False Coverage Rate</title>", "<p id=\"P100\">Finally, ##UREF##34##Weinstein and Ramdas (2020)## considered the problem of constructing <italic toggle=\"yes\">confidence intervals</italic> (CIs) that are valid for online hypothesis testing. In particular, they focus on control of the false coverage rate (FCR), which is the expected ratio of number of constructed CIs that fail to cover their respective parameters to the total number of constructed CIs. In the online hypothesis testing framework they considered, at each step the investigator observes independent data that are informative about the parameter of interest <italic toggle=\"yes\">θ<sub>t</sub></italic>, and must immediately make a decision whether to report a CI for <italic toggle=\"yes\">θ<sub>t</sub></italic> or not. If a CI is reported for <italic toggle=\"yes\">θ<sub>t</sub></italic>, then the the aim is to ensure that that the CI for <italic toggle=\"yes\">θ<sub>t</sub></italic> has FCR ≤ <italic toggle=\"yes\">α</italic> at all times <italic toggle=\"yes\">T</italic>. For further details of the proposed algorithms and their theoretical guarantees, see ##UREF##34##Weinstein and Ramdas (2020)##.</p>", "<title>Current shortcomings and future directions</title>", "<title>Online testing for small numbers of hypotheses</title>", "<p id=\"P101\">Online testing algorithms are most powerful in settings where there are a large number (i.e. <italic toggle=\"yes\">T</italic> &gt; 1000) of hypotheses that will eventually be tested. Thus the biggest advantage will likely be in settings such as A/B testing in large tech companies or in large-scale biological data repositories. However, while platform trials provide a framework for a trial to continue indefinitely in theory, in practice they will typically evaluate a maximum number of interventions in the low tens. Hence, there is a need for investigation of optimal online testing procedures when the maximum number of hypotheses to be tested is relatively low and when the correlation between hypotheses is known (e.g. because of a shared control arm). Separately, there is scope to further improve the power of online testing algorithms when combined with sequential testing of the individual hypotheses, for example by exploiting the fact that pre-specified group-sequential stopping boundaries may be used in a platform trial setting (see ##UREF##38##Zehetmayer, Posch and Koenig (2021)## for a recent proposal along these lines).</p>", "<title>Managing incentives across sponsors or products</title>", "<p id=\"P102\">There are some additional challenges in using online control methods in platform trials or in the IT industry. If different sponsors (e.g. pharmaceutical companies) are supporting a platform trial, they might be reluctant to have their intervention be tested at a notably more stringent level than other sponsors: it may be difficult to reconcile the most powerful overall procedure not being acceptable to individual sponsors. Similarly, if a large IT company imposes that experiments run across various products must all be subjected to oversight in the form of a common online FDR controlling procedure that acts across products, then it may be hard to convince individual product teams that their tests must be subject to a level determined by the results of experiments by other groups.</p>", "<title>Optimal choices of parameters for online algorithms</title>", "<p id=\"P103\">As seen in <xref rid=\"S8\" ref-type=\"sec\">Section 2</xref>, LORD++, SAFFRON and ADDIS depend on the choice of the initial wealth w0 as well as the sequence {<italic toggle=\"yes\">γ<sub>t</sub></italic>}. Further work could look at optimal choices of these parameters, given assumptions about the distribution of non-null <italic toggle=\"yes\">p</italic>-values. Exploring data-adaptive choices of time varying sequences (for SAFFRON and ADDIS) and (for ADDIS) with provable power increase would be another fruitful area of research. Future work could also look at optimal choices for the parameters for the other algorithms in <xref rid=\"S23\" ref-type=\"sec\">Section 5.1</xref>.</p>", "<title>Online batched testing</title>", "<p id=\"P104\">A number of open questions remain regarding the proposals of ##UREF##41##Zrnic et al. (2020)## for online batched testing. First, the framework could be extended to allow for asynchronous online batch testing, using the ideas of ##UREF##40##Zrnic, Ramdas and Jordan (2021)##. Second, it should be possible to derive online batched versions based on the offline counterpart of ADDIS, which would gain power in the presence of conservative nulls. Third, an open question is determining the trade off between the chosen batch size versus power in online batched testing.</p>", "<title>Online error rate control under dependence</title>", "<p id=\"P105\">One major shortcoming with almost all of the proposed online testing algorithms is their reliance on the assumption of independence of the null <italic toggle=\"yes\">p</italic>-values for provable FDR control, which is unlikely to always be case in real data applications. However, in terms of online FDR control under dependence, there have only been limited proposals in the literature. ##UREF##40##Zrnic, Ramdas and Jordan (2021)## showed that the LOND algorithm by ##UREF##17##Javanmard and Montanari (2015)## controls the FDR under positive dependence. In the online setting, arbitrary dependence of <italic toggle=\"yes\">p</italic>-values across all time is a rather pessimistic and unrealistic assumption, and thus in the asynchronous setting, ##UREF##40##Zrnic, Ramdas and Jordan (2021)## introduced the concept of arbitrary <italic toggle=\"yes\">local</italic> dependence and showed that online algorithms can be modified to control the FDR even with such dependence. See also ##UREF##11##Fisher (2021)##, who showed further results for control of the FDR under positive dependence in the minibatch setting. Finally, ##UREF##41##Zrnic et al. (2020)## showed how to control the FDR in the online batched setting under positive dependence. Future work could explore how to construct more powerful online algorithms under different forms of dependence, including when the correlation structure is known or estimated.</p>", "<title>Summary and Practical Guidance</title>", "<p id=\"P106\">##TAB##3##Table 4## gives a summary of the leading online testing methods discussed in this paper, comparing their assumptions as well as general pros and cons. In terms of practical guidance, we offer the following general suggestions: <list list-type=\"bullet\" id=\"L2\"><list-item><p id=\"P107\">A fundamental consideration is which type I error rate is most suitable to control given the experimental context and goals. As alluded to in <xref rid=\"S7\" ref-type=\"sec\">Section 1.2</xref>, this choice may be driven by the anticipated number of hypotheses to be tested, data dependencies and/or regulatory concerns.</p></list-item><list-item><p id=\"P108\">Given the type I error rate that the user wishes to control, there may be a variety of online testing algorithms to choose from. A key consideration is the assumptions around the <italic toggle=\"yes\">p</italic>-value dependencies, as shown in ##TAB##2##Table 3##. Algorithms that make stronger assumptions (i.e., assuming independence) will be more powerful, but this can come at the cost of inflated type I error rates if these assumptions do not hold. In practice, it may be difficult to anticipate or estimate the data dependencies in an experiment. In some settings, such as a platform trial with a common control, the correlation structure can be derived analytically. Otherwise, with enough data one can try to estimate the correlation empirically.</p></list-item><list-item><p id=\"P109\">The planned timing of hypothesis tests combined with the use of sequential testing may motivate the use of asynchronous or batched versions of online testing algorithms, as discussed in <xref rid=\"S23\" ref-type=\"sec\">Section 5.1</xref>. This links with the issue of the ordering of the hypothesis tests themselves: in some settings the ordering will be out of the analyst’s control, while in others it may be possible to use prior information about the probability of rejection to potentially gain power either implicitly by ordering the hypotheses (so that those that are a-priori more likely to be rejected are tested first) or by using prior weights (see <xref rid=\"S23\" ref-type=\"sec\">Section 5.1</xref>). In the batch setting (i.e., where the multiple hypotheses are available to be tested simultaneously) then the batched algorithms presented in <xref rid=\"S22\" ref-type=\"sec\">Section 5</xref> are recommended to achieve the best of both worlds of offline and online testing.</p></list-item><list-item><p id=\"P110\">As mentioned above, the setting of a small number of hypotheses (&lt; 1000) is a challenging one for online testing. Thus the biggest advantage in terms of power will be seen in settings with large-scale hypothesis testing, such as A/B testing. If at some point it becomes known that the number of hypothesis tests will be bounded by a finite number <italic toggle=\"yes\">M</italic> then it would make sense to maximise power by ensuring that the alpha-wealth is completely used up by the end of the <italic toggle=\"yes\">M</italic>-th hypothesis test.</p></list-item><list-item><p id=\"P111\">In general, simulation studies remain valuable for assessing the performance of an online testing algorithm given the experimental context and goals, particularly for evaluating power, as well as type I error rate considerations under departures from independence. We note that in terms of computational scalability, the algorithms presented in this paper all scale linearly with the number of hypotheses tested.</p></list-item></list></p>", "<title>Supplementary Material</title>" ]
[ "<title>Funding</title>", "<p>DS Robertson was supported by the Biometrika Trust, the UK Medical Research Council (MC_UU_0002/14) and the NIHR Cambridge Biomedical Research Centre (BRC1215-20014). The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health and Social Care (DHCS). For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising. JMS Wason was supported by a NIHR Research Professorship (NIHR301614). A Ramdas was supported by NSF DMS CAREER award 1916320.</p>", "<title>Data availability statement</title>", "<p id=\"P113\">The data used in <xref rid=\"S20\" ref-type=\"sec\">Section 4.1</xref> can be found at <ext-link xlink:href=\"https://zenodo.org/record/2396572\" ext-link-type=\"uri\">https://doi.org/10.5281/zenodo.1343578</ext-link>. Code to reproduce the simulation studies given in <xref rid=\"S15\" ref-type=\"sec\">Section 3</xref> can be found at <ext-link xlink:href=\"https://github.com/dsrobertson/online_testing\" ext-link-type=\"uri\">https://github.com/dsrobertson/online_testing</ext-link>.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Fig. 1</label><caption><p>An abstract online multiple testing framework. As time passes (left to right), new experiments testing different hypotheses are started and stopped, in a possibly indefinite manner. Each horizontal line represents a new experiment/hypothesis, and the length represents the number of samples collected. Decisions about each hypothesis must be made as soon as the corresponding experiment ends.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Fig. 2</label><caption><p>Diagrammatic representation of generalised alpha-investing (GAI), showing how the wealth W(t) at time <italic toggle=\"yes\">t</italic> changes depending on whether the hypothesis H<sub>t</sub> is rejected (i.e. whether the corresponding <italic toggle=\"yes\">p</italic>-value P<sub>t</sub> ≤ α) or not. Figure adapted from ##UREF##35##Xu and Ramdas (2022)##.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Fig. 3</label><caption><p>Test levels for LORD++, SAFFRON, ADDIS and monotone AI compared with uncorrected testing and alpha-spending. We set <italic toggle=\"yes\">T</italic> = 300, <italic toggle=\"yes\">α</italic> = 0.05 and the proportion of non-nulls <italic toggle=\"yes\">π</italic><sub>1</sub> = 0.5.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Fig. 4</label><caption><p>Power of LORD++, SAFFRON, ADDIS and monotone AI compared with uncorrected testing, the BH procedure and alpha-spending as the proportion of non-nulls <italic toggle=\"yes\">π</italic><sub>1</sub> varies. We set <italic toggle=\"yes\">T</italic> = 1000 and <italic toggle=\"yes\">α</italic> = 0.05. Results are based on 10<sup>4</sup> simulation replicates.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Fig. 5</label><caption><title>Schematic of the completed treatment arms in the STAMPEDE platform trial. ab = abiraterone, rt = radiotherapy.</title></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Fig. 6</label><caption><p>FDR of LORD++, SAFFRON, ADDIS and monotone AI compared with uncorrected testing, the BH procedure and alpha-spending as the proportion of non-nulls <italic toggle=\"yes\">π<sub>1</sub></italic> varies. The solid red horizontal line gives the target level of <italic toggle=\"yes\">α</italic> = 0.05. We set <italic toggle=\"yes\">T</italic> = 1000 and results are based on 10<sup>4</sup> simulation replicates.</p></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Number of rejections made by online FDR algorithms and various comparators using the IMPC datasets. SD = Sexual Dimorphism.</title></caption><table frame=\"below\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Genotype</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SD</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Uncorrected</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">35 575</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">20887</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>BH</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12 907</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">2084</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>ADDIS</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">12 558</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1713</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>SAFFRON</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">14268</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 705</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>LORD++</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8517</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1 193</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Monotone AI</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9 906</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">985</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Fixed Threshold</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">4158</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">969</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<bold>Alpha-spending</bold>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">795</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">60</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Reported <italic toggle=\"yes\">p</italic>-values for the STAMPEDE platform trial. SOC = Standard-of-care.</title></caption><table frame=\"below\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Trial arm</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><italic toggle=\"yes\">p</italic>-value</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>B</bold>: SOC + zoledronic acid</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.450</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>C</bold>: SOC + docetaxel</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.006</td></tr><tr style=\"border-bottom: dotted\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>E</bold>: SOC + zoledronic acid + docetaxel</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.022</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>D</bold>: SOC + celecoxib</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.847</td></tr><tr style=\"border-bottom: dotted\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>F</bold>: SOC + zoledronic acid + celecoxib</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.130</td></tr><tr style=\"border-bottom: dotted\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>G</bold>: SOC + abiraterone</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.001</td></tr><tr style=\"border-bottom: hidden\"><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"><bold>H</bold>: SOC + radiotherapy</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.266</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><title>Hypotheses rejected and current significance level <italic toggle=\"yes\">α</italic><sub>8</sub> of different algorithms using the results of the STAMPEDE trial, with the ordering as in ##TAB##1##Table 2##.</title></caption><table frame=\"void\" rules=\"cols\"><thead><tr style=\"border-bottom: solid thin\"><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Algorithm</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Rejections</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<italic toggle=\"yes\">α</italic>\n<sub>8</sub>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">uncorrected</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">C, E, G</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0500</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Alpha-spending</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">G</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0025</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">BH</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">C, G</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ADDIS</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">G</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0016</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">SAFFRON</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">C, G</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0165</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">LORD++</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.0002</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><title>Summary of leading methods for online error rate control, giving dependence assumptions and pros &amp; cons.</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"2\"/></colgroup><thead><tr style=\"border-top: hidden\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Error rate</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Algorithm</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dependence assumptions</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Pros &amp; Cons</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"4\" colspan=\"1\"><bold>FDR</bold> or <bold>mFDR</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">LORD++ [An online analogue of the BH procedure]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Independence of null <italic toggle=\"yes\">p</italic>-values for FDR control, conditional super-uniformity of null <italic toggle=\"yes\">p</italic>-values for mFDR control</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Extensions for prior weights, penalty weights, decaying memory, as well as local dependence (asynchronous and batch testing)</p>\n<p>+ Empirically robust to positive dependence of <italic toggle=\"yes\">p</italic>-values</p>\n<p>− Not robust to arbitrary dependence of <italic toggle=\"yes\">p</italic>-values</p>\n<p>− Typically lower power than SAFFRON or ADDIS</p></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SAFFRON [Adaptive algorithm based on an estimate of the proportion of true null hypotheses]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">As above</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Higher power than LORD++ if there is a significant fraction of non-nulls and the signals are strong</p>\n<p>+ Extensions for local dependence (asynchronous and batch testing)</p>\n<p>− Not robust to dependence of <italic toggle=\"yes\">p</italic>-values</p></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ADDIS [Combines adaptivity with discarding of conservative nulls]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">As above</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Higher power than SAFFRON when there are conservative nulls</p>\n<p>+ Extensions for local dependence (asynchronous testing)</p>\n<p>− Not robust to dependence of <italic toggle=\"yes\">p</italic>-values</p></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">LOND</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Controls FDR under positive dependence of <italic toggle=\"yes\">p</italic>-values</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Provable FDR control for positive dependence (the ‘PRDS’ assumption)</p>\n<p>− Substantially lower power than the algorithms above</p></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<bold>FDX</bold>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">supLORD</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Null <italic toggle=\"yes\">p</italic>-values are conditionally super-uniform</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Also controls the mFDR and FDR at both fixed times and stopping times</p>\n<p>+ User may choose the number of rejections after which we begin controlling FDX in exchange for more power</p>\n<p>− Unclear how robust to departures from conditional superuniformity</p></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<bold>FWER</bold>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Alpha-spending</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Robust to arbitrary dependence of <italic toggle=\"yes\">p</italic>-values</p>\n<p>− Very low power, rejects only a few hypotheses before becoming unable to reject any more hypotheses</p></td></tr><tr style=\"border-bottom: hidden\"><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ADDIS-spending [Combines adaptivity with discarding of conservative nulls]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Null <italic toggle=\"yes\">p</italic>-values are uniformly conservative and independent</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><p>+ Higher power than Alpha-spending</p>\n<p>+ Extensions for local dependence</p>\n<p>− Unclear how robust to departures from independence</p></td></tr></tbody></table></table-wrap>" ]
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overflow=\"scroll\"><mml:mtext>FWER</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>:</mml:mo><mml:mo>=</mml:mo><mml:mi>Pr</mml:mi><mml:mo stretchy=\"false\">[</mml:mo><mml:mi>V</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≥</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">]</mml:mo><mml:mo>.</mml:mo></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M8\" display=\"inline\" overflow=\"scroll\"><mml:mstyle displaystyle=\"true\"><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>∞</mml:mi></mml:msubsup><mml:mrow><mml:msub><mml:mi>α</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mi>α</mml:mi></mml:mrow></mml:mstyle></mml:math></inline-formula>", "<disp-formula id=\"FD7\"><mml:math id=\"M9\" display=\"block\" overflow=\"scroll\"><mml:mi>W</mml:mi><mml:mo 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stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:math></disp-formula>", "<disp-formula id=\"FD9\"><mml:math id=\"M11\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>φ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>ϕ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:mi>α</mml:mi><mml:mo>.</mml:mo></mml:math></disp-formula>", "<disp-formula id=\"FD10\"><mml:math id=\"M12\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>φ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>≤</mml:mo><mml:mi>min</mml:mi><mml:mo>{</mml:mo><mml:msub><mml:mi>ϕ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>b</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>,</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>ϕ</mml:mi><mml:mi>t</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>α</mml:mi><mml:mi>t</mml:mi></mml:msub></mml:mrow></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mi>b</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo>}</mml:mo><mml:mo>,</mml:mo></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M13\" display=\"inline\" overflow=\"scroll\"><mml:mi mathvariant=\"double-struck\">E</mml:mi><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi>V</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>+</mml:mo><mml:mi>W</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>R</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>∨</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mi>α</mml:mi></mml:math></inline-formula>", "<disp-formula id=\"FD11\"><mml:math id=\"M14\" display=\"block\" overflow=\"scroll\"><mml:mtext>FDP</mml:mtext><mml:mo>∗</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mstyle displaystyle=\"true\"><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>≤</mml:mo><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>j</mml:mi><mml:mo>∈</mml:mo><mml:msub><mml:mi>ℋ</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mrow><mml:msub><mml:mi>α</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:mrow></mml:mstyle></mml:mrow><mml:mrow><mml:mi>R</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>∨</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M15\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mtext>FDP</mml:mtext></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>LORD</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M16\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mtext>FDP</mml:mtext></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>SAFFRON</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M17\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mi>F</mml:mi><mml:mi>D</mml:mi><mml:mi>P</mml:mi></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>ADDIS</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M18\" display=\"inline\" overflow=\"scroll\"><mml:msubsup><mml:mrow><mml:mo>{</mml:mo><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub></mml:mrow><mml:mo>}</mml:mo></mml:mrow><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>∞</mml:mi></mml:msubsup></mml:math></inline-formula>", "<disp-formula id=\"FD12\"><mml:math id=\"M19\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>α</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>w</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:mstyle displaystyle=\"true\"><mml:munder><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>:</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>&lt;</mml:mo><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>≠</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mn>𝟙</mml:mn></mml:msub></mml:mrow></mml:munder><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mrow><mml:mi>t</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:mrow></mml:msub><mml:msub><mml:mi>b</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:mstyle><mml:mo>,</mml:mo></mml:math></disp-formula>", "<disp-formula id=\"FD13\"><mml:math id=\"M20\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mi>F</mml:mi><mml:mi>D</mml:mi><mml:mi>P</mml:mi></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>LORD</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mover accent=\"true\"><mml:mi>V</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>R</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>∨</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>:</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mstyle displaystyle=\"true\"><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>≤</mml:mo><mml:mi>t</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:msub><mml:mi>α</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:mrow></mml:mstyle></mml:mrow><mml:mrow><mml:mi>R</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>∨</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M21\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mi>F</mml:mi><mml:mi>D</mml:mi><mml:mi>P</mml:mi></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>LORD</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≤</mml:mo><mml:mi>α</mml:mi></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M22\" display=\"inline\" overflow=\"scroll\"><mml:mover accent=\"true\"><mml:mi>V</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M23\" display=\"inline\" overflow=\"scroll\"><mml:msubsup><mml:mrow><mml:mo>{</mml:mo><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub></mml:mrow><mml:mo>}</mml:mo></mml:mrow><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>∞</mml:mi></mml:msubsup></mml:math></inline-formula>", "<disp-formula id=\"FD14\"><mml:math id=\"M24\" display=\"block\" overflow=\"scroll\"><mml:msub><mml:mi>α</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>w</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>α</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mi>w</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mrow><mml:mi>t</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:msub><mml:mi>𝟙</mml:mi><mml:mrow><mml:mo>{</mml:mo><mml:mrow><mml:msub><mml:mi>τ</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>&lt;</mml:mo><mml:mi>t</mml:mi></mml:mrow><mml:mo>}</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:mi>α</mml:mi><mml:mstyle displaystyle=\"true\"><mml:munder><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>:</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>&lt;</mml:mo><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>≠</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:munder><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mrow><mml:mi>t</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mi>τ</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:mstyle><mml:mo>.</mml:mo></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M25\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mtext>FDP</mml:mtext></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>LORD</mml:mtext><mml:mo>+</mml:mo><mml:mo>+</mml:mo></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≤</mml:mo><mml:mi>α</mml:mi></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M26\" display=\"inline\" overflow=\"scroll\"><mml:mtext>FDP</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>V</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>R</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>∨</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M27\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub><mml:mo>∝</mml:mo><mml:mfrac><mml:mrow><mml:mi>log</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo>∨</mml:mo><mml:mn>2</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mi>t</mml:mi><mml:mi>exp</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:msqrt><mml:mrow><mml:mi>log</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:msqrt><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mfrac></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M28\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mtext>FDP</mml:mtext></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>LORD</mml:mtext><mml:mo>+</mml:mo><mml:mo>+</mml:mo></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M29\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mtext>FDP</mml:mtext></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>BH</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>s</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>n</mml:mi><mml:mo>⋅</mml:mo><mml:mi>s</mml:mi></mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mi>ℛ</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>s</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>|</mml:mo></mml:mrow></mml:mfrac></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M30\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mi>s</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mtext>BH</mml:mtext></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>max</mml:mi><mml:mrow><mml:mo>{</mml:mo><mml:mrow><mml:mi>s</mml:mi><mml:mo>:</mml:mo><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mtext>FDP</mml:mtext></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover></mml:mrow><mml:mrow><mml:mtext>BH</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>s</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≤</mml:mo><mml:mi>α</mml:mi></mml:mrow><mml:mo>}</mml:mo></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M31\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mi>F</mml:mi><mml:mi>D</mml:mi><mml:mi>P</mml:mi></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mtext>LORD </mml:mtext></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≤</mml:mo><mml:mi>α</mml:mi></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M32\" display=\"inline\" overflow=\"scroll\"><mml:msub><mml:mover accent=\"true\"><mml:mrow><mml:mi>F</mml:mi><mml:mi>D</mml:mi><mml:mi>P</mml:mi></mml:mrow><mml:mo stretchy=\"true\">^</mml:mo></mml:mover><mml:mrow><mml:mi>L</mml:mi><mml:mi>O</mml:mi><mml:mi>R</mml:mi><mml:mi>D</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>≤</mml:mo><mml:mi>α</mml:mi></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M33\" display=\"inline\" overflow=\"scroll\"><mml:msubsup><mml:mrow><mml:mo>{</mml:mo><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub></mml:mrow><mml:mo>}</mml:mo></mml:mrow><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>∞</mml:mi></mml:msubsup></mml:math></inline-formula>", "<disp-formula id=\"FD15\"><mml:math id=\"M34\" display=\"block\" overflow=\"scroll\"><mml:mi>Pr</mml:mi><mml:mo>{</mml:mo><mml:mi>P</mml:mi><mml:mo>/</mml:mo><mml:mi>c</mml:mi><mml:mo>≤</mml:mo><mml:mi>x</mml:mi><mml:mo>∣</mml:mo><mml:mi>P</mml:mi><mml:mo>≤</mml:mo><mml:mi>c</mml:mi><mml:mo>}</mml:mo><mml:mo>≤</mml:mo><mml:mi>x</mml:mi><mml:mspace width=\"0.2em\"/><mml:mspace width=\"0.2em\"/><mml:mtext>for</mml:mtext><mml:mspace width=\"0.2em\"/><mml:mtext>all</mml:mtext><mml:mspace width=\"0.2em\"/><mml:mi>x</mml:mi><mml:mo>,</mml:mo><mml:mi>c</mml:mi><mml:mo>∈</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>0</mml:mn><mml:mo>,</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>.</mml:mo></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M35\" display=\"inline\" overflow=\"scroll\"><mml:msubsup><mml:mrow><mml:mo>{</mml:mo><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mi>t</mml:mi></mml:msub></mml:mrow><mml:mo>}</mml:mo></mml:mrow><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow><mml:mi>∞</mml:mi></mml:msubsup></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M36\" display=\"inline\" 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[]
[]
[]
[]
[ "<supplementary-material id=\"SD1\" position=\"float\" content-type=\"local-data\"><label>Appendix</label></supplementary-material>" ]
[ "<fn-group><fn id=\"FN1\"><label>1</label><p id=\"P114\">Note that if violations of this condition are not too large in the sense that there exists some <italic toggle=\"yes\">ϵ</italic> &gt; 0 such that for every <italic toggle=\"yes\">x</italic> ∈ [0, 1] then it is straightforward to check that the FDR proofs still go through for the online algorithms presented in <xref rid=\"S10\" ref-type=\"sec\">Section 2.2</xref>, guaranteeing an FDR of <italic toggle=\"yes\">α</italic>(1 + <italic toggle=\"yes\">ϵ</italic>) instead.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"EMS185037-supplement-Appendix.pdf\" id=\"d64e4517\" position=\"anchor\"/>" ]
[{"person-group": ["\n"], "surname": ["Aharoni", "Rosset"], "given-names": ["E", "S"], "article-title": ["Generalized \u03b1-investing: definitions, optimality results and application to public databases"], "source": ["Journal of the Royal Statistical Society: Series B (Statistical Methodology)"], "year": ["2014"], "volume": ["76"], "fpage": ["771"], "lpage": ["794"]}, {"person-group": ["\n"], "surname": ["Berman", "Van den Bulte"], "given-names": ["R", "C"], "article-title": ["False discovery in A/B testing"], "source": ["Management Science"], "year": ["2021"]}, {"person-group": ["\n"], "surname": ["Bretz", "Maurer", "Xi"], "given-names": ["F", "W", "D"], "article-title": ["Replicability, reproducibility, and multiplicity in drug development"], "source": ["Chance"], "year": ["2019"], "volume": ["32"], "fpage": ["4"], "lpage": ["11"]}, {"person-group": ["\n"], "surname": ["Bretz", "Westfall"], "given-names": ["F", "PH"], "article-title": ["Multiplicity and replicability: Two sides of the same coin"], "source": ["Pharmaceutical statistics"], "year": ["2014"], "volume": ["6"], "fpage": ["343"], "lpage": ["344"]}, {"person-group": ["\n"], "surname": ["Cai", "Sun"], "given-names": ["TT", "W"], "article-title": ["Simultaneous testing of grouped hypotheses: Finding needles in multiple haystacks"], "source": ["Journal of the American Statistical Association"], "year": ["2009"], "volume": ["104"], "fpage": ["1467"], "lpage": ["1481"]}, {"person-group": ["\n"], "surname": ["Chen", "Arias-Castro"], "given-names": ["S", "E"], "article-title": ["On the power of some sequential multiple testing procedures"], "source": ["Annals of the Institute of Statistical Mathematics"], "year": ["2021"], "volume": ["73"], "fpage": ["311"], "lpage": ["336"]}, {"person-group": ["\n", "\n"], "surname": ["Chen", "Kasiviswanathan", "Chiappa", "Calandra"], "given-names": ["S", "S", "S", "R"], "source": ["Contextual Online False Discovery Rate Control"], "conf-name": ["Proceedings of the Twenty Third International Conference on Artificial Intelligence and Statistics"], "conf-sponsor": ["Proceedings of Machine Learning Research, PMLR"], "year": ["2020"], "volume": ["108"], "fpage": ["952"], "lpage": ["961"]}, {"person-group": ["\n"], "surname": ["Cook", "Dubey", "Lee", "Zhu", "Zhao", "Flaherty"], "given-names": ["T", "HV", "JA", "G", "T", "P"], "article-title": ["Cost-aware Generalized \u03b1-investing for Multiple Hypothesis Testing"], "source": ["arXiv preprint"], "year": ["2022"], "elocation-id": ["arXiv:2210.17514"]}, {"person-group": ["\n"], "surname": ["Dickerman", "Garc\u00eda-Alb\u00e9niz", "Logan", "Denaxas", "Hern\u00e1n"], "given-names": ["BA", "X", "RW", "S", "MA"], "article-title": ["Avoidable flaws in observational analyses: an application to statins and cancer"], "source": ["Nature medicine"], "year": ["2019"], "volume": ["25"], "fpage": ["1601"], "lpage": ["1606"]}, {"person-group": ["\n"], "surname": ["D\u00f6hler", "Meah", "Roquain"], "given-names": ["S", "I", "E"], "article-title": ["Online multiple testing with super-uniformity reward"], "source": ["arXiv preprint"], "year": ["2021"], "elocation-id": ["arXiv:2110.01255"]}, {"person-group": ["\n"], "surname": ["Fischer", "Roig", "Brannath"], "given-names": ["L", "MB", "W"], "article-title": ["An Adaptive-Discard-Graph for online error control"], "source": ["arXiv preprint"], "year": ["2023"], "elocation-id": ["arXiv:2301.11711"]}, {"person-group": ["\n"], "surname": ["Fisher"], "given-names": ["A"], "article-title": ["SAFFRON and LORD Ensure Online Control of the False Discovery Rate Under Positive, Local Dependence"], "source": ["arXiv preprint"], "year": ["2021"], "elocation-id": ["arXiv:2110.08161"]}, {"person-group": ["\n"], "surname": ["Fisher"], "given-names": ["AJ"], "source": ["Online Control of the False Discovery Rate under \u201cDecision Deadlines\u201d"], "conf-name": ["International Conference on Artificial Intelligence and Statistics"], "conf-sponsor": ["PMLR"], "year": ["2022"], "fpage": ["8340"], "lpage": ["8359"]}, {"person-group": ["\n"], "surname": ["Foster", "Stine"], "given-names": ["DP", "RA"], "article-title": ["\u03b1-investing: a procedure for sequential control of expected false discoveries"], "source": ["Journal of the Royal Statistical Society: Series B (Statistical Methodology)"], "year": ["2008"], "volume": ["70"], "fpage": ["429"], "lpage": ["444"]}, {"person-group": ["\n"], "surname": ["Goodman", "Fanelli", "Ioannidis"], "given-names": ["SN", "D", "JP"], "article-title": ["What does research reproducibility mean?"], "source": ["Science translational medicine"], "year": ["2016"], "volume": ["8"], "elocation-id": ["341ps12"]}, {"person-group": ["\n"], "surname": ["Howard", "Ramdas", "McAuliffe", "Sekhon"], "given-names": ["SR", "A", "J", "J"], "article-title": ["Time-uniform, nonparametric, nonasymptotic confidence sequences"], "source": ["The Annals of Statistics"], "year": ["2021"], "volume": ["49"], "fpage": ["1055"], "lpage": ["1080"]}, {"person-group": ["\n"], "surname": ["James", "Sydes", "Clarke", "Mason", "Dearnaley", "Spears", "Ritchie", "Parker", "Russell", "Attard", "de Bono"], "given-names": ["ND", "MR", "NW", "MD", "DP", "MR", "AWS", "CC", "JM", "G", "J"], "article-title": ["Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial"], "source": ["The Lancet"], "year": ["2016"], "volume": ["387"], "fpage": ["1163"], "lpage": ["1177"]}, {"person-group": ["\n"], "surname": ["Javanmard", "Montanari"], "given-names": ["A", "A"], "article-title": ["On online control of false discovery rate"], "source": ["arXiv preprint"], "year": ["2015"], "elocation-id": ["arXiv:1502.06197"]}, {"person-group": ["\n"], "surname": ["Javanmard", "Montanari"], "given-names": ["A", "A"], "article-title": ["Online Rules for Control of False Discovery Rate and False Discovery Exceedance"], "source": ["Annals of Statistics"], "year": ["2018"], "volume": ["46"], "fpage": ["526"], "lpage": ["554"]}, {"person-group": ["\n"], "surname": ["Johari", "Koomen", "Pekelis", "Walsh"], "given-names": ["R", "P", "L", "D"], "article-title": ["Always valid inference: Continuous monitoring of a/b tests"], "source": ["Operations Research"], "year": ["2021"]}, {"person-group": ["\n"], "surname": ["Karp", "Mason", "Beaudet", "Benjamini", "Bower", "Braun", "Brown", "Chesler", "Dickinson", "Flenniken"], "given-names": ["NA", "J", "AL", "Y", "L", "RE", "SD", "EJ", "ME", "AM"], "article-title": ["Prevalence of sexual dimorphism in mammalian phenotypic traits"], "source": ["Nature Communications"], "year": ["2017"], "volume": ["8"], "elocation-id": ["15475"]}, {"person-group": ["\n"], "surname": ["Katsevich", "Ramdas"], "given-names": ["E", "A"], "article-title": ["Simultaneous high-probability bounds on the false discovery proportion in structured, regression and online settings"], "source": ["The Annals of Statistics"], "year": ["2020"], "volume": ["48"], "fpage": ["3465"], "lpage": ["3487"]}, {"person-group": ["\n"], "surname": ["Liou", "Hornburg", "Robertson"], "given-names": ["L", "M", "DS"], "article-title": ["Online FDR Control across multiple RNAseq experiments"], "source": ["Bioinformatics"], "year": ["2023"], "volume": ["39"]}, {"person-group": ["\n"], "surname": ["Liou", "Robertson"], "given-names": ["L", "DS"], "source": ["OnlineFDRex-plore"], "year": ["2021"], "date-in-citation": ["Accessed: 2022-06-15"], "comment": ["\n"], "ext-link": ["http://shiny.mrc-bsu.cam.ac.uk/apps/onlineFDRexplore/"]}, {"person-group": ["\n"], "surname": ["Parker", "James", "Brawley", "Clarke", "Hoyle", "Ali", "Ritchie", "Attard", "Chowdhury", "Cross", "Dearnaley"], "given-names": ["CC", "ND", "CD", "NW", "AP", "A", "AWS", "G", "S", "W", "DP"], "article-title": ["Radiotherapy to the primary tumour for newly diagnosed, metastatic prostate cancer (sTAMPEDE): a randomised controlled phase 3 trial"], "source": ["The Lancet"], "year": ["2018"], "volume": ["392"], "fpage": ["2353"], "lpage": ["2366"]}, {"person-group": ["\n"], "surname": ["Ramdas", "Yang", "Wainwright", "Jordan"], "given-names": ["A", "F", "MJ", "MI"], "article-title": ["online control of the false discovery rate with decaying memory"], "source": ["Advances in Neural Information Processing Systems"], "year": ["2017"], "volume": ["30"], "fpage": ["5650"], "lpage": ["5659"]}, {"person-group": ["\n"], "surname": ["Ramdas", "Zrnic", "Wainwright", "Jordan"], "given-names": ["A", "T", "M", "M"], "source": ["sAFFRoN: an Adaptive Algorithm for online Control of the False Discovery Rate"], "conf-name": ["Proceedings of the 35th International Conference on Machine Learning"], "conf-sponsor": ["Proceedings of Machine Learning Research"], "year": ["2018"], "volume": ["80"], "fpage": ["4286"], "lpage": ["4294"]}, {"person-group": ["\n"], "surname": ["Rebjock", "Kurt", "Januschowski", "Callot"], "given-names": ["Q", "B", "T", "L"], "article-title": ["online false discovery rate control for anomaly detection in time series"], "source": ["Advances in Neural Information Processing Systems"], "year": ["2021"], "volume": ["34"], "fpage": ["26487"], "lpage": ["26498"]}, {"person-group": ["\n"], "surname": ["Robertson", "Wason"], "given-names": ["DS", "JMS"], "article-title": ["Online control of the false discovery rate in biomedical research"], "source": ["arXiv preprint"], "year": ["2018"], "elocation-id": ["arXiv:1809.07292"]}, {"person-group": ["\n"], "surname": ["Robertson", "Liou", "Ramdas", "Karp"], "given-names": ["DS", "L", "A", "NA"], "article-title": ["onlineFDR: Online error rate control"], "year": ["2021"], "pub-id": ["10.18129/B9.bioc.onlineFDR"]}, {"person-group": ["\n"], "surname": ["Storey"], "given-names": ["JD"], "article-title": ["A direct approach to false discovery rates"], "source": ["Journal of the Royal Statistical Society: Series B (Statistical Methodology)"], "year": ["2002"], "volume": ["64"], "fpage": ["479"], "lpage": ["498"]}, {"person-group": ["\n"], "surname": ["Tian", "Ramdas"], "given-names": ["J", "A"], "article-title": ["ADDIS: an adaptive discarding algorithm for online FDR control with conservative nulls"], "source": ["Advances in Neural Information Processing Systems"], "year": ["2019"], "volume": ["32"]}, {"person-group": ["\n"], "surname": ["Tukey"], "given-names": ["JW"], "source": ["The collected works of John W Tukey Volume VIII Multiple comparisons: 1948-1983"], "publisher-name": ["Chapman and Hall"], "year": ["1953"]}, {"person-group": ["\n"], "surname": ["\u0160id\u00e1k"], "given-names": ["Z"], "article-title": ["Rectangular confidence regions for the means of multivariate normal distributions"], "source": ["Journal of the American Statistical Association"], "year": ["1967"], "volume": ["62"], "fpage": ["626"], "lpage": ["633"]}, {"person-group": ["\n"], "surname": ["Weinstein", "Ramdas"], "given-names": ["A", "A"], "source": ["Online control of the false coverage rate and false sign rate"], "conf-name": ["International Conference on Machine Learning"], "conf-sponsor": ["PMLR"], "year": ["2020"], "fpage": ["10193"], "lpage": ["10202"]}, {"person-group": ["\n"], "surname": ["Xu", "Ramdas"], "given-names": ["Z", "A"], "part-title": ["Dynamic algorithms for online multiple testing"], "source": ["Mathematical and Scientific Machine Learning"], "publisher-name": ["PMLR"], "year": ["2022"], "fpage": ["955"], "lpage": ["986"]}, {"person-group": ["\n"], "surname": ["Yang", "Ramdas", "Jamieson", "Wainwright"], "given-names": ["F", "A", "K", "M"], "article-title": ["A framework for Multi-A(rmed)/B(anit) testing with online FDR control"], "source": ["Advances in Neural Information Processing Systems"], "year": ["2017"], "volume": ["30"], "fpage": ["5959"], "lpage": ["5968"]}, {"person-group": ["\n"], "surname": ["Zeevi", "Astashenko", "Benjamini"], "given-names": ["Y", "S", "Y"], "article-title": ["Ignored evident multiplicity harms replicability-adjusting for it offers a remedy"], "source": ["arXiv preprint"], "year": ["2020"], "elocation-id": ["arXiv:2006.11585"]}, {"person-group": ["\n"], "surname": ["Zehetmayer", "Posch", "Koenig"], "given-names": ["S", "M", "F"], "article-title": ["Online control of the False Discovery Rate in group-sequential platform trials"], "source": ["Statistical Methods in Medical Research"], "year": ["2021"], "volume": ["31"], "fpage": ["2470"], "lpage": ["2485"]}, {"person-group": ["\n"], "surname": ["Zhao", "Small", "Su"], "given-names": ["Q", "DS", "W"], "article-title": ["Multiple testing when many "], "italic": ["p"], "source": ["Journal of the American Statistical Association"], "year": ["2019"], "volume": ["114"], "fpage": ["1291"], "lpage": ["1304"]}, {"person-group": ["\n"], "surname": ["Zrnic", "Ramdas", "Jordan"], "given-names": ["T", "A", "MI"], "article-title": ["Asynchronous online Testing of Multiple Hypotheses"], "source": ["J Mach Learn Res"], "year": ["2021"], "volume": ["22"], "fpage": ["33"], "lpage": ["1"]}, {"person-group": ["\n"], "surname": ["Zrnic", "Jiang", "Ramdas", "Jordan"], "given-names": ["T", "D", "A", "M"], "source": ["The Power of Batching in Multiple Hypothesis Testing"], "conf-name": ["International Conference on Artificial Intelligence and Statistics"], "conf-sponsor": ["PMLR"], "year": ["2020"], "fpage": ["3806"], "lpage": ["3815"]}]
{ "acronym": [], "definition": [] }
60
CC BY
no
2024-01-13 23:49:37
Stat Sci. 2023 Nov 1; 38(4):557-575
oa_package/61/4e/PMC7615519.tar.gz
PMC7615520
37022834
[ "<title>Introduction</title>", "<p id=\"P2\">Gestational diabetes mellitus (GDM) is defined by the World Health Organization (WHO) as carbohydrate intolerance resulting in hyperglycemia - high blood glucose - of variable severity with onset or first recognition during pregnancy [##REF##9686693##1##]. It is one of the most common non-communicable medical complications during pregnancy. The prevalence of GDM is increasing rapidly worldwide. In 2021, the average prevalence of GDM was 16.7% globally, and was highest in Southeast Asia at 25.9%, as reported based on the population, the diagnostic criteria used, and geographical locations [##REF##25884746##2##–##REF##32822601##6##]. GDM is associated with both short- and long-term adverse health consequences for mothers and children. During pregnancy, GDM is associated with an increased risk of pre-eclampsia, increased fetal growth leading to macrosomia, shoulder dystocia, birth trauma and neonatal hypoglycaemia [##REF##15951574##7##, ##REF##19797280##8##]. Women with GDM also have a 50% risk of developing type 2 diabetes in the decade following their pregnancy [##REF##19465232##9##, ##REF##24341443##10##]. This makes GDM a condition of great public health interest in the fight against the global epidemic of type 2 diabetes.</p>", "<p id=\"P3\">During a healthy pregnancy, particularly in the second half of pregnancy, placental hormones favour a state of insulin resistance, resulting in increased insulin secretion from the pancreatic β-cells [##REF##9540023##11##]. There is a higher risk of developing hyperglycaemia for women in whom insulin secretion is inadequate, or those with higher peripheral insulin resistance [##REF##24341443##10##].</p>", "<p id=\"P4\">The most widely accepted test for diagnosing GDM is the 75-gram oral glucose tolerance test (OGTT), recommended by the WHO [##REF##24847517##12##] and UK National Institute for Health and Care Excellence (NICE) [##UREF##2##13##]. Using the WHO/IADPSG criteria, GDM is diagnosed if the woman has either a fasting plasma glucose level of 5.1 mmol/L or above, 1-hour ≥10.0 mmol/L, or 2-hour ≥ 8.5 mmol/L. However, these thresholds are not globally agreed. Many counties, including the UK, recommend their own national thresholds to diagnose GDM. This diversity in diagnosis is also reflected in a lack of universal clinical management and monitoring targets.</p>", "<p id=\"P5\">After diagnosis, glycaemic management in GDM is based on self-collected glucose, most commonly through fingerstick capillary blood testing. Different organizations use different blood glucose targets in women with GDM. For example, NICE 2015 (and updated in 2020) [##UREF##2##13##–##UREF##4##15##] recommends targets of fasting &lt; 5.3 mmol/L and 1-hour postprandial &lt;7.8 mmol/L. Women with fasting glucose levels of 7.0 mmol/L or above are advised to start medication treatment immediately. Women with fasting glucose &lt; 7.0 mmol/L are initially given lifestyle advice, such as diet and regular exercise. Diet and medication plans should be reviewed by a clinician every two to four weeks, either during hospital visits, telephone calls or remote-monitoring platforms [##REF##29559428##16##]. If blood glucose targets are not met within 1-2 weeks, women should be offered medication (e.g., metformin or/and insulin) [##UREF##4##15##, ##UREF##5##17##]. In comparison, the American Diabetes Association recommends similar glucose targets for women with GDM as follows: fasting glucose less than or equal to 95 mg/dL, 1 hour after eating less than or equal to 140 mg/dL (equivalent to 7.8 mmol/L), and 2 hours after eating less than or equal to 120 mg/dL (equivalent to 8.6 mmol/L) [##REF##21193625##18##].</p>", "<p id=\"P6\">Due to the vital role of glycaemic monitoring and the changing physiology as pregnancy progresses, women with GDM are asked to monitor their blood glucose levels daily. The monitoring results are generally labelled as fasting pre-meal, one-hour post-meal and at bedtime.</p>", "<p id=\"P7\">To better support women performing glycaemic monitoring, and to provide clinicians and patients with clinically traceable measures, digital health technologies have been developed for GDM. The U.S. Food and Drug Administration (FDA) defines digital health as mobile health (m-Health), health information technology, wearable devices, telehealth and telemedicine, and personalized medicine [##UREF##6##19##]. The power of digital innovations and machine learning aims to facilitate the prevention, early diagnosis and management of people’s health in hospital, community and home settings.</p>", "<p id=\"P8\">##FIG##0##Fig. 1## demonstrates potential scenarios of how digital health and machine learning could be fused into the health and care infrastructure for people with gestational diabetes. There are three rings: (1) the dark-blue outer ring demonstrates the components of the digital health framework of gestational diabetes in public health and care, including the maternal and neonatal healthcare cost, public health and global partnerships, law, regulation and policy, digital health innovation, technology certificate, national screening services and scientific research; (2) the light-blue ring shows three healthcare environments, including primary and community care, hospital care as well as the diabetes screening services before and after the pregnancy; and (3) the white inner ring demonstrates the scenarios of personal gestational diabetes self-monitoring at home, including weight tracking, blood glucose self-test and monitoring, diet control and medical interventions, such as metformin pills or insulin injections. These three rings are comprehensive to each other and demonstrate three levels of antenatal and postnatal GDM care infrastructure for patients with GDM. Blood glucose monitoring is often linked to wearable sensors in mobile devices, such as smartphones, watches, and waistbands. The ability to collect, collate and analyse data from different sensors for activity tracking, food intake quantification, blood glucose monitoring and medication management could open up new possibilities to help people manage GDM. But there is a gap for data scientists, engineers and clinicians to fill. Personalized, explainable, and trusted AI and ML models are needed to assist patients and clinicians in clinical management with the aim of improving patients’ lifestyles and short-term and long-term clinical outcomes.</p>", "<p id=\"P9\">For conditions such as GDM, digital health technologies can provide real-time glucose monitoring, thus enabling timely diagnosis, treatment and personalised advice on food intake, exercise and medication. Current methods for glucose measurement in women with GDM only provide a snapshot of the change in glucose levels. Ideally, glucose levels should be monitored in real-time, using a non-invasive and unobtrusive method, such as continuous glucose monitoring.</p>", "<p id=\"P10\">A review on gestational diabetes by Saravanan [##REF##32822601##6##] suggests that a shift is needed to move from the perception of a short-term condition that confers an increased risk of large babies to a potentially modifiable long-term condition that contributes to the growing burden of childhood obesity and cardio-metabolic disorders in women and their offspring.</p>", "<p id=\"P11\">In this paper, we provide comprehensive reviews of (i) glucose monitoring technologies for women with GDM; (ii) the types of glucose sensor technologies currently available and their potential for application in GDM; and (iii) digital health technologies and machine learning algorithms for blood glucose prediction, medication advice and lifestyle management in GDM, with comparison to the current state-of-the-art technologies used in type 1 and type 2 diabetes monitoring. We then finally discussed potential future work in this field to improve the health and care of women with GDM.</p>" ]
[ "<title>Methods based on optical detection</title>", "<p id=\"P26\">Unlike electrochemical methods, physical approaches to detect glucose levels are based on measuring specific properties of the glucose molecule. Among the physical approaches, the optical sensing method is one of the most common methods for CGM due to its potential benefits of increased stability and low [##UREF##16##36##]. The optical approaches mainly include infrared (IR) spectroscopy, Raman spectroscopy, photoacoustic spectroscopy, fluorescence spectroscopy, and optical coherence tomography (OCT) [##UREF##17##37##, ##UREF##18##38##]. The following sections will give an overview of how these technologies are used in continuous glucose sensing.</p>", "<p id=\"P27\">IR spectroscopy usually consists of near-infrared (NIR) and mid-infrared (MIR) spectroscopy depending on the light wavelength – the short-band 780-1500 nm and 4000-400 cm-1 ranges are usually used for NIR and MIR, respectively. For noninvasive studies, reflectance light-emitting diode (LED) arrays that are readily available and low-cost are commonly used for NIR as the sources, and the sensing location can be fingertip, forearm or upper arm. For MIR, glucose has absorption peaks in several regions, and the 1200-1000 cm-1 range has received the most attention in sensor studies because it is related to the skeletal vibrations of glucose. Compared with NIR absorption bands that are typically combined bands, weaker and broader, the MIR range absorption bands are relatively sharp, more selective and have a stronger signal. For IR spectroscopy, the common challenge is the interfering molecules that have absorption spectra similar to glucose; examples of these molecules include lactate, urea, and sugars.</p>", "<p id=\"P28\">Raman spectroscopy, as its name indicates, employs Raman scattering in order to observe vibrational modes in glucose molecules [##REF##17028718##39##]. Usually, a single-wavelength source with visible or NIR wavelengths is sufficient to produce the entire Raman spectrum, as only the frequency shift is measured. The resulting frequency shift due to the scattering is sensitive to the vibrational modes of the molecule and independent of the excitation photon frequency. And therefore, the Raman spectrum for glucose can be quite clearly distinguished from other biological compounds. Furthermore, the Raman signal can be amplified by several orders of magnitude with the technique of surface-enhanced Raman scattering (SERS). However, the main challenge of Raman spectroscopy is the small cross-section, which can be ten orders of magnitude smaller than the fluorescence cross-section, resulting in the Raman scattering signal being masked by interfering fluorescence signals.</p>", "<p id=\"P29\">As mentioned above, both IR and Raman spectroscopy detect glucose concentration through the direct interaction between light and glucose. In contrast, fluorescence sensing does not measure glucose directly but measures the signal from molecules that can reversely bind to glucose. These molecules are called exogenous fluorophores; they are engineered to form a complex with glucose molecules and only fluoresce in the presence of glucose. As a result, the fluorescent light intensity will depend on the glucose concentration, since more fluorophores are active when there is more glucose bound with them.</p>", "<p id=\"P30\">OCT is a measurement method that uses an interferometer with low coherence light – typically light in the NIR range due to its nature of miniaturisation and low cost. The OCT system consists of one reference and one sample arm for the light, a moving window to vary the path length, and a photodetector for the light. The light scattered back from the tissue is combined with light from the reference arm, and the interference signal is sent to the photodetector. The reflective index of the interstitial fluid will change when the glucose concentration changes, which in turn changes the scattering coefficient. This change in scattering coefficient and concomitant variation in the interferogram is used to determine the glucose concentration. The main challenges of OCT for glucose monitoring are that the measured change in the scattering coefficient is small and sensitive to motion artefacts.</p>" ]
[]
[ "<title>Discussion and Future Directions</title>", "<p id=\"P88\">Digital health and AI technologies offer potential new approaches to improve clinical outcomes and patient experience for women with GDM. By combining digital health techniques and machine learning methods with blood glucose measurements, we can transfer the hard-to-interoperate blood glucose values to actionable insights for intervention [##REF##27566735##112##].</p>", "<p id=\"P89\">Thanks to the recent developments in GDM management and the need for remote monitoring through the COVID-19 pandemic, several innovations in digital health for GDM are now available to provide patient-centred blood glucose, diet, medication and behavioural management during pregnancy. Whilst there are only a handful of monitoring platforms that have been clinically evaluated, digital platforms are associated with higher satisfaction rates than standard clinical practice. However, the following remain unmet challenges still await to be addressed: (1) scalability and sustainability of known interventions [##REF##15951574##7##]; (2) high cost and other barriers to implementation [##REF##29559428##16##]; (3) engaging women with lifestyle changes during pregnancy [##UREF##26##51##, ##REF##24798080##113##]; (4) achieving equitable coverage for all women [##REF##22462760##46##]; and (5) using new technologies (e.g., artificial intelligence, smartphones/tablet, telemedicine) in promoting improved glucose monitoring [##REF##32535090##114##, ##UREF##52##115##].</p>", "<p id=\"P90\">To resolve these challenges, one question is often asked: can digital health technologies help solve the GDM management crisis? The shortages of clinical resources and the high cost of GDM self-monitoring (either via CGM or daily fingerstick test) created a barrier to cost health inequality. One direction is to consider remote home monitoring (“virtual ward”) and virtual consultation to ease the pressure of shortages in clinical resources. This direction can take advantage of the advances in digital technologies of smartphones, wearables, Apps and machine learning to speed up the adoption of remote monitoring within the national healthcare services. The other direction is to develop risk-based low-cost GDM services that are fit for mothers with different financial and clinical resources. This direction can take advantage of the development of wearable blood glucose sensors and predictive patient monitoring models, which have been reviewed in Sections II and IV of this paper.</p>", "<p id=\"P91\">From the other perspective, studies in mobile health technologies in blood glucose monitoring have shown the improvement of patient satisfaction in their patient care, but thus far have not shown significant clinical outcome improvement. This is largely due to the lack of clinically-plausible digital health technologies for medication and lifestyle interventions. Meanwhile, large multi-centre or national GDM studies are needed to help identify sub-group of GDM patients thereby improving and redefining the GDM care pathway.</p>", "<p id=\"P92\">The current key players in the GDM monitoring and management market are hospitals, universities, and companies in the pharmaceutical industry, insurance companies, sensor technology manufacturers and other emerging companies in consumer markets, third-party services and women themselves. These innovators and service providers aim to use digital health technologies to reduce inefficiencies, improve access, reduce costs, increase quality, and make medicine more personalized for patients. Early identification, referral and management of pregnant women at increased risk may offer opportunities for prevention. Innovations for pre-GDM and post-pregnancy health monitoring are also urgently needed; these innovations can enable individuals and healthcare providers to estimate the risk of gestational diabetes and type 2 diabetes and provide timely intervention.</p>", "<p id=\"P93\">There are other challenges limiting the digital health innovations for GDM monitoring. Firstly, despite the advances in AI-enabled technologies, there remain issues in model interpretability, trustworthiness, fairness and ethics for end-users and service providers. Beyond the technical and technology challenges, evidence-based and quantitative analysis of improvement in patient health and economic costs need to be evaluated in epidemiology studies for specified populations (community, city, state, country, global, or specific to patients with certain diseases). Secondly, the accuracy and cost of sensor technologies in glucose monitoring have yet to be improved. CGM fills a gap that exists in diabetes monitoring and treatment. It provides continuous readings and thereby can continuously analyze and respond to an individual's glucose levels. Another advantage of CGM comes from its nature as “wearable”. Nevertheless, patients need to change their sensors every three to seven days, making it an effective but expensive solution that many people and health systems cannot afford. There is still a lack of applications in monitoring blood glucose that can be fused into finger-tip periodic blood glucose sensors.</p>", "<p id=\"P94\">In the following, we suggest some promising directions for future research: (i) early diagnosis of GDM: proven genetic and/or placental extracellular vesicle bound biomarkers should be taken into consideration for the early diagnosis of GDM [##REF##32822601##6##, ##REF##31164969##116##]. The pre-diabetes population will benefit from this improvement [##REF##31441246##117##]; (ii) Management of gestational diabetes during pregnancy: This should be personalised on the basis of underlying pathogenesis and response to different management strategies. Future GDM screening strategies should include specific treatment guidelines for patients with companion diseases, such as obesity and cardiovascular disease. Therefore, patients at high risk of complications can be informed and treated in a timely manner: machine learning is an ideal tool for this development; (iii) Postnatal type 2 diabetes and cardiovascular disease prevention: national level postnatal screening programmes, large-scale clinical research and digital health applications for postnatal monitoring are urgently needed to delay the development of type 2 diabetes and cardiovascular disease. (iv) Health economic research: global prevalence of GDM varies; the rate of GDM was shown doubled in pregnant South East Asian women than in Caucasian women [##UREF##53##118##]. National and local health economic analyses are needed for developing country-specific cost-effectiveness models that incorporate the cost of any new technologies, alongside treatment costs and long-term healthcare costs for the health system, as well as directly for the mother and child. This will assist policymakers and service providers in prioritising whether novel technologies make financial sense in different care systems around the world. (v) Low-cost blood glucose monitoring solutions are urgently needed for low-and-middle-income countries. (vi) Development of other potential wearable techniques include optical-based blood glucose estimation methods on wearable or portable devices and wearable contact lenses for blood glucose monitoring. (vii) Expanding the physiology knowledge map in GDM to understand and quantitatively address the association among diabetes, pregnancy, and human metabolism, as well as other health conditions, such as hypertension, sleep disorder, postnatal depression, and cardiovascular diseases.</p>", "<p id=\"P95\">In conclusion, AI and ML are promising, emerging areas for the monitoring and management of women with gestational diabetes. While ML and AI have proven to be useful in research and clinical practices for patient monitoring using risk stratification, patient subgroup discovery, and natural language processing-based outcome prediction models [##UREF##54##119##–##UREF##61##130##], similar approaches for GDM are yet to be developed. The exciting field of collecting data from different sensors for activity tracking, food intake quantification, blood glucose monitoring and medication management could open up new possibilities to help people manage GDM. However, there are still many unanswered questions for data scientists, engineers and clinicians. Personalized, explainable, and trustful AI and ML models are needed to assist patients and clinicians in improving patients’ lifestyles and short-term and long-term clinical outcomes. There is an urgent need to develop digital health technologies and explainable AI methods to identify patients at different risk groups at an earlier stage (preventive medicine) and provide clinicians with a reactive treatment plan using predictive monitoring models.</p>" ]
[]
[ "<p id=\"P1\">Innovations in digital health and machine learning are changing the path of clinical health and care. People from different geographical locations and cultural backgrounds can benefit from the mobility of wearable devices and smartphones to monitor their health ubiquitously. This paper focuses on reviewing the digital health and machine learning technologies used in gestational diabetes – a subtype of diabetes that occurs during pregnancy. This paper reviews sensor technologies used in blood glucose monitoring devices, digital health innovations and machine learning models for gestational diabetes monitoring and management, in clinical and commercial settings, and discusses future directions. Despite one in six mothers having gestational diabetes, digital health applications were underdeveloped, especially the techniques that can be deployed in clinical practice. There is an urgent need to (1) develop clinically interpretable machine learning methods for patients with gestational diabetes, assisting health professionals with treatment, monitoring, and risk stratification before, during and after their pregnancies; (2) adapt and develop clinically-proven devices for patient self-management of health and well-being at home settings (“virtual ward” and virtual consultation), thereby improving clinical outcomes by facilitating timely intervention; and (3) ensure innovations are affordable and sustainable for all women with different socioeconomic backgrounds and clinical resources.</p>", "<title>Index Terms</title>" ]
[ "<title>Blood Glucose Monitoring Techniques for Gestational Diabetes</title>", "<p id=\"P12\">Blood glucose monitoring plays a vital role in the early detection, diagnosis, treatment and management of women who have gestational diabetes. Health providers use blood glucose values to check and adjust the effect of treatments, such as the effectiveness of diet and exercise and the dosage of medications. There are two main methods for ambulatory glycemic testing: intermittent capillary blood glucose technologies and continuous glucose monitoring technologies (CGM). CGM can be subdivided into intermittently scanned (flash) and real-time continuous (CGM) sensors.</p>", "<p id=\"P13\">These two kinds of sensors are different in four aspects: i) intermittent blood glucose monitoring measures discrete glucose levels accurately from capillary blood, whereas continuous monitoring provides multiple glucose levels of fair accuracy from the interstitial fluid beneath the skin, which approximates blood glucose levels; ii) with standard intermittent monitoring, current blood glucose levels do not predict future glucose levels; but with continuous monitoring, trends in glucose levels are often predicted to enable the insulin pump to provide a precise amount of insulin accordingly; iii) with intermittent monitoring, blood glucose results can be used directly without data processing; but with continuous monitoring, data analysis is required to extract fluctuations of GL, for example, mean blood glucose results at different events, such as before or after breakfast; and iv) an intermittent blood glucose monitor requires patient’s input for every reading, whereas a continuous monitor records the time-series blood glucose value synchronously.</p>", "<p id=\"P14\">It is challenging for patients to self-monitor their blood glucose using fingerstick testing four to six times a day; however, this is especially important for women who are on multiple daily insulin injections to balance the risk of hyperglycaemia and hypoglycaemia. Finger stick testing is inconvenient and painful for women, with limitations including accuracy, specificity, and inappropriate usage. Because of cost and lack of evidence, continuous blood glucose monitoring is an alternative, but it is not used broadly in clinical practice for gestational diabetes management.</p>", "<p id=\"P15\">This section will explain the technical aspects of intermittent and continuous sensor technologies in detail.</p>", "<title>Periodic Monitoring using Fingertip Blood Tests</title>", "<p id=\"P16\">In order to gain an accurate picture of blood glucose trends, women in pregnancy are requested to perform several fingerstick tests each day. The test is typically performed by piercing the skin – usually on the fingertip – with a lancet device to obtain a small volume of blood, and then the glucose concentration is determined with a glucose meter (##FIG##1##Fig. 2a##). The development of the first blood glucose meter dates back to the 1970s after developments in urinary glucose testing and blood glucose dry-reagent test strips [##REF##22872934##23##, ##REF##3898972##24##]. The first blood glucose meters combined dry chemistry test strips (Dextrostix) with reflectance photometry to measure blood glucose, of which the paper strip is treated with enzyme reagents that give approximate results for glucose concentration with one drop of whole capillary or venous blood. Since then, significant progress has been achieved in the development of blood glucose meters, such as reducing blood sample size, obtaining blood samples from alternate sites, and improving test time, display, data storage, and calibration (2b) [##REF##22872934##23##, ##UREF##10##25##]. Nowadays, there are a large number of devices on the market, with meters developed by Roche Diagnostics, Lifescan, Medisense, Bayer, and Therasense among the most popular [##UREF##10##25##].</p>", "<p id=\"P17\">Though popular and easy to use, self-monitoring of blood glucose using finger-stick test strips has limitations, including accuracy, specificity, and inappropriate usage, as discussed by Olansky et al. [##REF##20351231##26##]. To be safe and of clinical value, blood glucose meters should measure blood glucose levels accurately and precisely. In terms of accuracy, the standard developed by the FDA in 2016 for blood glucose meters for over-the-counter use requires 95% of data pairs of BGM measurement and a reference measurement to be within 15% for BG values &gt;100 mg/dL (5.55 mmol/L), which is similar to the standard issued by the International Organization for Standardization (ISO) 15197:2013 [##UREF##11##27##, ##REF##29898901##28##].</p>", "<title>Continuous Blood Glucose Monitoring using Wearable Sensors</title>", "<p id=\"P18\">With advances and the development of technologies in electronics, manufacturing, materials and sensors, glucose monitoring has developed rapidly. Technology enabling accurate, continuous, long-term and noninvasive glucose monitoring has become possible, with blood no longer the only medium for glucose monitoring.</p>", "<p id=\"P19\">Compared with finger-stick blood testing, continuous glucose monitoring (CGM) – either from real-time use or intermittently viewed – provides insights about the direction, magnitude, duration, frequency, and fluctuations in glucose levels, which enables sufficient information that is clinically valuable [##REF##15855600##29##]. CGM can reduce risks of hypoglycemia, hyperglycemia, and glycemic variability and improve the quality of life for patients with blood glucose imparity, including patients with gestational diabetes. CGM via an implantable, transdermal sensor has become the gold standard method for monitoring continuous glucose levels in pregnant women with type 1 diabetes. CGM has been widely studied and is now being used in clinical settings [##UREF##12##30##]. However, prolonged use of CGM is invasive and may cause vascular damage or infection [##REF##15855600##31##]. No articles have been published on their long-term performance.</p>", "<p id=\"P20\">The evidence for CGM use in pregnancy comes mostly from women with type 1 diabetes, and in the UK, the technology is currently not recommended for women with other forms of diabetes [##UREF##5##17##]. There are several barriers that will need to be addressed before CGM can be considered for women with GDM. The first barrier is cost. Depending on the length of time it is used, real-time CGM can cost up to £2000 per pregnancy (2021 NHS list price). With up to 65000 women having GDM in the UK each year, the potential cost increase of adopting CGM for GDM could be &gt; £100 M per year. The second barrier is the limited understanding of managing GDM using glycaemic time in range. The optimal time in range for women with GDM is unknown [##REF##15855600##29##]. Finally, it is unclear whether outcomes would be improved for women and their babies with GDM by adopting CGM; thus, adequately powered clinical trials are needed in this area.</p>", "<p id=\"P21\">However, given the trend of reducing the cost of technologies, ongoing technological developments and research in this area, it is possible in the future that CGM may become an option for women with GDM. The remainder of this section of this review will focus on noninvasive continuous glucose monitoring techniques with the potential for application in GDM.</p>", "<p id=\"P22\">Noninvasive continuous glucose sensing can be classified into three categories: electrochemical methods, optical methods, and non-optical methods, including microwave methods. Each of these technologies will be elaborated as below.</p>", "<title>Enzyme-based electrochemical sensors</title>", "<p id=\"P23\">Enzyme-based electrochemical sensing combines enzymatic detection with electrochemical measurements, and it can yield higher detection accuracy than each of the single techniques. The enzymatic sensor is performed on bio-fluids that contain lower glucose concentrations than blood, for example, interstitial fluid, tears, saliva, and sweat [##UREF##13##32##] (##FIG##2##Fig. 3##). For example, the well-known Google contact lens is such a sensor, designed to detect glucose levels in tears. Alternatively, interstitial fluid has been used as the medium to extract glucose onto the skin surface by using the technique of reverse iontophoresis with an enzymatic glucose sensor. GlucoWatch® (Cygnus Inc., Redwood, CA, USA) is a commercial product that adopted reverse iontophoresis as the way to extract glucose samples.</p>", "<p id=\"P24\">One of the main advantages of such technology is that it is noninvasive, unobtrusive, and real-time. This technology can be realized via flexible epidermal sensors that can be fabricated into body-compliant wearable platforms, such as a patch, wrist band, temporary tattoo, and integrated wireless electronics for practical wearable applications [##REF##29108571##34##].</p>", "<p id=\"P25\">However, there are two disadvantages of such methods. One is that there is a time lag between the blood glucose level and the glucose in interstitial fluid; the other is that such a method by applying potential onto the human skin (or other epithelial surfaces) can cause irritation [##UREF##15##35##]. The latest research and development of epidermal electrochemical glucose sensors can be found in more detail in the review paper by Kim et al. [##REF##29108571##34##]. A more elaborative review of enzyme-based electrochemical glucose sensors, including materials, device structures, fabrication processes, and system engineering, can be found in the review by Lee [##UREF##14##33##].</p>", "<title>Other noninvasive glucose detection technologies</title>", "<p id=\"P31\">Except for the commonly studied optical sensing approaches, other noninvasive methods, such as electrical impedance spectroscopy and microwave sensing methods, have also been explored, as shown in ##FIG##3##Fig. 4##. The electrical impedance spectroscopy (EIS) method has the advantages of being low-cost and user-friendly. The design of such systems consists of bioimpedance sensors, signal measurement strategies, modelling and parameter estimation methods to extract blood glucose levels, and portable system designs [##UREF##15##35##]. While microwave technology measures the dielectric properties of aqueous glucose with a microwave sensor, generally, a patch antenna which is compact, cost-effective, painless and has the potential to provide a more accurate measurement. For example, inspired by vasculature anatomy topologies, Hanna et al. developed a tunable electromagnetic multi-sensing system that is noninvasive and wearable for continuous glucose monitoring [##REF##32577523##40##]. Such systems achieved a high correlation between system’s physical parameters and blood glucose levels [##UREF##16##36##, ##UREF##17##37##].</p>", "<p id=\"P32\">In general, the advantages of optical and microwave methods lie in their highly non-invasive nature and continuous monitoring without stimulating discomfort to the human body. However, the measured value may not be highly correlated with the actual blood glucose value as the linear range is narrow, so subsequent algorithm correction is required. More detailed information on various sensing technologies for glucose monitoring can also be found in the reviewer papers on continuous blood glucose monitoring [##UREF##16##36##, ##UREF##17##37##].</p>", "<title>Emerging Monitoring Devices and Management Platforms for Gestational Diabetes</title>", "<p id=\"P33\">Based on the periodic fingerstick and continuous blood glucose monitoring technologies introduced in Section II, various platforms for monitoring and managing gestational diabetes have been implemented globally in both clinical and non-clinical applications, and in either commercial or research settings. In this section, we will review a few exemplary ones that started to make an impact on the digital health industry for gestational diabetes.</p>", "<title>Devices and Applications Trailed for Clinical Observations and Interventions</title>", "<p id=\"P34\">The NIH ClinicalTrials.gov is a database of privately and publicly funded clinical studies conducted worldwide, managed by the NIH U.S. National Library of Medicine. To provide insight into digital health technologies used in clinical practices worldwide, we screened 538 studies (accessed on 8 August 2021) using the keyword “gestational diabetes” on the NIH ClinicalTrials.gov database. After shortlisting clinical trials using keywords, and removing clinical trials that were not completed or not started, we shortlisted 16 observational studies and 36 interventional studies that used digital health and mobile health technologies for patient monitoring. Protocols were reviewed under two categories: observational studies and clinical interventions.</p>", "<p id=\"P35\">People living with GDM require self-management to maintain blood glucose levels, including diet and exercise control, as well as through medication intervention. ##TAB##0##Table 1## summarises the technologies in the primary purpose, outcome measures, monitoring method, patient size, country of use, and completion status. These studies focus on the technologies for blood glucose monitoring, medical intervention, and lifestyle management for weight control. Readers interested in clinical trial details can read the full details of the above trials at the NIH ClinicalTrials.gov. Systematic clinical reviews in gestational diabetes monitoring systemic clinical reviews [##UREF##0##4##, ##REF##30291088##43##–##REF##32736942##45##] and studies on clinical outcomes [##REF##22462760##46##, ##UREF##22##47##] are also helpful reading materials. Countries with different prevalence levels of GDM, limitations in doctor resources, or different income levels, such as low- and-medium income countries, often require cost-effective and different digital health solutions to address their challenges. Country-specific reviews [##REF##28469095##3##, ##UREF##23##48##, ##UREF##24##49##] can provide a comprehensive source of information that addresses risk factors, diagnosis criteria in racial variance and geographical differences that suit epidemiology studies.</p>", "<p id=\"P36\">Among the clinical trials listed in ##TAB##0##Table 1##, a few studies have been commercialized and put into clinical practice. For example, in the UK, “<italic toggle=\"yes\">GDm-Health</italic>”, was developed from 2014 -16 [##UREF##25##50##] and subsequently licensed to Sensyne Health plc in 2017. In a randomised controlled trial, \"TREAT-GDm\", GDm-Health was deployed on Android mobile phones that can link to Bluetooth-enabled blood glucose meters. To use this self-monitoring platform, patients need to tag their capillary blood readings into six mealtime tags (pre- and post- breakfast, lunch and dinner) and enter their medication dose if applicable. Collected blood glucose measurements are then sent to the GDm-Health server by mobile network and made visible to clinicians. Feedback is provided in the form of colour coding of individual readings: red for high, green for target, and blue for low blood glucose measurements. Summaries of blood glucose measurements (tabulated and graphical) and reminders are also provided as appropriate. The above work has been published [##UREF##2##13##, ##REF##29559428##16##, ##UREF##26##51##–##UREF##29##54##] with a technology appraisal on the NICE website [##UREF##25##50##]. This innovation has been approved for clinical usage by NICE. As of 2021, GDm-Health is available in 56 Trusts, representing 47% of NHS Trusts in England.</p>", "<p id=\"P37\">MobiGuide [##UREF##30##55##, ##REF##24876573##56##] is an evidence-based decision-support system developed in 2014 and then modified in 2017. It can provide personalised decision support based on patients' personal health records, including data from hospital medical records, mobile biosensors, data entered by patients, and recommendations and abstractions output by MobiGuide. This system can be used by patients with gestational diabetes (via mobile MobiGuide) and their care provider (via central MobiGuide). Their study analyzed usage patterns and opinions collected via questionnaires of the 10 atrial fibrillation and 20 (GDM patients and their care providers. The results confirmed using the MobiGuide system have resulted in diagnosis changes for 2/10 atrial fibrillation patients and anticipated changes in therapy for 11/20 GDM patients.</p>", "<p id=\"P38\">The Pregnant+ smartphone application was developed in 2015 to motivate women to have a healthy diet, and be physically active [##REF##27417764##57##]. Similar to GDm-Health, it allows the automatic transfer of blood glucose measures from the glucose meter to the smartphone. A green or red face indicates a normal or high blood glucose level (hyperglycemia). There is no colour code for the low blood glucose level (hypoglycemia). The trial protocol was published in 2017 [##UREF##31##58##]. In the trial, women answered questionnaires during pregnancy and were followed up three months postpartum. Trial results were published in 2018 and 2019 [##UREF##32##59##, ##REF##29329023##60##]. Results suggested that using the Pregnant+ app did not affect the 2-hour glucose level at routine postpartum OGTT. And after controlling for parity, the difference in the emergency caesarean section was not statistically significant.</p>", "<p id=\"P39\">Apart from blood glucose monitoring, several studies focus on lifestyle interventions, such as diet and physical activity control. The Institute of Medicine guidelines for gestational weight gain are based on pre-pregnancy BMI. Close attention to food intake is necessary during pregnancy to avoid excessive gestational weight gain while ensuring strict glycemic control. Approximately 80% of women with GDM can reach their glycemic goals with diet and lifestyle modifications alone. Lifestyle interventions are helpful in weight control and may impact pregnancy outcomes.</p>", "<p id=\"P40\">The behavioural lifestyle intervention (PEARS) RCT in a lifestyle intervention study for women overweight and obesity [##UREF##33##61##, ##REF##26625980##62##]. A total of 278 pregnant women (BMI 25–39.9 kg/m2) were randomized to the intervention (n = 278), or a control group (n = 287). Results showed that there were no differences between the groups at baseline. Compared with the control group, the intervention group had improved dietary intake post-intervention. Physical activity (MET-minutes/week) was higher in the intervention group post-intervention. App use was associated with a lower glycaemic index and less energy from free sugars, but not with physical activity.</p>", "<p id=\"P41\">The Mobile-Based Lifestyle Intervention in Women with Glucose Intolerance after Gestational Diabetes Mellitus (MELINDA) study is a Belgian multi-centre RCT in seven hospitals (236 women).[##REF##32823771##63##] The aims of this study are: (1) to evaluate the prevalence and risk factors of glucose intolerance after a recent history of GDM; and (2) to evaluate the efficacy and feasibility of a telephone- and mobile-based lifestyle intervention in women with glucose intolerance after GDM. Women in the intervention group will receive a blended program based on one face-to-face education session and further follow-up through a mobile application and monthly telephone advice. Women in the control group will receive follow-up as in normal routine with referral to primary care. Participants will receive an OGTT one year after baseline. The primary endpoint is the frequency of weight goal achievement (≥5% weight loss if pre-pregnancy BMI ≥ 25 Kg/m<sup>2</sup> or return to pre-gravid weight if BMI &lt; 25 Kg/m<sup>2</sup>). At each visit, blood samples are collected, anthropometric measurements are obtained, and self-administered questionnaires are completed. Recruitment began in May 2019 and expect to finish in June 2022.</p>", "<p id=\"P42\">Electronic Monitoring Of Mom’s Schedule (eMOMs) platforms have three clinical studies in Finland. The trial protocol [##REF##32346648##64##] addressed a feasibility randomized controlled study for women with high pre-pregnancy BMI to improve postpartum weight, blood sugar, and breastfeeding. A total of 72 women were included, 24 per group. The design of this trial has combined breastfeeding and the National Diabetes Prevention Program. eMOMS compares the feasibility and efficacy of three interventions on six-month post-partum weight loss among women with a BMI &gt;=25. Patients were recruited at two clinical sites (rural and urban). Program costs will be compared to that of traditionally scheduled group meetings. The study was completed in October 2021, but the results were not published.</p>", "<p id=\"P43\">Hola Babe and GlucoseMama are the two products that have been used in random clinical trials. The results of these trials were not published, but product information is available on their website for products.</p>", "<p id=\"P44\">All the above platforms are based on the fingerstick blood test and a telemedicine framework. Chen’s study [##REF##14738172##65##] used CGM on 57 women with GDM, 47 in Israel and ten in California. Data derived from the MiniMed CGM System were compared to fingerstick glucose measurements (6–8 times a day) in 72-hour windows. The time of food intake, insulin injections and hypoglycemic events are recorded in the system and used to monitor the health status of GDM. The study authors suggested that CGM is helpful for adjusting diabetes therapy and can accurately detect high blood glucose and hypoglycemic events that may go unrecognized by intermittent blood glucose monitoring.</p>", "<title>Consumer-driven Devices and Applications</title>", "<p id=\"P45\">Along with devices and platforms for clinical purposes, there is a broader commercial market for devices and applications for non-clinical purposes. This section reviews the consumer-level commercial devices and applications used for diabetes. We used the keyword of gestational diabetes to search top-ranked Apps run on iOS using an online search engine (<ext-link xlink:href=\"http://www.deepaso.com\" ext-link-type=\"uri\">www.deepaso.com</ext-link>). The search date is 18<sup>th</sup> November 2021, and the results were compared to 18<sup>th</sup> May 2021. We limited our search to only English and regions with a high prevalence of GDM [##REF##26742932##66##]. The overlap of regions with high prevalence and country-specific ranking data in the search engine are Singapore (SGP), United Arab Emirates (UAE), Thailand (THAI), Malaysia (MAL), United Kingdom (UK), Belgium (BEL) and India (IND).</p>", "<p id=\"P46\">As shown in ##TAB##1##Table 2##, we listed the top 10 apps across these regions under three categories: Medical (M), Food &amp; Drink (FD), Health &amp; Fitness (HF), and lifestyle (LS). It is noted that the ranking is dynamic and can be misleading when an app is promoted at the searching date. Additionally, the search results included Apps designed for both GDM and general diabetes. The devices and apps that are applicable for GDM overlap with those for diabetes management.</p>", "<p id=\"P47\">This review lists the top 10 Apps in three categories listed in these seven countries. Several apps in the medical category are ranked high across different counties, such as mySugr- Diabetes Tracker Log, One Touch Reveal, Glucose Buddy Diabetes Tracker, Medisage Phill Reminder, Glucose – Blood Sugar racker, and MySweetGestation. A few of the FD or LS category Apps are ranked high across these seven countries. Within the Medical and Health &amp; Fitness categories, the main features of apps include (i) glucose monitoring, (ii) exercise monitoring or advice, (iii) food intake recommendation and (iii) medicine-taking reminders.</p>", "<p id=\"P48\">Arguably, the App from the non-medical category is consistently ranked high in different countries and could be popular across these countries.Whereas the Apps belonging to the medical category can be country-specific because of the nature of their development.</p>", "<p id=\"P49\">The mobile device applications primarily focus on glucose monitoring, exercise tracking, food intake recording, and medication reminders.</p>", "<title>Blood glucose monitoring</title>", "<p id=\"P50\">As discussed in section II, consumer-level glucose monitoring uses capillary blood glucose meters or CGM systems. The former requires a user to prick their finger to release a small amount of blood and then place the blood on a test strip readable by the meter. It usually requires the user to manually input those readings in the App, although meters with Bluetooth and NFC are also available. These systems are often associated with reminder features. In contrast, CGM systems can bundle the CGM sensor, smartphone, and smartwatch together. Most existing apps for diabetes management target type 2 diabetes. Some Apps also offer advanced features based on blood glucose levels, including setting alerts for high and low glucose and predicting future glucose levels.</p>", "<title>Exercise trackers</title>", "<p id=\"P51\">These are used to record the user’s exercise and other physical activity. Some apps sync the third party’s apps, e.g., the Apple Health app, to track steps and other physical activity. The exercise tracker is easier to work with smartwatches and other wearable devices for daily use.</p>", "<title>Food intake</title>", "<p id=\"P52\">Food tracking typically provides nutritional information (calories, fat, carbohydrates, cholesterol, added sugar content, hidden ingredients, etc.) based on a private food database and/or barcode scanner. This is often useful for customized food to improve blood glucose control. For nonstandardized portion sizes of food, a user can estimate more accurately with “hint” provided by Apps, e.g., using the photo of incremental portion size provided in the Apps as a reference.</p>", "<title>Medication reminders</title>", "<p id=\"P53\">Apps in medication reminders are non-diabetic general medication reminders or diabetes-specialised reminders. The former is typically embedded into the common calendar and can be customised by a user. The latter could include recording medications and insulin, calculating insulin doses, and setting up warnings for drug interaction. An advanced feature of medication trackers is to analyse drug responses according to glucose levels.</p>", "<p id=\"P54\">In ##TAB##2##Table 3##, we provide the features of Apps of Medical (M) and Health &amp; Fitness (HF) categories that are not country-specific and ranked high in at least two countries. Specifically, we removed two Apps, MySweetGestation [##REF##31669627##68##] and myGestationalDiabetes, the former is designed to be an interactive educational tool for both patients and physicians in the field, and the latter’s website does not provide support for our evaluation regarding the features we are interested.</p>", "<p id=\"P55\">Most diabetes management Apps are good for patients who have been recently diagnosed. The significant advantages of medical diabetes management Apps include: (i) offering a report with more insight into diabetes that can be sent to a healthcare provider to assess the management outcomes, and (ii) setting up an alert system that can automatically send messages to emergency contacts. The diabetes management Apps from the HF category are typically specialised in food, exercise, and lifestyle tracking and recommendation designed for daily use. The built-in trackers in the HF category Apps can be synced in medical diabetes management Apps.</p>", "<p id=\"P56\">There are many reviews that list top-ranked Apps for diabetes management. In this paper, we aim to overview the proportion of GDM Apps in the diabetes management Apps and provide some insight into whether the diabetes management Apps and devices are sufficient for GDM monitoring. Our evaluation may be limited because we used only one App-ranking search engine, evaluated the IOS Apps only, and searched for the keyword of gestational diabetes only.</p>", "<title>Machine Learning Algorithms for GDM Monitoring and Management</title>", "<p id=\"P57\">Data-driven machine learning algorithms and models are trained to detect patterns and hierarchical casualties in training data and predict future results or make decisions under uncertainty [##UREF##45##96##]. Statistical and machine learning models are widely used in medical and healthcare data, including generalized linear models (such as logistic regression and linear regression), Bayesian and probability models, deep neural networks, nonparametric models (exemplar-based methods, kernel methods, and trees, forests, bagging and boosting), graph neural networks, generative adversarial networks, transformers, and reinforcement learning. Following the United States Food and Drug Administration (FDA) regulations, artificial intelligence and machine learning-based software are classified as medical devices [##UREF##46##97##].</p>", "<p id=\"P58\">In this section, we focus on reviewing machine learning methods and algorithms that have been used in the rapidly expanding field of \"Clinical AI\" for gestational diabetes and methods that suit a broader family of diabetes (type 1 and type 2 diabetes) that are feasible for GDM monitoring and patient management. These methods are transferable due to (1) the similarity of type 2 diabetes and GDM, and (2) CGM can be used in GDM but is mainly used for patients with type 1 diabetes for timely insulin intervention.</p>", "<p id=\"P59\">##TAB##3##Table 4## summarizes the machine learning models used in gestational studies or models that can potentially be transferred to GDM. There are merely any clinical studies in GDM or type 2 diabetes using CGM. Considering the similarity between GDM and type 2 diabetes, we included models that were developed for hypoglycemia prediction in type 1 and type 2 diabetes studies and the continuous blood glucose prediction using CGM in type 1 diabetes. We will first introduce physiology and hybrid models for measuring and predicting blood glucose levels, and then discuss machine learning models in detail.</p>", "<title>ML Approaches for Monitoring and Management of Blood Glucose</title>", "<p id=\"P60\">Machine learning methods used in blood glucose sensor data among different types of diabetes are largely similar, with the mutual aim of facilitating blood glucose monitoring, representing patient electronic health records, and providing timely medication intervention and lifestyle advice to manage diabetes in a personalised and predictive manner. For the prediction of blood glucose, as shown in ##FIG##4##Fig. 5##, there are, in general, two types of prediction models: the physiology-based model and the data-driven machine learning model [##UREF##47##98##, ##REF##31042157##99##]. As shown in ##FIG##4##Fig. 5##, we used the taxonomy to summarise the model types, and then provided a few exemplary studies with details in this sub-section.</p>", "<title>Physiological Models and Hybrid Method</title>", "<p id=\"P61\">Physiological models in GDM aim to simulate mother’s and offspring’s glucose-insulin system. There is no physiological model specially developed for mothers with GDM yet, which needs to take offspring’s growth (energy consumption) into consideration. However, it is worth mentioning the need for such models and reviewing existing physiological models that have been developed for general diabetes purposes.</p>", "<p id=\"P62\">Millsap [##UREF##48##100##] provided the mathematical analysis of lumped models for the glucose-insulin system, which are made for linear and quadratic inhibition of insulin release. It is a semi-empirical model that the trajectories of the models in the hodograph and time planes are determined, and a comparison of the inhibitory processes is presented.</p>", "<p id=\"P63\">In the physiological models based on a study by Hovarka [##REF##15382830##101##], the hypothesis is that glucose excursions are influenced by the glucose absorption process, and can be represented as follows: </p>", "<p id=\"P64\">Where <italic toggle=\"yes\">t</italic><sub>max,G</sub> (min) is the time of the maximum appearance rate of glucose in the accessible glucose compartment, <italic toggle=\"yes\">CHO<sub>IN</sub></italic> is the number of carbohydrates ingested, and <italic toggle=\"yes\">CHO<sub>BIO</sub></italic> (dimensionless) is carbohydrate bioavailability. In this model, t was categorised into four periods of 6 h, and was labelled as Nocturnal (01:00 to 06:59 h), <italic toggle=\"yes\">Breakfast</italic> (07:00 to 12:59 h), <italic toggle=\"yes\">Lunch</italic> (13:00 to 18:59 h), and <italic toggle=\"yes\">Dinner</italic> (19:00 h to 00:59).</p>", "<p id=\"P65\">##FIG##5##Fig. 6## demonstrates a hybrid model that comprises physiological models based on insulin and carbohydrate and a grammatical evolution model. In the data-driven grammatical evolution model [##UREF##49##102##], the grammar is a population-based heuristic search algorithm that performs an evolutionary process through selection, recombination, and mutation. This method uses a variable-length linear genome to govern how a Backus Naur Form grammar definition is mapped to a program, and expressions and arbitrary complexity programs may evolve. Then the sinusoidal function is added to account for the circadian variations in patients' physiology in the final model with maximum day-to-day 20% amplitude variations.</p>", "<title>Machine Learning Models</title>", "<title>Blood Glucose Monitoring for GDM</title>", "<p id=\"P66\">Machine learning technologies for GDM monitoring are premature compared to existing work in type 1 and type 2 diabetes. The main reasons are that the global GDM population is much smaller than the mainstream diabetes population and that type 1 patients widely use CGM compared to GDM patients. Studies that have used ML methods can be divided into those aiming to predict who will develop GDM and those aiming to improve GDM management during or after pregnancies, as listed in ##TAB##3##Table 4##. For mothers with GDM, machine learning models are mainly used to improve blood glucose management, predict clinical outcomes before and after childbirth deliveries, and estimate postnatal risks of type 2 diabetes.</p>", "<p id=\"P67\">The following studies focused on the prediction and risk evaluation of GDM for early diagnosis.</p>", "<p id=\"P68\">H. W. Liu et al.’s [##UREF##34##69##] study used risk scores to predict gestational diabetes in early pregnancy in Tianjin, China. An established population-based prospective cohort of 19,331 pregnant women registered as pregnant before the 15th gestational week. In total, 1484 (7.6%) women developed GDM. The dataset was randomly divided into a training set (70%) and a test set (30%). In this study, the eXtreme gradient boosting (XGBoost) method was employed to predict the presence of GDM. The logistic model was also developed for comparison purposes. Risk factors collected at registration were examined and used to construct the prediction model in the training dataset, including pre-pregnancy body mass index, maternal age, fasting plasma glucose at registration, and alanine aminotransferase. The XGBoost model achieved a higher area under the receiver operating characteristic curve (AUROC) than the logistic model (0.742 vs 0.663, p &lt; 0.001), while the logistic model tended to overestimate the risk at the highest risk level (Hosmer–Lemeshow test p-value: 0.243 vs 0.099).</p>", "<p id=\"P69\">N. S. Artzi et al. [##UREF##35##70##] used boosting models for the prediction of GDM based on 588,622 pregnancies in Israel’s nationwide electronic health records. Gradient boosting models predicted GDM with AUROC = 0.85. Results were validated on different geographical validation sets in Israel to emulate real-world performance. Interrogating the boosting models using Sharply value for feature selection, authors developed a risk score table for pre-GDM diagnosis based on nine risk factors.</p>", "<p id=\"P70\">The Diagnosis of Gestational Diabetes Mellitus (GDM-AI) project [##UREF##36##71##] implemented an AI model that compared nine algorithms in GDM diagnosis. This is the first prospective and multi-centre clinical study that supports the GDM diagnosis for pregnant women in a resource-restrained setting by using only fasting blood glucose measurement, patient age, and a smartphone connected to the internet. This system was trained on 12,304 pregnant outpatients with their consent, who received a test for GDM in the obstetrics and gynaecology department of the First Affiliated Hospital of Jinan University between November 2010 and October 2017. GDM was diagnosed according to the American Diabetes Association 2011 diagnostic criteria [##REF##21193625##18##]. Age and fasting blood glucose were chosen as critical parameters. Five-fold cross-validation was used for the internal dataset and an external validation dataset that included 1655 cases from the Prince of Wales Hospital, Chinese University of Hong Kong. The AUROC of the external validation dataset for support vector machine (SVM), random forest, AdaBoost, k-nearest neighbours (kNN), Naïve Bayes (NB), decision tree, logistic regression (LR), XGBoost, and gradient boosting decision tree (GBDT) were 0.780, 0.657, 0.736, 0.669, 0.774, 0.614, 0.769, 0.742, and 0.757, respectively. SVM was selected as the method among all nine algorithms. Results showed that the specificity for SVM retained 100% in the external validation set with an accuracy of 88.7%.</p>", "<p id=\"P71\">Machine learning methods have also been used in finding biomarkers in gestational diabetes. L. Yoffe et al. [##REF##31539877##72##] used a logistic regression model to investigate the role of circulating microRNAs in the plasma of pregnant women in their first trimester. Two populations were included in the study to enable population-specific as well as cross-population inspection of expression profiles. Each microRNA was tested for differential expression in GDM vs control samples. Using both microRNAs in a logistic regression model, the study achieved an AUC value of 0.91. The authors then applied the multivariate models, which achieved an accuracy of mean AUROC = 0.77.</p>", "<p id=\"P72\">As shown in ##TAB##3##Table 4##, several studies used ML with CGM data in blood glucose prediction for pregnant women with T1 and T2 diabetes. However, ML applications using CGM data in GDM diabetes are limited.</p>", "<p id=\"P73\">In Pustozerov et al.’s [##REF##29317385##74##] study, linear regression models with lasso regularisation were developed for postprandial glucose response prediction with CGM readings. In this model, the AUC60, AUC120, BG60, Peak BG, the amount and kind of consumed food, the start time of food intake, physical activity, duration of sleep, and the blood glucose were used for model training. Models were evaluated using the correlation coefficient between actual and predicted values (R), root mean square error (RMSE), mean absolute value (MAE) and mean absolute percentage error (MAPE). The prediction results for BG levels 1 hour after food intake were RSME=0.87 mmol/L, MSE=0.69 mmol/L, and MAPE=12.8%, which correspond to an adequate prediction accuracy for BG control decisions. The system was evaluated using the measurement of glucose levels for seven days using the iPro2 CGM with Enlite sensors (Medtronic, Minneapolis, MN, U.S) and independently calibrated with the Accu-Check Performa Nano blood glucose meter (Roche Diabetes Care, Indianapolis, IN, USA) with a minimum of four measurements per day. Linear regression was chosen due to its good interpretability, simplicity, rapid tuning, and adequate accuracy compared to other methods. Two years later, Pustozerov et al.[##UREF##37##75##] developed another machine learning model using decision tree gradient boosting for postprandial glucose response prediction in women with GDM.</p>", "<p id=\"P74\">Similar to their study in 2018, this model uses meal-related glycemic index data derived from a mobile App diary, information on previous meals, EHR and patient behavioural questionnaires. This study shows a significant improvement in prediction accuracy compared to their earlier study. Authors reported the best performance model for the prediction of the incremental area under the blood glucose curve two hours after food intake had the following characteristics: R = 0.631, MAE = 0.373 mmol/L*h for the model not using data on current blood glucose; R = 0.644, MAE = 0.371 mmol/L*h for the model using data on the current blood glucose levels; and R = 0.704, MAE = 0.341 mmol/L*h for the model utilizing data on the continuous blood glucose trends before the meal. Based on Shapley additive explanations method, feature ranking results suggested the meal glycemic load, amount of carbohydrates in the meal, type of meal (e.g., breakfast), amount of starch and amount of food consumed 6 hours before the current meal were the most important contributors in the models.</p>", "<title>Beyond: Continuous Blood Glucose Prediction Methods in Type 1 and Type 2 Diabetes</title>", "<p id=\"P75\">Thanks to the development of CGM in recent years, there are a significant number of studies in the field of blood glucose prediction using continuous blood glucose measurements, especially for patients with type 1 diabetes. Whilst this paper focuses on machine learning algorithms for GDM management, CGM research to date has mostly focused on type 1 diabetes. Thus we present a review of continuous blood glucose prediction for type 1 and type 2 diabetes that could potentially be adapted for use in GDM patients. Three major machine learning model architectures, including the deep convolutional neural network model (##FIG##6##Fig. 7##), time-series recurrent neural network (RNN) model (##FIG##7##Fig. 8##), and reinforcement learning (##FIG##8##Fig. 9##) architectures are shown to demonstrate machine learning pipelines and their hyperparameters in model designs.</p>", "<p id=\"P76\">Refiman [##REF##19885110##77##] proposed the autoregressive models to (i) explore the correlations in time-series glucose data and (ii) make blood glucose predictions. Results based on nine type 1 diabetic subjects collected over a continuous 5-day period indicated that, for a 30-minute prediction horizon, individually tuned models yielded 97.6 to 100.0% of data in the clinically acceptable zones A and B. In contrast, cross-subject, portable models yielded 95.8 to 99.7% of data in zones A and B. Due to the small number of patients in this study, the accuracy of the autoregressive model needs to be evaluated in a larger patient cohort.</p>", "<p id=\"P77\">Mhaskar et al. [##UREF##39##81##] developed a convolutional neural network (CNN) model to identify the trends of hypoglycemic (0–70 mg/dL), euglycemic (70–180 mg/dL), or hyperglycemic (180–450 mg/dL) based on the 5-min blood glucose prediction. A “judge” network is then used to determine a final prediction based on the outputs of the prediction results for hypoglycemic, euglycemic and hyperglycemic conditions. Methods are evaluated on 25 type 1 diabetes patients’ 160 BG time series data, taken at 5-minute intervals. Diffusion geometry is used to train the networks in a manner analogous to manifold learning. Based on 50% of the training data, this model correctly predicted 96.43% in the hypoglycemic range, 97.96% in the euglycemic range, and 85.29% in the hyperglycemic range.</p>", "<p id=\"P78\">Zhu [##REF##30946685##86##] used casual dilated CNN layers and WaveNet algorithms to forecast the future glucose levels of patients with type 1 diabetes. The output from the previous layer is the input of the subsequent dilated convolutional layer. The process is repeated until obtaining the final output layer. Then the output is fed into a 1 × 1 convolutional layer followed by the Softmax layer. The model was evaluated on OhioT1DM dataset (6 adolescent subjects) and achieved an RSME of 21.73±2.52 md/dl.</p>", "<p id=\"P79\">As shown in ##FIG##6##Fig. 7##, Li [##REF##31369390##79##] demonstrates a more complex deep neural network (DNN) architecture called GluNet. This model consists of four parts: pre-processing, DNN, post-processing, and label transformation and recovery. The input data exemplars are CGM time-series measurements <italic toggle=\"yes\">G</italic>, insulin <italic toggle=\"yes\">I</italic> and meal <italic toggle=\"yes\">M,</italic> ; other input factors are optional. As shown in ##FIG##6##Fig. 7##, there are five processes in the preprocessing for data representation: P1 rules out outliers in <italic toggle=\"yes\">G, I, M</italic>; P2 interpolates <italic toggle=\"yes\">G</italic> when the missing data gap is not large; P3 fills or estimates the missing data in <italic toggle=\"yes\">I</italic> and <italic toggle=\"yes\">M</italic>; P4 calculates other factors that should be included as input to the DNN, for instance, plasma insulin estimation <italic toggle=\"yes\">Pi</italic> and glucose rate of appearance <italic toggle=\"yes\">Ra</italic>; and P5 aligns all factors with the same timeline and use them as input to the DNN. The aligned BG time series <italic toggle=\"yes\">Gt</italic> is also sent to the label transform, and quantised <italic toggle=\"yes\">Gt</italic> is used as the category target in training.</p>", "<p id=\"P80\">##FIG##7##Fig. 8## demonstrates an RNN architecture developed by Beauchamp [##REF##34068808##85##] using long short-term memory (LSTM) and a deep residual network for type 1 diabetes management with CGM. The grey star represents the bolus at time t + 10. For the bolus recommendation scenario, the events outlined in red or orange are not allowed in inertial examples. This model was evaluated on the OhioT1DM dataset (12 adolescent subjects) with RSME=13.76.</p>", "<p id=\"P81\">In addition to supervised and unsupervised machine learning, reinforcement learning is another branch of machine learning methods used for blood glucose prediction. As shown in ##FIG##8##Fig. 9##, Zhu [##REF##32899979##87##] proposed a novel insulin bolus advisor which uses deep reinforcement learning and continuous glucose monitoring to optimize insulin dosing at mealtime. In particular, an actor-critic model based on a deep deterministic policy gradient is designed to compute mealtime insulin doses. The proposed system architecture uses a two-step learning framework, in which a population model is first obtained and then personalized by subject-specific data. Prioritized memory replay is adopted to accelerate the training process in clinical practice. To evaluate the algorithm, an FDA-approved UVA / Padova T1 diabetes simulator was used to perform an in-silico trial on ten adult subjects and ten adolescent subjects. Compared to a standard bolus calculator, the deep reinforcement learning insulin bolus advisor improved the average percentage time in the target range (70–180 mg/dL) from 74.1%±8.4% to 80.9%±6.9% (p&lt;0.01) and 54.9%±12.4% to61.6%±14.1% (p&lt;0.01) in the adult and adolescent cohorts, respectively.</p>", "<p id=\"P82\">Goldner’s [##REF##28851713##107##] study describes a machine learning method to predict projected blood glucose using 1,923,416 BG measurements from 14,706 people with noninsulin-treated T2 diabetes collected from the One Drop mobile app. Contextual information (CI) on health metrics, including weight and A1c, are included in the demographics. Inputs to each BG prediction included a prior BG and available CI. The model did not distinguish whether BGs with similar CIs were from the same or different users. Forecast horizons were set by the time since the prior BG and varied from 10 minutes to several days. Machine learning methods were not specified in the paper. The median and mean absolute error of holdout predictions were 14.2 and 21.3 mg/dL, respectively, with 91% of predictions within +/-50 mg/dL. Maternal hyperglycemia during pregnancy and delivery is associated with neonatal hypoglycemia and fetal distress. Frequent glucose monitoring is essential to reduce the risk of severe hypoglycemia. Women with well-controlled diabetes and within-range fetal testing may be managed expectantly between 39 and 40 weeks of gestation. However, women with diabetes-related complications, poor glycemic control, or prior stillbirth should be considered for delivery between 36 and 38 weeks of gestation. Readers can find more information on the predictive methods used for hypoglycemia in patients with type 1 diabetes in the review paper [##REF##33466659##108##].</p>", "<title>Medication and Pregnancy Outcome Management</title>", "<p id=\"P83\">Due to the individual variability and complex glucose dynamics, optimizing the doses of insulin delivery to minimize the risk of hyperglycemia and hypoglycemia is still a challenge in both CGM and intermittent fingerstick glucose monitoring.</p>", "<p id=\"P84\">Velardo et al. [##REF##33688832##94##] used machine learning models to identify when a woman with GDM needs to switch to from dietary control to medications (insulin or metformin). Through the analysis of 411,785 blood glucose measurements of 3029 patients, a logistic regression model that can predict the timing of initiation of pharmacological treatment was developed. The authors repeated this experiment on 100 different random permutations of the main dataset between training and validation data using a 70% training and 30% validation split. At each iteration, to avoid biasing the algorithm toward the overrepresented class (diet–diet), this was randomly downsampled to the number of women in the underrepresented class (diet–drug). The lasso function was used with its alpha parameter set to.75 (corresponding to elasticnet regression) and 5-fold cross-validation. After 100 experimental repetitions, they obtained an average area under the receiver operating characteristic curve of 0.80 (SD 0.02) and an algorithm that allows the flexibility of setting the operating point rather than relying on a static heuristic method, which is currently used in clinical practice.</p>", "<p id=\"P85\">Due to the higher levels of blood glucose in mothers with GDM, offspring will have a higher risk of large-for-gestational-age (LGA) and hyperglycaemia. Using data from a large multi-centre cohort, Gibbons et al [##UREF##44##92##] created a risk prediction model for LGA infants using logistic regression and naïve Bayes models. Models were developed combining the risks of hyperglycaemia (assessed in three forms: IADPSG GDM yes/no, GDM subtype, OGTT z score quintiles), demographic and clinical variables as potential predictors. Using data from the Hyperglycaemia and Adverse Pregnancy Outcome (HAPO) study [##UREF##22##47##], authors compared the predictive ability and stability between the models. The two approaches resulted in similar estimates of LGA risk.</p>", "<p id=\"P86\">In addition to a higher risk of having LGA offspring, excessive gestational weight gain is also associated with poorer pregnancy outcomes [##REF##32822601##6##]. Lu et al. [##REF##35340404##93##] developed machine learning models on 97 patients with GDM to demonstrate a proof-of-the-concept work of caesarean section prediction and to explore the role of temporal blood glucose in predicting caesarean birth. Logistic regression, SVM and Boosting trees were used in model development. The Logistic regression model with Lasso regulator achieved an AUROC of 0.857 ± 0.008. The study also suggested that temporal blood glucose measurements may improve the prediction subject to further validation.</p>", "<p id=\"P87\">SineDie is a smartphone application with AI that was used during the COVID-19 pandemic [##UREF##51##109##]. Authors of this paper suggested that it can provide hyperglycaemia prediction and therapy planning, classify and analyse ketonuria, diet transgressions, and blood glucose values, and make recommendations regarding diet or insulin treatment. It automatically prescribed diet therapy modifications, identified the need for insulin treatment and proposed insulin dose changes to doctors. Publication [##REF##28495347##110##] showed that the Expectation Maximization clustering algorithm is first used to group BG measurements in three meal tags: breakfast, lunch, or dinner. Then decision tree is firstly applied to assign each reading into five mealtime tags: \"breakfast preprandial\",\"breakfast postprandial\", \"lunch postprandial\",\"dinner postprandial\", or \"other\", which are then used to classify patients with hyperglycaemia. A randomized clinical trial of 25 GDM patients showed an 88.6% reduction in face-to-face visits and a 27.4% reduction in the time devoted by clinicians to patients’ evaluations. Taking height, weight, and age into account can help advise the patient's initial diet therapy and suggest the total calorie intake distributed in carbohydrate units throughout the day. To calculate the total calorie intake, the authors used the Harris-Benedict equation [##REF##16576330##111##]. This system did not use activity factors or impose any calorie restrictions for obese women. The endocrinologist can modify the personalized diet prescription for each patient. Authors suggested the SineDie system detected all situations requiring therapy adjustment, generating safe recommendations without providing methods used in the decision-making system.</p>" ]
[ "<title>Acknowledgement</title>", "<p>Huiqi Lu is supported by the Royal Academy of Engineering Daphne Jackson Trust Fellowship grant, an EPSRC Healthcare Technologies Challenge Award (EP/N020774/1), and an Oxford John Fell Fund (0011028). Xiaorong Ding is supported by Sichuan Science and Technology Program (Grant No. 2021YFH0179). Jane Hirst is supported by the UKRI Future Leaders Fellowship grant. Jenny Yang is a Marie Sklodowska-Curie Fellow, under the European Union’s Horizon 2020 research and innovation programme (Grant agreement: 955681, “MOIRA”). This work was supported in part by InnoHK Project Programme 3.2: Human Intelligence and AI Integration (HIAI) for the Prediction and Intervention of CVDs: Warning System at Hong Kong Centre for Cerebro-cardiovascular Health Engineering (COCHE). The views expressed are those of the authors and not necessarily those of InnoHK. This research was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.</p>" ]
[ "<fig position=\"float\" id=\"F1\"><label>Fig. 1</label><caption><title>Digital health for antenatal and postnatal health and care in hospital, community and home care environments</title></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Fig. 2</label><caption><title>Self-monitoring glucose meters</title><p>(a) fingertip blood glucose testing with mobile connections [##UREF##7##20##, ##UREF##8##21##], (b) four generations of blood glucose meters (c. 1987-2005): Top left: Reflolux S (Accu-Chek III in the U.S.), by Boehringer Mannheim, 2-minute read time, based on reflectance; top right: ExacTech Card, by MediSense, 30-second read time, electrochemical test stripe; bottom left: FreeStyle, by TheraSense, 15-second read time, electrochemical test stripe; bottom right: Freestyle Mini, by Abbott, 7-second test time, electrochemical test stripe. [##UREF##9##22##].</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Fig. 3</label><caption><title>Noninvasive enzyme-based glucose monitoring sensing systems through different contact agents and body sensors</title><p>(a) contact lens glucose sensor on tear, (b) saliva glucose monitoring strip on saliva, (3) needle-type glucose sensor on insulin sensitivity factor (ISF), and (d) wearable glucose monitoring patch on sweat. [##UREF##14##33##]</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Fig. 4</label><caption><title>Noninvasive continuous glucose sensing techniques</title><p>(a) skin-like glucose biosensor [##UREF##19##41##], (b) wearable-band type near infrared (NIR) optical biosensor [##UREF##20##42##], (c) sensing through fluorescent labelling [##UREF##18##38##], and (d) microwave sensors [##REF##32577523##40##].</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Fig. 5</label><caption><title>Taxonomy of models for blood glucose prediction.</title></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Fig. 6</label><caption><title>An example of a hybrid approach using the physiological model and data-driven model [##UREF##38##76##]</title></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Fig. 7</label><caption><title>The architecture of GluNet [##REF##31369390##79##].</title></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Fig. 8</label><caption><title>LSTM time-series prediction model with deep residual network [##REF##34068808##85##]</title></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Fig. 9</label><caption><title>The block diagram of the proposed DRF model with the actor-critic architecture, reproduced without changes from [##REF##32899979##87##]</title></caption></fig>" ]
[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><title>Patient monitoring technologies in GDM clinical studies: primary purpose, outcome measures, monitoring methods, country of use, and reviewer comments</title></caption><table frame=\"hsides\" rules=\"rows\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Application/First Authors (Year) [reference]</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Study title</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Location, Phase (study size)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Outcome Measures</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Study type: Primary purpose</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Treat-GDM</bold> (2013-2016) [##UREF##27##52##, ##UREF##28##53##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trial of Remote Evaluation and Treatment of Gestational Diabetes Mellitus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UK, N/A (203)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glycosylated haemoglobin; mean Blood glucose levels for fasting, preprandial and postprandial readings; percentage of ‘on target’ blood glucose readings; effectiveness of monitoring; maternal outcomes; maternal weight gain; birthweight; birth injury; neonatal hypoglycaemia.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioural: Health services research, self home blood glucose monitoring</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>PEARS</bold> (2013-2016) [##REF##26625980##62##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pregnancy, Exercise And nutrition Research study with app support</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ireland, N/A (750)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Effectiveness of a smartphone-assisted targeted healthy lifestyle intervention; support antenatal management of overweight and obese pregnant population in preventing GDM.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Hola Bebe</bold> (2014-2017)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mobile Health App to Reduce Diabetes in Latina Women</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, Phase 1 (18)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Self-efficacy for healthy eating; physical activity; body weight.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioural: Prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>MobiGuide</bold> (2014) [##UREF##30##55##, ##REF##24876573##56##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Group and Mobile Care for Gestational Diabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spain, N/A (22)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Patient data automatically or manually entered in App; decision support system generates feedback to patients and clinicians based on clinical guidelines.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Pregnant</bold>+ (2015-2017) [##UREF##31##58##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A Mobile Smartphone Application to Promote a Healthy Diet and Physical Activity Among Pregnant Women With GDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Norway, N/A (238)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OGGT; dietary intake; motivation for eating healthy; physical activity; motivation for physical activity; depression; complications of pregnancy; mode of delivery and complications at birth for the mother; complications for the newborn.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: Prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Pregnant</bold>+ (2019-2022)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Effects of Dietary and Weight Management on Pregnancy Outcomes in Mobile Medical Platform</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China, N/A (2000)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Incidence of gestational diabetes mellitus, gestational hypertension, caesarean section and premature birth.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioural: Intensive dietary; weight management; standard care</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Pregnant</bold>+ (2021-2022)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Development and Testing of a Mobile Health Application for Management of Gestational Diabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nepal, N/A (60)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal blood glucose levels at 6 weeks postpartum; neonatal birth weight; induction of labour; caesarean delivery; self-monitoring adherence; usability of telemonitoring; app acceptability and usability.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioural: Supportive care.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>University of Colorado Hospital</bold> (2015-2021)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">User Testing and Feedback for a Mobile Health Program for Postpartum Women: A Pilot Study</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, N/A (200)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Usability of the application; engagement with the application; navigability of the application; acceptability of the application</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Observational</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Peking Union Medical College Hospital</bold> (2016-2020)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">the Effect of Mobile Medical Used for the Standardized Management of Gestational Diabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China, N/A (400)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glycaemic qualification rate; pregnancy outcome.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioural: Prevention, intensive dietary, weight management and standard care</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>iHealth</bold> (2017-2018)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Evaluating the Feasibility of Using M-Health to Improve Serum Glucose Logs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, N/A (8)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glucose log completeness; patient satisfaction.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>FAB</bold> (2017-2020)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fit After Baby: Increasing Postpartum Weight Loss in Women at Increased Risk for Cardiometabolic Disease</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, N/A (34)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Changes in weight loss, postpartum weight retention, waist circumference fasting insulin, fasting glucose HbA1c; adherence to self-monitoring; Satisfaction; use of App; number of interactions with lifestyle coaches</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: Prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>LIVING</bold> (2017-2021)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lifestyle Intervention IN Gestational Diabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bangladesh and India, N/A (1612)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Change of glycemic category; changes in fasting blood glucose; change in body weight; change in waist circumference; change in systolic blood pressure; change in physical activity level; change in dietary habits.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: Prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>SweetMama</bold> (2017-2021) [##UREF##50##104##, ##REF##32627582##105##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SweetMama: Testing of a Novel Technology for Diabetes Education and Support to Pregnant Women</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, N/A (80)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Focus group and individual user feedback; feasibility; user interactivity data; usability testing; diabetes self-efficacy; patient activation; difference in HbA1C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: Supportive care</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>GlucoseMama</bold> (2018-2019)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Group and Mobile Care for Gestational Diabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, N/A (22)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduction in the number of pharmacotherapy for treatment, neonates born large, and infants with neonatal hypoglycemia; increased number of screened in the postpartum period for type 2 diabetes.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: supportive care</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>eMoMs</bold> (2019-2022)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Usability Study of the Sensors and eMoM GDM Application</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Finland, N/A (34)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acceptability and usability of sensors; acceptability of prototype app; technical functionality.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Observational</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>MELINDA</bold> (2019-2022) [##REF##32823771##63##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mobile-based Lifestyle Intervention in Women With Glucose Intolerance After Gestational Diabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Belgium, N/A (236)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Metabolic syndrome; insulin resistance Matsuda; insulin resistance HOMA-IR beta-cell function ISSI-2 index; beta-cell function insulinogenic index; beta-cell function HOMA-B; weight loss</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioural: Prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>GUIDES</bold> (2021-2022)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gestational Diabetes in Uganda and India Improving Screening and Self-management</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">India; Uganda, N/A (20000)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDM diagnosis; mean fasting blood glucose; adverse perinatal outcomes (composite measure); HbAlc</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>SMARThealth</bold> (2019-2020) [##REF##34816187##103##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SMARThealth Pregnancy: Feasibility &amp; Acceptability of a Complex Intervention for High-risk Pregnant Women in Rural India</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">India, N/A (258)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruitment rate; retention rate; number of home visits; number of women with GDM; postpartum follow-up; number of hypertensive disorders; number of severe anaemia; mean postpartum haemoglobin; mean postpartum; Systolic and diastolic blood pressure.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: Prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Johns Hopkins Health System and University</bold> (2021-2024)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pragmatic Randomized Clinical Trial to Limit Weight Gain in Pregnancy and Prevent Obesity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">USA, N/A (380)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total gestational weight gain; number of participants who gained excess weight; incidence of GDM; postpartum weight retention; infant weight; proportion of low birth weight infants</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interventional: Health services research</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><title>Top 10 IOS apps ranked seven countries with high GDM prevalence</title></caption><table frame=\"hsides\" rules=\"rows\"><thead><tr><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Countries</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Medical (M)</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Health &amp; Fitness (HF)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food &amp; Drink (FD)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">lifestyle (LS)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>SGP</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L1\"><list-item><p>Glucose tracker ++</p></list-item><list-item><p>MySweetGestation</p></list-item><list-item><p>gluQUO: Control your Diabetes</p></list-item><list-item><p>Pregnant with diabetes</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L2\"><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>My Diabetes Diet &amp; Meal Plan</p></list-item><list-item><p>Klinio: Diabetic Diet Log</p></list-item><list-item><p>Habits: Gestational Diabetes</p></list-item><list-item><p>Cori – Better Diabetes</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Melinda</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>UAE</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L3\"><list-item><p>FreeStyle LibreLink – AE</p></list-item><list-item><p>Alma Health</p></list-item><list-item><p>Glucose Buddy Diabetes Tracker</p></list-item><list-item><p>Medisage Phill Reminder</p></list-item><list-item><p>MySweetGestation</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L4\"><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>Habits: Gestational Diabetes</p></list-item><list-item><p>Carb Manager: Keto Diat App</p></list-item><list-item><p>Calorie Counting App</p></list-item><list-item><p>Withings Health Mate</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>THAI</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L5\"><list-item><p>DMThai Diary</p></list-item><list-item><p>Medisafe Phill Reminder</p></list-item><list-item><p>Glucose Buddy Diabetes Tracker</p></list-item><list-item><p>mySugr- Diabetes Tracker Log</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L6\"><list-item><p>Habits: Gestational Diabetes</p></list-item><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>Life Fasting Progress Tracker</p></list-item><list-item><p>Withings Health Mate</p></list-item><list-item><p>One Drop Diabetes Management</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">My Diabetic Meal Planner</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>MAL</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L7\"><list-item><p>Glucose Buddy Diabetes Tracker</p></list-item><list-item><p>Medisafe Phill Reminder</p></list-item><list-item><p>Glucose – Blood Sugar racker</p></list-item><list-item><p>Diabetes: M</p></list-item><list-item><p>Pregnant with Diabetes</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L8\"><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>Habits: Gestational Diabetes</p></list-item><list-item><p>Carb Manager: Keto Diet App</p></list-item><list-item><p>Life Fasting Progress Tracker</p></list-item><list-item><p>One Drop Diabetes Management</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>UK</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L9\"><list-item><p>GDm-Health</p></list-item><list-item><p>mySugr- Diabetes Tracker Log</p></list-item><list-item><p>Glucose tracker ++</p></list-item><list-item><p>Hedia -Personal Diabetes App</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L10\"><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>Diabetes Diary</p></list-item><list-item><p>Mumoactive Diabetes</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L11\"><list-item><p>Diabetes Recipe App</p></list-item><list-item><p>Diabetes Diet FREE</p><list list-type=\"simple\" id=\"L12\"><list-item><label>–</label><p>Proper Nutrition for the Diabetic</p></list-item></list></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>BEL</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L13\"><list-item><p>One Touch Reveal</p></list-item><list-item><p>FreeStyle LibreLink – BE</p></list-item><list-item><p>mySugr-DiabetesTracker</p></list-item><list-item><p>LogMySweetGestation</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L14\"><list-item><p>Habits: Gestational Diabetes</p></list-item><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>Withings Health Mate</p></list-item><list-item><p>Pacer Pedometer &amp; Step Tracker</p></list-item><list-item><p>Carb Manager: Keto Diat App</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>IND</bold>\n<xref rid=\"TFN1\" ref-type=\"table-fn\">*</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><list list-type=\"order\" id=\"L15\"><list-item><p>mySugr- Diabetes Tracker Log</p></list-item><list-item><p>One Touch Reveal</p></list-item><list-item><p>Glucose Buddy Diabetes Tracker</p></list-item><list-item><p>Medisafe Phill Reminder</p></list-item><list-item><p>Glucose – Blood Sugar racker</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<list list-type=\"order\" id=\"L16\"><list-item><p>Habits: Gestational Diabetes</p></list-item><list-item><p>myGestationalDiabetes</p></list-item><list-item><p>BeatO Biabetes Management</p></list-item><list-item><p>Withings Health Mate</p></list-item><list-item><p>One Drop Diabetes Management</p></list-item></list>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><title>Features of different diabetes management apps</title></caption><table frame=\"hsides\" rules=\"rows\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">App Name and Type</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glucose Monitoring with CGM</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exercise Response</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food Tracking</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Medication Reminders</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interaction with Doctors</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>mySugr- Diabetes Tracker Log (M)</bold> [##REF##31169427##67##]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Diabetes: M (M)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Glucose Buddy Diabetes Tracker (M)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Medisage Phill Reminder (M)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Habits: Gestational Diabetes (HF)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Carb Manager: Keto Diet App (HF)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Withings Health Mate (HF)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>One Drop Diabetes Management (HF)</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><title>Machine learning models used in gestational diabetes and type 1 and type 2 diabetes studies</title></caption><table frame=\"hsides\" rules=\"rows\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"2\" colspan=\"1\">Aim</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Model</th><th valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">Previously Used For:</th><th valign=\"top\" align=\"left\" rowspan=\"2\" colspan=\"1\">Publication</th></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Type 1 <xref rid=\"TFN2\" ref-type=\"table-fn\">*</xref></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Type 2 <xref rid=\"TFN2\" ref-type=\"table-fn\">*</xref></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDM</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"6\" colspan=\"1\">\n<bold>Early Diagnosis of GDM including biomarker detection</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Boosting Models</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##34##69##–##UREF##36##71##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Random Forest</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##36##71##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Generalized Linear Model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##36##71##, ##REF##31539877##72##, ##UREF##41##84##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">K-Nearest Neighbour</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##36##71##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bayesian, include Naïve Bayes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##36##71##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Support Vector Machine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##36##71##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"4\" colspan=\"1\">\n<bold>Hypoglycemia Prediction</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Random forest</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##25316712##73##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Support Vector Machine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##25316712##73##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">k-nearest neighbour</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##25316712##73##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bayesian, include Naïve Bayes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##25316712##73##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"11\" colspan=\"1\">\n<bold>Continuous Blood Glucose Prediction</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Linear regression with Lasso</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##29317385##74##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Boosting Model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##37##75##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Physiological models</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##38##76##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Autoregressive Model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##19885110##77##–##REF##31369390##79##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bayesian, include Naïve Bayes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##32009223##80##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Deep Neural Network</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##31369390##79##, ##UREF##39##81##, ##UREF##40##83##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Convolutional Neural Network</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##32091990##78##, ##REF##31369390##79##, ##REF##34068808##85##, ##REF##30946685##86##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforcement Learning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##32899979##87##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Long Short Term Memory</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##32091990##78##, ##REF##34068808##85##, ##UREF##42##88##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Support Vector Regressor</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##31369390##79##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Transfer Learning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##43##89##–##REF##35415440##91##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"2\" colspan=\"1\">\n<bold>Pregnancy Outcome Prediction</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Logistic Regression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##44##92##, ##REF##35340404##93##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bayesian, include Naïve Bayes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##UREF##44##92##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Boosting Models</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35340404##93##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tree-based Models</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35340404##93##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Support Vector Machine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35340404##93##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"3\" colspan=\"1\">\n<bold>Medication and Clinical Review Management</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Logistic Regression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##36104698##82##, ##REF##33688832##94##, ##REF##35808300##95##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Random Forest</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##36104698##82##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Boosting Models</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##36104698##82##, ##REF##35808300##95##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"4\" colspan=\"1\">\n<bold>Postnatal Type 2 Diabetes Prediction</bold>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Logistic Regression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35788016##106##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Support Vector Machine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35788016##106##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Boosting Models</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35788016##106##]</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neural Network</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">✓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[##REF##35788016##106##]</td></tr></tbody></table></table-wrap>" ]
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[ "<fn-group><fn id=\"FN1\" fn-type=\"COI-statement\"><p id=\"P96\">All conflicts of interest: LM is supported by the NIHR Oxford Biomedical Research Centre and is a part-time employee of EMIS plc.</p></fn></fn-group>", "<table-wrap-foot><fn id=\"TFN1\"><label>*</label><p id=\"P97\">SGP: Singapore, UAE: United Arab Emirates, THAI: Thailand, MAL: Malaysia, UK: United Kingdom, BEL: Belgium, IND: India</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"TFN2\"><label>*</label><p id=\"P98\">Type 1 and type 2 applications were only used when there were limited applications in gestational diabetes</p></fn></table-wrap-foot>" ]
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[{"label": ["[4]"], "person-group": ["\n"], "surname": ["Martis R"], "given-names": ["CC"], "article-title": ["Treatments for women with gestational diabetes mellitus: an overview of Cochrane systematic reviews"], "source": ["Cochrane Db Syst Rev"], "year": ["2018"], "comment": ["REVIEW no. 8"]}, {"label": ["[5]"], "source": ["International Diabetes Federation \"IDF diabetes altas\""], "comment": ["[Online] Avialable: "], "ext-link": ["https://diabetesatlas.org/data"]}, {"label": ["[13]"], "collab": ["NICE"], "source": ["Diabetes in pregnancy: management from preconception to the postnatal period"], "year": ["2020"], "comment": ["[Online]. Available: "], "ext-link": ["https://www.nice.org.uk/guidance/ng3/chapter/1-recommendations#antenatal-care-for-women-with-diabetes-2"]}, {"label": ["[14]"], "collab": ["NICE"], "source": ["Diabetes in pregnancy overview"], "year": ["2018"], "comment": ["[Online]. Available: "], "ext-link": ["http://pathways.nice.org.uk/pathways/diabetes-in-pregnancy"]}, {"label": ["[15]"], "collab": ["NICE"], "source": ["Gestational Diabetes: Risk assessment, testing, diagnosis and management"], "year": ["2018"], "comment": ["[Online]. Available: "], "ext-link": ["http://pathways.nice.org.uk/pathways/diabetes-in-pregnancy"]}, {"label": ["[17]"], "person-group": ["\n"], "surname": ["Bhatia"], "given-names": ["M"], "article-title": ["Clinical implications of the NICE 2015 criteria for gestational diabetes mellitus"], "source": ["J Clin Med"], "year": ["2018"], "month": ["Oct"], "volume": ["7"], "issue": ["10"]}, {"label": ["[19]"], "source": ["What is digital health"], "year": ["2020"], "comment": ["[Online]. Available: "], "ext-link": ["https://www.fda.gov/medical-devices/digital-health-center-excellence"]}, {"label": ["[20]"], "person-group": ["\n"], "surname": ["Loerup"], "given-names": ["L"], "article-title": ["GDm-Health: a pilot study examining acceptability of mobile phone assisted remote blood glucose monitoring for women with gestational diabetes"], "source": ["Diabetic Med"], "year": ["2014"], "month": ["Mar"], "volume": ["31"], "fpage": ["147"]}, {"label": ["[21]"], "source": ["GDm-Health app wins innovation award"], "comment": ["[Online]. Avialable: "], "ext-link": ["https://www.wrh.ox.ac.uk/news/gdm-health-app-wins-innovation-award"]}, {"label": ["[22]"], "source": ["Blood glucose monitoring"], "comment": ["[Online]. Avialable: "], "ext-link": ["https://en.wikipedia.org/wiki/Blood_glucose_monitoring"]}, {"label": ["[25]"], "person-group": ["\n"], "surname": ["Benjamin"], "given-names": ["EM"], "article-title": ["Self-monitoring of blood glucose: The basics"], "source": ["Clinical Diabetes"], "year": ["2002"], "volume": ["20"], "issue": ["1"], "fpage": ["45"], "lpage": ["47"]}, {"label": ["[27]"], "source": ["Self-monitoring blood glucose test systems for over-the-counter use: Guidance for industry and good and drug administration staff"], "comment": ["[Online] Available: "], "ext-link": ["https://www.fda.gov/regulatory-information/search-fda-guidance-documents/self-monitoring-blood-glucose-test-systems-over-counter-use"]}, {"label": ["[30]"], "person-group": ["\n"], "surname": ["Didyuk"], "given-names": ["O"], "article-title": ["Continuous glucose monitoring devices: Past, present, and future focus on the history and evolution of technological innovation"], "source": ["Journal of Diabetes Science and Technology"], "volume": ["0"], "issue": ["0"], "elocation-id": ["1932296819899394"]}, {"label": ["[32]"], "person-group": ["\n"], "surname": ["Thulasi"], "given-names": ["AA"], "article-title": ["Portable impedance measurement device for wweat based glucose detection"], "source": ["Int Conf Wearab Impl"], "year": ["2017"], "fpage": ["63"], "lpage": ["66"]}, {"label": ["[33]"], "person-group": ["\n"], "surname": ["Lee"], "given-names": ["H"], "article-title": ["Enzyme-based glucose sensor: From invasive to wearable device"], "source": ["Adv Healthc Mater"], "year": ["2018"], "month": ["Apr"], "day": ["25"], "volume": ["7"], "issue": ["8"]}, {"label": ["[35]"], "person-group": ["\n"], "surname": ["Huang", "Zhang", "Wu"], "given-names": ["JM", "Y", "J"], "article-title": ["Review of non-invasive continuous glucose monitoring based on impedance spectroscopy"], "source": ["Sensor Actuat a-Phys"], "year": ["2020"], "month": ["Aug"], "day": ["15"], "volume": ["311"]}, {"label": ["[36]"], "person-group": ["\n"], "surname": ["van Enter", "von Hauff"], "given-names": ["BJ", "E"], "article-title": ["Challenges and perspectives in continuous glucose monitoring"], "source": ["Chem Commun"], "year": ["2018"], "month": ["May"], "day": ["18"], "volume": ["54"], "issue": ["40"], "fpage": ["5032"], "lpage": ["5045"]}, {"label": ["[37]"], "person-group": ["\n"], "surname": ["Siddiqui"], "given-names": ["SA"], "article-title": ["Pain-free blood glucose monitoring using wearable sensors: Recent advancements and future prospects"], "source": ["IEEE reviews in biomedical engineering. Research Support, Non-U.S. Gov\u2019t; Review"], "year": ["2018"], "volume": ["11"], "fpage": ["21"], "lpage": ["35"]}, {"label": ["[38]"], "person-group": ["\n"], "surname": ["Jernelv"], "given-names": ["IL"], "article-title": ["A review of optical methods for continuous glucose monitoring"], "source": ["Appl Spectrosc Rev, Review"], "year": ["2019"], "month": ["Aug"], "volume": ["54"], "issue": ["7"], "fpage": ["543"], "lpage": ["572"]}, {"label": ["[41]"], "person-group": ["\n"], "surname": ["Chen"], "given-names": ["YH"], "article-title": ["Skin-like biosensor system via electrochemical channels for noninvasive blood glucose monitoring"], "source": ["Science Advances"], "year": ["2017"], "month": ["Dec"], "volume": ["3"], "issue": ["12"]}, {"label": ["[42]"], "person-group": ["\n"], "surname": ["Rachim", "Chung"], "given-names": ["VP", "WY"], "article-title": ["Wearable-band type visible-near infrared optical biosensor for non-invasive blood glucose monitoring"], "source": ["Sensor Actuat B-Chem"], "year": ["2019"], "month": ["May"], "day": ["1"], "volume": ["286"], "fpage": ["173"], "lpage": ["180"]}, {"label": ["[44]"], "person-group": ["\n"], "surname": ["Raman"], "given-names": ["P"], "article-title": ["Different methods and settings for glucose monitoring for gestational diabetes during pregnancy"], "source": ["Cochrane Db Syst Rev"], "year": ["2017"], "issue": ["10"]}, {"label": ["[47]"], "person-group": ["\n"], "surname": ["Freathy"], "given-names": ["RM"], "article-title": ["Hyperglycemia and adverse pregnancy outcome (HAPO) study"], "source": ["Diabetes"], "year": ["2010"]}, {"label": ["[48]"], "person-group": ["\n"], "surname": ["Nguyen"], "given-names": ["M"], "article-title": ["Systematic evaluation of Canadian diabetes smartphone applications for people with type 1, type 2 and gestational diabetes"], "source": ["Can J Diabetes"], "year": ["2021"], "month": ["Mar"], "volume": ["45"], "issue": ["2"]}, {"label": ["[49]"], "person-group": ["\n"], "surname": ["Ding"], "given-names": ["GF"], "article-title": ["Evaluation of continuous glucose monitoring (CGM) on gestational diabetes mellitus in China"], "source": ["Diabetes"], "year": ["2012"], "month": ["Jun"], "day": ["1"], "volume": ["61"], "fpage": ["A588"]}, {"label": ["[50]"], "collab": ["NICE"], "source": ["Health App: GDm-Health for people with gestational diabetes"], "volume": ["131"], "comment": ["[Online] Available: "], "ext-link": ["https://www.nice.org.uk/advice/mib"]}, {"label": ["[51]"], "person-group": ["\n"], "surname": ["Hirst"], "given-names": ["JE"], "article-title": ["Preventing childhood obesity starts during pregnancy"], "source": ["Lancet"], "year": ["2015"], "month": ["Sep"], "day": ["12"], "volume": ["386"], "issue": ["9998"], "fpage": ["1039"], "lpage": ["40"]}, {"label": ["[52]"], "person-group": ["\n"], "surname": ["Hirst"], "given-names": ["JE"], "article-title": ["GDm-health: development of a real-time smartphone solution for the management of women with gestational diabetes mellitus (GDM)"], "source": ["Bjog-Int J Obstet Gy"], "year": ["2015"], "month": ["Apr"], "volume": ["122"], "fpage": ["403"]}, {"label": ["[53]"], "person-group": ["\n"], "surname": ["Loerup"], "given-names": ["L"], "article-title": ["A comparison of blood glucose metrics to assess the feasibility of a digital health system for management of women with gestational diabetes: the GDm-Health study"], "source": ["Diabetic Med"], "year": ["2015"], "month": ["Mar"], "volume": ["32"], "fpage": ["18"], "lpage": ["19"]}, {"label": ["[54]"], "person-group": ["\n"], "surname": ["Dyson"], "given-names": ["PA"], "article-title": ["GDm-Health Plus: Development of a remote behavioural lifestyle management system for women with gestational diabetes"], "source": ["Diabetic Med"], "year": ["2018"], "month": ["Mar"], "volume": ["35"], "fpage": ["171"]}, {"label": ["[55]"], "person-group": ["\n"], "surname": ["Peleg"], "given-names": ["M"], "article-title": ["MobiGuide: a personalized and patient-centric decision-support system and its evaluation in the atrial fibrillation and gestational diabetes domains"], "source": ["User Model User-Adap"], "year": ["2017"], "month": ["Jun"], "volume": ["27"], "issue": ["2"], "fpage": ["159"], "lpage": ["213"]}, {"label": ["[58]"], "person-group": ["\n"], "surname": ["Borgen"], "given-names": ["I"], "article-title": ["Smartphone application for women with gestational diabetes mellitus: a study protocol for a multicentre randomised controlled trial"], "source": ["Bmj Open"], "year": ["2017"], "month": ["Mar"], "volume": ["7"], "issue": ["3"]}, {"label": ["[59]"], "person-group": ["\n"], "surname": ["Borgen"], "given-names": ["I"], "article-title": ["Effect of the pregnant plus smartphone application in women with gestational diabetes mellitus: a randomised controlled trial in Norway"], "source": ["Bmj Open"], "year": ["2019"], "month": ["Nov"], "volume": ["9"], "issue": ["11"]}, {"label": ["[61]"], "person-group": ["\n"], "surname": ["Ainscough"], "given-names": ["KM"], "article-title": ["Nutrition, behavior change and physical activity outcomes from the PEARS RCT-An mHealth-supported, lifestyle intervention among pregnant women with overweight and obesity"], "source": ["Front Endocrinol)"], "year": ["2019"], "volume": ["10"], "fpage": ["938"]}, {"label": ["[69]"], "person-group": ["\n"], "surname": ["Liu"], "given-names": ["HW"], "article-title": ["Machine learning risk score for prediction of gestational diabetes in early pregnancy in Tianjin, China"], "source": ["Diabetes-Metab Res"], "year": ["2021"], "month": ["Jul"], "volume": ["37"], "issue": ["5"]}, {"label": ["[70]"], "person-group": ["\n"], "surname": ["Artzi"], "given-names": ["NS"], "article-title": ["Prediction of gestational diabetes based on nationwide electronic health records"], "source": ["Nature Medicine"], "year": ["2020"], "volume": ["26"], "issue": ["1"], "fpage": ["71"], "lpage": ["76"]}, {"label": ["[71]"], "person-group": ["\n"], "surname": ["Shen"], "given-names": ["JY"], "article-title": ["An innovative artificial intelligence-based App for the diagnosis of gestational diabetes mellitus (GDM-AI): development study"], "source": ["Journal of Medical Internet Research"], "year": ["2020"], "month": ["Sep"], "day": ["15"], "volume": ["22"], "issue": ["9"]}, {"label": ["[75]"], "person-group": ["\n"], "surname": ["Pustozerov"], "given-names": ["EA"], "article-title": ["Machine learning approach for postprandial blood glucose prediction in gestational diabetes mellitus"], "source": ["Ieee Access"], "year": ["2020"], "volume": ["8"], "fpage": ["219308"], "lpage": ["219321"]}, {"label": ["[76]"], "person-group": ["\n"], "surname": ["Contreras"], "given-names": ["I"], "article-title": ["Personalized blood glucose prediction: A hybrid approach using grammatical evolution and physiological models"], "source": ["PLoS ONE"], "year": ["2017"], "volume": ["12"]}, {"label": ["[81]"], "person-group": ["\n"], "surname": ["Mhaskar", "Pereverzyev", "Van Der Walt"], "given-names": ["HN", "SV", "MD"], "article-title": ["A deep learning approach to diabetic blood glucose prediction"], "source": ["Frontiers in Applied Mathematics and Statistics"], "year": ["2017"], "volume": ["3"]}, {"label": ["[83]"], "person-group": ["\n"], "surname": ["Faruqui"], "given-names": ["SHA"], "article-title": ["Development of a deep learning model for dynamic forecasting of blood glucose level for type 2 diabetes mellitus: Secondary analysis of a randomized controlled trial"], "source": ["JMIR mHealth and uHealth"], "year": ["2019"], "volume": ["7"]}, {"label": ["[84]"], "person-group": ["\n"], "surname": ["Muche", "Olayemi", "Gete"], "given-names": ["AA", "OO", "YK"], "article-title": ["Predictors of postpartum glucose intolerance in women with gestational diabetes mellitus: a prospective cohort study in Ethiopia based on the updated diagnostic criteria"], "source": ["BMJ Open"], "year": ["2020"], "volume": ["10"], "elocation-id": ["e036882"]}, {"label": ["[88]"], "person-group": ["\n"], "surname": ["Aliberti"], "given-names": ["A"], "article-title": ["A multi-patient data-driven approach to blood glucose prediction"], "source": ["IEEE Access"], "year": ["2019"], "volume": ["7"], "fpage": ["69311"], "lpage": ["69325"]}, {"label": ["[89]"], "person-group": ["\n"], "surname": ["Deng"], "given-names": ["Y"], "article-title": ["Deep transfer learning and data augmentation improve glucose levels prediction in type 2 diabetes patients"], "source": ["NPJ Digital Medicine"], "year": ["2021"], "volume": ["4"]}, {"label": ["[92]"], "person-group": ["\n"], "surname": ["Gibbons"], "given-names": ["KS"], "article-title": ["Prediction of large-for-gestational age infants in relation to hyperglycemia in pregnancy \u2013 A comparison of statistical models"], "source": ["Diabetes Res Clin Pr"], "year": ["2021"], "volume": ["178"], "elocation-id": ["108975"]}, {"label": ["[96]"], "person-group": ["\n"], "surname": ["Murphy"], "given-names": ["KP"], "source": ["Machine learning: A probabilistic perspective"], "publisher-name": ["Massachusetts Institute of Technology"], "year": ["2012"]}, {"label": ["[97]"], "source": ["Artificial Intelligence and Machine Learning in Software as a Medical Device"], "publisher-name": ["U. S. F. a. D. Administration"], "year": ["2021"]}, {"label": ["[98]"], "person-group": ["\n"], "surname": ["Oviedo"], "given-names": ["S"], "article-title": ["A review of personalized blood glucose prediction strategies for T1DM patients"], "source": ["International Journal for Numerical Methods in Biomedical Engineering"], "year": ["2017"], "volume": ["33"], "issue": ["6"], "elocation-id": ["e2833"]}, {"label": ["[100]"], "person-group": ["\n"], "surname": ["Millsaps", "Pohlhausen"], "given-names": ["K", "K"], "article-title": ["A mathematical model for glucose-insulin interaction"], "source": ["Mathematical Biosciences"], "year": ["1975"], "month": ["04"], "day": ["01"], "volume": ["23"], "issue": ["3"], "fpage": ["237"], "lpage": ["251"], "comment": ["1975"]}, {"label": ["[102]"], "person-group": ["\n"], "surname": ["Ryan", "Collins", "Neill"], "given-names": ["C", "J", "MO"], "article-title": ["Grammatical evolution: Evolving programs for an arbitrary language"], "source": ["Springer Berlin Heidelberg"], "year": ["1998"], "fpage": ["83"], "lpage": ["96"]}, {"label": ["[104]"], "person-group": ["\n"], "surname": ["Yee"], "given-names": ["LM"], "article-title": ["SweetMama: Usability testing of a novel mobile application for diabetes education and support during pregnancy"], "source": ["Am J Obstet Gynecol"], "year": ["2020"], "volume": ["222"], "issue": ["1"], "fpage": ["S474"], "lpage": ["S475"]}, {"label": ["[109]"], "person-group": ["\n"], "surname": ["Albert"], "given-names": ["L"], "article-title": ["Managing gestational diabetes mellitus using a smartphone application with artificial intelligence (SineDie) during the COVID-19 pandemic: Much more than just telemedicine"], "source": ["Diabetes Res Clin Pr"], "year": ["2020"], "month": ["Nov"], "volume": ["169"]}, {"label": ["[115]"], "person-group": ["\n"], "surname": ["Hodgkinson"], "given-names": ["JA"], "article-title": ["Is self monitoring of blood pressure in pregnancy safe and effective?"], "source": ["Bmj-Brit Med J"], "year": ["2014"], "month": ["Nov"], "day": ["18"], "volume": ["349"]}, {"label": ["[118]"], "person-group": ["\n"], "surname": ["Li"], "given-names": ["R"], "article-title": ["Graph signal processing, Graph neural network and graph learning on biological data: A systematic review"], "source": ["IEEE Rev Biomed Eng"], "year": ["2022"]}, {"label": ["[119]"], "person-group": ["\n"], "surname": ["Zhou", "Yang", "Shi", "Ma"], "given-names": ["B", "G", "Z", "S"], "article-title": ["Natural language processing for smart healthcare"], "source": ["IEEE Rev Biomed Eng"], "year": ["2022"]}, {"label": ["[120]"], "person-group": ["\n"], "surname": ["Giuste"], "given-names": ["F"], "article-title": ["Explainable artificial intelligence methods in combating pandemics: A systematic review"], "source": ["IEEE Rev Biomed Eng"], "year": ["2022"]}, {"label": ["[121]"], "person-group": ["\n"], "surname": ["Kahankova"], "given-names": ["R"], "article-title": ["Review of recent advances and future developments in fetal phonocardiography"], "source": ["IEEE Rev Biomed Eng"], "year": ["2022"], "month": ["Jun"], "day": ["2"]}, {"label": ["[123]"], "person-group": ["\n"], "surname": ["Chen"], "given-names": ["D"], "article-title": ["Deep learning and alternative learning strategies for retrospective real-world clinical data"], "source": ["npj Dig Med"], "year": ["2019"], "volume": ["2"], "fpage": ["1"], "lpage": ["5"]}, {"label": ["[125]"], "person-group": ["\n"], "surname": ["Choi"], "given-names": ["E"], "article-title": ["RETAIN: An interpretable predictive model for healthcare using reverse rime attention mechanism"], "source": ["Adv Neural Inf Process Syst"], "year": ["2016"], "volume": ["29"], "fpage": ["3504"], "lpage": ["3512"]}, {"label": ["[126]"], "person-group": ["\n"], "surname": ["Oliver"], "given-names": ["C"], "article-title": ["Longitudinal patient stratification of electronic health records with flexible adjustment for clinical outcomes"], "source": ["ML4H"], "year": ["2021"]}, {"label": ["[128]"], "person-group": ["\n"], "surname": ["Rajkomar"], "given-names": ["A"], "article-title": ["Scalable and accurate deep learning with electronic health records"], "source": ["NPJ Digital Med"], "year": ["2018"], "volume": ["1"], "fpage": ["18"]}, {"label": ["[130]"], "person-group": ["\n"], "surname": ["Li"], "given-names": ["L"], "article-title": ["Identification of type 2 diabetes subgroups through topological analysis of patient similarity"], "source": ["Sci Transl Med"], "year": ["2015"], "volume": ["7"], "elocation-id": ["311ra174"]}]
{ "acronym": [], "definition": [] }
130
CC BY
no
2024-01-13 23:49:37
IEEE Rev Biomed Eng. 2023 Feb 7; 17:98-117
oa_package/f2/35/PMC7615520.tar.gz
PMC7819213
33478550
[ "<title>Background</title>", "<p id=\"Par5\">Juvenile offenders in residential care are a population marked by highly elevated rates of trauma, psychopathology and other psychosocial problems [##REF##18664994##1##, ##UREF##0##2##], while child welfare youth often show delinquent behavior in addition to similarly elevated rates of psychosocial treatment needs [##REF##26886603##3##, ##UREF##1##4##]. Furthermore, a substantial number of juveniles involved with the juvenile justice system are so called crossover youth, meaning they have also been involved with child welfare authorities [##UREF##2##5##]. There have thus been political discussions to prioritize treatment needs and rehabilitation of juvenile offenders over punishment and not place them in juvenile correction facilities, but in the most appropriate residential treatment setting (e.g., [##UREF##3##6##]). This would involve sharing resources and institutions with child welfare youth who have been placed in out-of-home care because of, for example, maltreatment or neglect.</p>", "<p id=\"Par6\">The Swiss juvenile justice system has an explicit focus on rehabilitation, education and treatment of delinquent juveniles [##UREF##4##7##]. In general, juvenile delinquency is viewed as a symptom for developmental adjustment problems and juvenile delinquents are viewed as a population in need of protection and guidance more than, and above, punishment alone. As in the adult system, there is a two-pronged approach separating punishment (“<italic>Strafen”</italic>) and interventions based on treatment needs (“<italic>Massnahmen”</italic>). For example, following a delinquent act, the court can order a juvenile to restitution or another punishment and/or, given the personal and social circumstances that might have contributed to the delinquent act (e.g., ongoing lack of parental supervision, mental health issues, developmental problems), an open-ended foster family or residential placement to address these issues [##UREF##4##7##]. This means minors can be placed in child welfare and juvenile justice institutions because of delinquent behavior (juvenile justice measure), child protection reasons (civil law measure, e.g., maltreatment, neglect, or parental absence, psychopathology or drug abuse) or other reasons (e.g., special needs, special education) [##UREF##4##7##, ##UREF##5##8##].</p>", "<p id=\"Par7\">In Switzerland, out-of-home placement of children and juveniles is usually a measure of last resort, after other interventions within the family of origin have failed or a placement is deemed necessary to protect the child’s wellbeing and development [##UREF##4##7##, ##UREF##6##9##]. Children and adolescents are usually placed based on their age, gender and treatment needs and thus, unlike in many other countries, child welfare youth and juvenile delinquents can reside in the same facilities [##UREF##5##8##]. Switzerland thus offers an opportunity to study potential effects of shared placement of child welfare and juvenile justice youth. However, despite this ongoing practice, very little is known about minors placed in care in Switzerland in general [##UREF##6##9##], and there is no systematic knowledge on any effects of shared placement of juvenile offenders with child welfare youth to date.</p>", "<p id=\"Par8\">The aim of the current paper is to map the demographic, crime-related and psychosocial characteristics of child welfare and juvenile justice youths in residential care in Switzerland, and subsequently examine its relationship with offending behavior in adulthood. This knowledge will not only inform us which factors to emphasize on in the assessment and treatment of these youngsters, but could also help better match the adolescents’ needs with the institution’s treatment options.</p>", "<title>Adolescents in out-of-home care in Switzerland</title>", "<p id=\"Par9\">To this day, there are no official statistics on the total number of minors in foster care families or other out-of-home placement facilities in Switzerland, but estimates range from 22,000 to 30,000 children and adolescents [##UREF##7##10##]. Child protection is regulated by local authorities. Foster sector case management is less regulated and influenced by local structures, availability of local treatment options, and the individual qualifications of the case workers [##UREF##6##9##]. However, since the beginning of this century, steps have been taken to improve reporting, professionalism and quality control. In 2007, for example, a new juvenile criminal code (<italic>Jugendstrafgesetz,</italic> JStG) has entered into effect. All adolescents placed in a child welfare or juvenile justice institution through juvenile justice authorities have to be placed in an institution approved by the Swiss Federal Office of Justice (<italic>Bundesamt für Justiz</italic> [BJ]). To be approved, the institution have to fulfill certain quality and reporting standards [##UREF##8##11##] which, under new juvenile criminal law, includes regular assessments to document ongoing appropriateness of the placement [##UREF##4##7##]. In 2013, around 200 newly regionally consolidated professionalized and interdisciplinary Authorities for Child and Adult Protection (<italic>Kindes- und Erwachsenschutzbehörden</italic> [KESB]) have replaced the 1420 lay authorities which were organized on a municipal level [##UREF##9##12##]. At the same time, new federal legal regulations for foster placements (<italic>Pflegekindverordnung</italic> [PAVO]) have taken effect and the BJ has begun to collect and share best practices, general information and statistical analyses on youth placed in institutions or foster families on a new online platform (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.casadata.ch\">www.casadata.ch</ext-link>). The Swiss Federal Office of Statistics (<italic>Bundesamt für Statistik</italic> [BFS]) yearly publishes the number, age and gender of sentenced minors (e.g., [##UREF##10##13##]. Similarly, the National Conference for Child and Adult Protection (<italic>Konferenz für Kindes- und Erwachsenenschutz</italic> [KOKES]) now publish yearly numbers of child protection articles in court rulings (e.g., termination of parental rights or removal of custody), giving an idea of the number of new residential placements based on civil law. However, to date no information on the exact nature of the civil law intervention or any information on the children it aims to protect has been released.</p>", "<title>Mental health problems of youth in the child welfare system</title>", "<p id=\"Par10\">A recent meta-analysis of studies from the U.S. and Europe showed that among children and adolescents in the child welfare system, 49% met criteria for a current mental disorder [##REF##26886603##3##]. More in detail, 27% were diagnosed with a conduct (CD) or oppositional defiant disorder (ODD) and 11% met the criteria for an attention-deficit/hyperactivity disorder (ADHD). The population also had high rates of internalizing problems, with prevalence estimates for anxiety at 18%, depressive disorders at 11% and posttraumatic stress disorder (PTSD) at 4%, with higher prevalence of internalizing disorders among girls and more externalizing disorders among boys. In comparison, worldwide-pooled prevalence of mental health disorders within adolescents in the general population is estimated at 13%, with anxiety disorder at 7%, any disruptive disorder (i.e., CD or ODD) at 6%, ADHD at 3%, and any depressive disorder at 3% across geographic location [##REF##25649325##14##].</p>", "<p id=\"Par11\">These numbers have been even higher among youth placed in residential care, ranging from 49 to 76% [##UREF##11##15##–##UREF##12##17##], with high rates of comorbidity in all studies. Additionally, these adolescents show elevated rates of chronic illness [##REF##15956866##18##], and childhood trauma [##UREF##13##19##], which in turn have been associated with worse mental health status into adulthood [##REF##22222293##20##, ##REF##22329484##21##]. For example, in a study with a Norwegian sample of youth in residential placement, the 71% of adolescents who had experienced maltreatment were even more likely to show CD, general anxiety disorder, dysthymia and major depressive disorder as well as more attempted suicides [##REF##26003821##22##]. In addition, substance use as well as depression have been associated with increased rates of juvenile delinquency in youth in the child welfare system [##UREF##1##4##] and there is a substantial body of research linking past maltreatment experiences with delinquent behavior in adolescence [##REF##22403901##23##, ##REF##18082884##24##]; within the child welfare system, delinquency rates for youth with a history of maltreatment are approximately 47% greater than their non-maltreated counterparts [##UREF##14##25##]. In one recent U.S. study, for example, a history of maltreatment increased the risk of arrest by 55% and of committing a violent crime by 96% [##UREF##15##26##]. Finally, it is estimated that more than a third of youth in child welfare are known to the juvenile justice system [##UREF##2##5##, ##REF##18082884##24##].</p>", "<p id=\"Par12\">The limited data on Swiss samples of youth in out-of-home placements have not differentiated between child welfare and juvenile justice youth but have shown similar high rates of mental health treatment needs and high comorbidity, with overall 74% of children and adolescents fulfilling criteria for one, and 60% fulfilling criteria for more than one mental disorder in residential care populations [##UREF##16##27##]. Furthermore, 25% of them suffered from complex psychiatric disorders with emotional and behavioral symptoms and elevated rates of delinquency.</p>", "<title>Mental health problems of youth in the juvenile justice system</title>", "<p id=\"Par13\">Similar to their counterparts in the child welfare system, youth in the juvenile justice system often come from backgrounds of poverty, family dysfunction, and maltreatment [##REF##25418616##28##, ##REF##24887073##29##]. Between 70 and 95% of detained youth were found to have mental health problems [##REF##18664994##1##, ##REF##20416149##30##, ##REF##23454219##31##]. A meta-analysis of youth in juvenile detention and correctional facilities has shown that they are about 10 times more likely to suffer from psychosis than the general adolescent population [##REF##18664994##1##], and there is a high prevalence of previous trauma and PTSD [##UREF##17##32##, ##REF##19103704##33##]. Rates of substance use are extremely high, with dependence and abuse affecting between 40 and 70% of juvenile offenders in custody [##REF##23454219##31##]. A systematic review among detained male adolescents found mean prevalence estimates of 70% for any mental disorders, with CD and substance use disorders (SUDs) being the most frequent [##REF##20416149##30##]. Although their numbers are much smaller, girls in the juvenile justice system tend to be younger and have more severe mental health problems than their male counterparts [##REF##18664994##1##, ##UREF##18##34##]. Fazel and colleagues [##REF##18664994##1##] found that almost 30% of girls in detention qualified for a diagnosis of major depression compared to 11% of boys, almost 20% presented with ADHD, (12% among boys). Both girls and boys shared similar elevated rates of CD at 53% of the sample, but girls present with higher rates of comorbid externalizing and internalizing disorders. These findings are troubling, since meta-analytic results have shown that presenting with an externalizing disorder or comorbidity increases the risk of recidivism for juvenile delinquents by around 20% [##UREF##19##35##].</p>", "<title>Risk of adult criminal behavior</title>", "<p id=\"Par14\">A substantial body of research links past maltreatment and neglect with juvenile delinquency and (violent) offending in adulthood [##UREF##20##36##]. Simultaneously, studies show an elevated risk of adult criminal involvement among former foster care youth [##REF##22239390##37##, ##UREF##21##38##]. To date, the evidence regarding the influence of out-of-home placement on future delinquency is inconclusive [##UREF##22##39##, ##UREF##23##40##]. For example, it has been shown that any out-of-home placement increases the likelihood of delinquency in adolescence and into young adulthood, especially if there is a history of placement instability [##UREF##14##25##, ##UREF##24##41##, ##UREF##25##42##]. Type of placement matters as well, and for some adolescents, kinship or family foster care has been associated with better outcomes compared to residential placement in group homes [##UREF##20##36##]. However, all those findings are confounded by the fact that placement outside the home is usually reserved for the most severe cases of detected maltreatment, while more severe mental health, substance use and conduct problems are associated with both residential placement over family foster care and more placement instability, all of which independently influence likelihood of future delinquency [##REF##18082884##24##, ##UREF##21##38##, ##UREF##26##43##–##REF##21871374##45##]. Furthermore, a Swedish population-based study only found negative effects of placement in care on adult criminality for boys first placed between ages 13–18, but not for girls or younger boys [##UREF##23##40##].</p>", "<p id=\"Par15\">Recent national data showed that 8% of adolescents born in Switzerland in 1992 were convicted as adolescents, and that having a juvenile conviction was associated with a six-fold increased odds of an adult conviction [##UREF##10##13##]. Among convicted Swiss adolescents, 26% were re-convicted as young adults. Additional analyses showed that being male, having multiple juvenile convictions, being over 16 years of age at first conviction as well as having been convicted for more severe crimes increased the risk of adult criminal conviction. However, the study did not differentiate between type of adult criminal conviction (violent or non-violent) and there is no knowledge on risk of adult criminal conviction among young adults who were in residential care as adolescents.</p>", "<p id=\"Par16\">Based on the aforementioned information, the aim of the current study threefold. First, to examine similarities and differences in demographic markers, previous offending (self-report and official conviction) and treatment needs (psychiatric profile, substance use) between adolescents placed in residential care by either child protection or juvenile justice authority. Second, to investigate whether the adolescents committed by child protection or juvenile justice authority differ in their long-term risk for any, violent, and non-violent young adulthood criminal convictions. Third, to examine if this relationship persists after controlling for well-known risk factors for adult criminal conviction (gender, age at beginning of placement, trauma, past self-reported delinquency, past convictions) or mental health treatment needs in adolescent residential care.</p>" ]
[ "<title>Methods</title>", "<title>Study procedures and sample description</title>", "<p id=\"Par17\">The sample was drawn from the larger Swiss Study for Clarification and Goal-Attainment in Youth Welfare and Juvenile Justice Institutions, (<italic>Modellversuch Abklärung und Zielerreichung in stationären Massnahmen</italic> [MAZ.]) involving the standardized monitoring and evaluation of mental health problems of youths in child welfare and juvenile justice institutions throughout Switzerland between 2007 until 2012 [##UREF##5##8##], as well as criminal justice follow-up data until the end of 2017 In this study, all child welfare and juvenile justice institutions accredited by the BJ were invited to participate. The final sample of 64 participating facilities (35% of eligible facilities) were representative for the different types of institutions in Switzerland (e.g., large versus small institutions, institutions with or without internal schools, and internal versus external access to treatment programs) as well as the heterogeneous group of youths who reside in them. Adolescents who resided in one of the participating facilities were asked to participate if they had been living in the institution for more than one month prior to the assessment and were able to complete the French, German or Italian assessment tools. Assessments consisted of clinical interviews by trained psychologists, computer assisted self-reports as well as ratings from institutional caregivers. Hence, assessment was not conducted at entrance per se, but could also have been taken place after the adolescent was already in the institution for a while (<italic>M</italic> = 13.1 months; <italic>SD</italic> = 13.3).</p>", "<p id=\"Par18\">Institutional staff approached adolescents and their legal guardians and explained the aims and nature of the study. A total of 592 (32%) adolescents from the participating institutions, in the French- (20 facilities), German- (38 facilities) and Italian-speaking (6 facilities) parts of Switzerland participated in the study. To check the representativeness of the study sample, institutional caregivers were asked to rate some adolescents who did not participate in the study (N = 46) on the Child Behavior Checklist (CBCL) [##UREF##27##46##] or the Young Adult Behavior Checklist (YABCL) [##UREF##28##47##]. Matched comparisons (i.e., on age and gender) between adolescents who did and did not participate in the study showed no differences in the frequency of scoring in the clinical range on the internalizing-, externalizing- and total problems scales of the CBCL or the YABCL. This suggests that the participating sample was representative for youth in the aforementioned participating institutions. For more details on study methodology see [##UREF##5##8##]. The Ethics Review Committees of Basel, Lausanne (Switzerland) and Ulm (Germany) approved the study.</p>", "<title>Participants</title>", "<p id=\"Par19\">First, for the current study, adolescents had to be placed under either civil or criminal law. Second. participants under 10 years of age were excluded since under current Swiss law, there are no juvenile justice commitments before that age. Third, the upper limit was set at 18 years of age which is usually when child welfare placements end, and all participants placed in institutions for young adult offenders were excluded since there are no child welfare placements in those institutions. Hence, the total available sample was reduced to a study sample of 354 adolescents (252 child welfare and 102 juvenile justice youths; 223 boys and 131 girls) between 10 and 18 years of age at entry to one of 58 institutions (mean age = 14.5, SD = 1.8). More information on participant demographics by committing authority are printed in Table ##TAB##0##1## (see “<xref rid=\"Sec14\" ref-type=\"sec\">Results</xref>” section).</p>", "<title>Measures</title>", "<title>Predictors</title>", "<p id=\"Par20\">Committing authority was a binary variable specifying whether youth were in a commitment at wave 1 based on child protection reasons by a child welfare authority (civil law measure, N = 252) or by a juvenile criminal court (juvenile justice measure, N = 102).</p>", "<title>Outcomes</title>", "<p id=\"Par21\">Adult criminal conviction data was obtained from the BFS until the end of 2017, up to 10 years after the initial assessment of the study. We assessed convictions for the two more serious types of offenses (<italic>Verbrechen, Vergehen</italic>), excluding the most minor category of offenses (<italic>Übertretungen</italic>), which under Swiss law are all offenses only punishable by fine (see Art. 103 Swiss Criminal Code). Violent offenses were classified following the definitions used by the BFS and included all offenses that included actual or threatened harm against persons, such as all forms of assault, robbery or coercion. Non-violent offenses were all other offenses above the aforementioned severity threshold, including violence against property or serious drug offenses.</p>", "<title>Other risk factors for adult criminal convictions</title>", "<p id=\"Par22\"><italic>Gender</italic> was a binary variable coded as 0 = female, 1 = male; <italic>Age at Beginning of Current Placement</italic> was assessed in years.</p>", "<p id=\"Par23\"><italic>Mental health problems and trauma</italic> were assessed using the Massachusetts Youth Screening Instrument-Version 2 (MAYSI-2), one of the most widely used tools for mental health screening for youth entering the juvenile justice system [##UREF##29##48##]. It consists of a 52-items self-report questionnaire screening for potential emotional or behavioral problems (e.g., suicidal ideation and aggressive behavior) that could require further (psychiatric) evaluation and has shown to be reliable and valid in diverse samples of detained youths [##UREF##30##49##]. The MAYSI-2 is currently used in detention, intake probation, and/or corrections facilities in about 44 states in the USA as well as a in growing number of institutions in Europe, including Switzerland [##UREF##29##48##, ##REF##27716175##50##]. In the current study, computerized versions of the French, German and Italian questionnaire were used. Respondents rated all items with yes (1 point) or no (0 points), resulting in seven scales, of which the current study used five [##UREF##29##48##]: alcohol/drug use, angry-irritable, depressed-anxious, suicide ideation, and traumatic experiences. Thought disturbance and somatic complaints were not included in the current study. Trauma exposure questions vary by gender and are not included in the scoring of cautions and warnings related to the screening.</p>", "<p id=\"Par24\">Data on <italic>previous juvenile convictions</italic> of crimes committed before wave 1 was obtained from the BFS. The information was recoded into a binary variable (yes/no) and again excluded the most minor category of offenses. <italic>Severity of past delinquency</italic> was assessed through a German self-report questionnaire on offending behavior [##UREF##31##51##]. All analyses were adjusted for <italic>Time at Risk</italic>, which was calculated as time in months that the respondent had been over 18 years of age and thus subject to adult criminal law.</p>", "<title>Statistical analysis</title>", "<p id=\"Par25\">Analyses were performed using Mplus version 8.2 [##UREF##32##52##] and SPSS version 25 [##UREF##33##53##]. First, the sample was grouped into two groups of adolescents as described above. Then, univariate difference tests between the two groups were tested either with χ2 difference scores (for categorical variables) or one-way analysis of variance (ANOVA; for continuous variables, see Table ##TAB##0##1##). All analyses with continuous variables used a Holm-Bonferroni sequential correction to adjust for multiple comparisons and Levene’s correction for unequal variances if necessary [##UREF##34##54##].</p>", "<p id=\"Par26\">Next, to estimate risk of adult criminal conviction by committing authority we conducted three sets of logistic regressions in Mplus, investigating any, violent and non-violent convictions separately. In each set of regressions, unadjusted models were estimated first, before risk factors (i.e., gender, age at commitment, number of previous convictions, severity of previous self-reported offending, time at risk and time since intake), trauma and mental health treatment needs (alcohol/drug use, angry-irritable, depressed-anxious, suicide ideation) were added to predict outcomes. Multilevel analyses showed intraclass correlations of study variables ranging from 0.08 to 0.57 by placement facility. To account for clustering of the data within facilities, all logistic regression analyses thus used a complex sampling procedure with cluster robust standard errors [##REF##27149401##55##]. Participants with missing data were included in the model estimations using Full Information Maximum Likelihood (FIML) techniques and MLR estimation with Montecarlo integration for binary outcomes was used [##UREF##35##56##]. Model fit was assessed with the Sample size adjusted Bayesian Information Criterion (BIC) and the Akaike information criterion (AIC).</p>" ]
[ "<title>Results</title>", "<title>Demographic characteristics of adolescents in residential placements</title>", "<p id=\"Par27\">The first aim of the current study was to examine similarities and differences in demographic markers, current treatment needs (psychiatric profile, substance use) and previous offending (self-report and official conviction) between adolescents placed in residential care by either child protection or juvenile justice authority. As shown in Table ##TAB##0##1##, results showed that juvenile justice youth were predominantly male, while there was an equal distribution of gender within the welfare youth. There were some geographical differences in placement authority, with an increased proportion of juvenile justice youth coming from the French speaking region of Switzerland compared to welfare youth, while there were no juvenile justice youth from the Italian speaking region. Juvenile justice youth were also older at the time of the study as well as when they were first placed in out-of-home care compared to welfare youth. There were no other differences in institutionalization history or in planned duration of current placement; differences in types of institution are likely due to differences in age and gender between the two groups. There were few differences in current mental health treatment needs and previous self-reported trauma exposure between juvenile justice and welfare youth. However, juvenile justice youth had higher mean levels of self-reported alcohol and drug use. Finally, there were differences in history of offending on both the self-reports as well as official records. Specifically, juvenile justice youth scored higher on all forms of delinquency and had more previous convictions, both violent and non-violent. There were no differences in age of first conviction and importantly, among those who were placed by a juvenile justice authority, 22.5% had no previous criminal conviction.</p>", "<title>Risk of adult criminal conviction by committing authority</title>", "<p id=\"Par28\">To investigate whether the adolescents committed by child welfare or juvenile justice authority differed in their risk for adult criminal conviction we calculated three sets of logistic regressions (second aim). Results of these analyses are presented in Table ##TAB##1##2##. Unadjusted models showed that committing authority predicted adult criminal conviction overall (model 1a), as well as violent (model 2a) and non-violent convictions (model 3a) separately, with adolescents being committed through juvenile justice authority showing increased risk on all outcomes.</p>", "<p id=\"Par29\">However, when controlling for other risk factors of adult criminal conviction, i.e., gender, age at beginning of current placement, severity of previous delinquency, previous conviction, time at risk and time since intake, as well as mental health treatment needs and traumatic experiences (third aim), committing authority no longer had an effect on risk of adult conviction. Specifically, the adjusted models showed that being male and more time at risk was associated with an increased risk for any adult conviction (model 1b) as well as for violent (model 2b) and non-violent convictions (model 3b). Similarly, having a previous conviction increased odds for all forms of adult convictions, while in contrast there was no association between self-reported severity of past delinquency and any of the outcomes. In terms of mental health treatment needs, more alcohol and drug use increased risk of general adult conviction, and traumatic experiences were associated with an increased likelihood of non-violent adult conviction within this high-risk sample. There was no association between trauma, mental health and risk of adult violent conviction.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par30\">Adolescents in residential care are marked by multiple disadvantages before and during placement, as well as consequently in young adulthood. They show elevated rates of trauma, psychopathology and other psychosocial problems, and an elevated risk of involvement in both juvenile delinquency and adult criminal behavior [##REF##18664994##1##–##UREF##1##4##]. At least one third of youth in child welfare are also known to the juvenile justice system [##UREF##2##5##, ##REF##18082884##24##].</p>", "<p id=\"Par31\">In Switzerland, adolescents are placed in residential care because of delinquent behavior (juvenile justice measure) or for child protection reasons (civil law measure, e.g., maltreatment, neglect, or parental absence, psychopathology or drug abuse), meaning they might reside in the same institutions based on their educational or treatment needs [##UREF##4##7##, ##UREF##5##8##]. The current study capitalized on this opportunity to examine effects of shared placement of juvenile offenders with child welfare youth in Switzerland and investigated long-term adjustment in the form of adult criminal conviction. Similarities and differences in demographic markers, current treatment needs (trauma, psychiatric profile, substance use) and previous offending behavior (self-report and official conviction) between both groups were examined, and it was investigated if these demographic and crime-related risk factors, and mental health treatment needs while in residential care influenced risk for adult criminal conviction. Especially investigating the influence of mental health treatment needs on risk of adult offending is of high practical relevance, since it might present an important avenue for intervention.</p>", "<p id=\"Par32\">Results of the current study showed overall few differences in mental health treatment needs between child welfare and juvenile justice youth, and no association between placement authority and risk of adult criminal conviction after accounting for other risk factors and mental health treatment needs. Univariate analyses of group differences showed that, while juvenile justice youth had higher levels of substance use, there were no differences in past traumatic experiences, angry-irritable or depressed-anxious problems on the MAYSI-2. As expected, both groups differed in previous offending behavior as well as in previous convictions, with juvenile justice youth scoring higher on all indicators. It is important to note however, that even among juvenile justice youth, 22.5% had no previous conviction. This indicates that juvenile justice authorities do mandate placements based on educational or treatment needs independent of substantiated delinquent behavior, as intended by Swiss law. The lack of differences in mental health problems or planned duration of placements between the two groups is also an indicator that placement decisions are based mainly on treatment needs and not as a means to discipline juvenile justice youth.</p>", "<p id=\"Par33\">In terms of demographic factors, we found age, gender and nationality differences, as well as some regional differences. While the increased proportion of males among the juvenile justice youth corresponds to international samples of juvenile offenders [##REF##18664994##1##, ##UREF##18##34##], the age differences and differences in nationality merit some closer attention. While groups did not differ in their number of placements or age at first conviction, juvenile justice youth were older at their first placement as well as at the time of the study. Furthermore, although the results regarding nationality are difficult to interpret,<xref ref-type=\"fn\" rid=\"Fn1\">1</xref> juveniles with a non-Swiss nationality were more prevalent in the juvenile justice than in the child welfare sample. This could indicate that older adolescents and adolescents with a non-Swiss nationality represent a subgroup where the consequences of dysfunction at home or treatment needs appear later compared to welfare youth whose needs seems to get noticed by authorities earlier. Whether this is associated with the severity of the situation or represents a different reaction of the adolescents to similar scenarios has to be the focus of future research.</p>", "<p id=\"Par35\">Furthermore, the results of the logistic regressions showed several associations between risk factors and adult criminal conviction, as well as differing associations by type of adult criminal conviction. As in the Swiss national data [##UREF##10##13##], gender was the strongest predictor of adult criminal conviction, with males showing between 4.6 to 7.1 times increased odds for an adult conviction. Same as in the national sample, in the present high-risk population previous juvenile convictions were associated with an increased likelihood of general, violent and non-violent conviction. However, these findings were not repeated in self-reports of juvenile delinquency, with self-reported severity of previous delinquency having no association with any of the outcomes when other factors were taken into account. While our results showed that placement authority did not influence likelihood of adult conviction after accounting for other risk factors, this difference indicates that there might be labeling processes by contact with the juvenile justice system that results in legal convictions. However, they do not seem to influence reasons for institutionalization. Further investigation of mechanisms behind these results should be the focus of future research.</p>", "<p id=\"Par36\">Lastly, our results showed an association between traumatic experiences and non-violent adult conviction up to ten years later, as well as an association between alcohol and drug use and general adult conviction. The latter is consistent with the results of previous studies in which substance use problems were found to be related to (adult) (re)offending [##UREF##36##57##–##REF##19839964##60##]. Despite a small effect size, it could still be important to screen for alcohol and drug problems in adolescents upon entering the residential care institution, so these problems can be taken into account in treatment in order to prevent long-term negative outcomes, such as delinquency in adulthood [##REF##19926231##59##, ##REF##16445547##61##, ##UREF##38##62##]. Regarding trauma, though the effect sizes were also comparatively small, our findings correspond to the body of research showing a connection between childhood trauma, delinquency and adult criminal involvement [##UREF##39##63##–##UREF##41##65##], whilst noting that this does not apply to all forms of childhood traumatic experiences [##UREF##42##66##]. Hence traumatic experiences and psychosocial stress should also be included into standard screening and assessment, and taken into account in the treatment of juvenile and adult offenders [##UREF##42##66##]. Evidence-based trauma-therapeutic interventions as well as trauma-pedagogic care concepts should be embedded into child welfare and juvenile justice settings. Trauma-informed care is a conceptual framework and milieu therapeutic approach that relates to the understanding of and responsiveness to trauma exposure [##UREF##43##67##]. It conceptualizes problem behaviour in the context of an individual’s traumatic exposure and contains anticipating and avoiding practices which increase the risk of traumatic re-enactment [##REF##28339563##68##, ##UREF##44##69##]. Guiding principles of trauma informed care include: safety, trustworthiness and transparency, peer support, collaboration and mutuality, empowerment and choice, and cultural, historical and gender issues (see also the infographic on the website of the Office of Public Health Preparedness and Response [OPHPR] of the Center for Disease and Control Prevention [CDC]: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cdc.gov/cpr/infographics/6_principles_trauma_info.htm\">https://www.cdc.gov/cpr/infographics/6_principles_trauma_info.htm</ext-link>).<xref ref-type=\"fn\" rid=\"Fn2\">2</xref> This trauma informed approach has also been examined in the more specific context of the juvenile justice system [##REF##28165266##70##, ##REF##31910832##71##]. Although the combination of evidence-based trauma-therapeutic interventions in combination with trauma informed care concepts is highly promising for the treatment of, for example juvenile offenders (but also adolescents in the child welfare system), more research is warranted to examine its impact on offending behavior/recidivism as well as other adolescent/adult functional outcomes.</p>", "<title>Limitations</title>", "<p id=\"Par38\">The current study must be seen in the light of several limitations. A first set of limitations relate to the research design of the larger MAZ. study from which this sample was drawn [##UREF##5##8##]. First, the classification child welfare versus juvenile justice youth was based on the placement ground in the institution at baseline assessment of the study. However, research in the field of crossover youth has shown that a percentage of adolescents appear in both systems during their childhood/young adulthood [##UREF##2##5##]. Second, in our sample, we know that a part of the adolescents was in out-of-home care before and could still be found in both systems after the study. Unfortunately, we were unable to carry out a comprehensive, accurate residential care trajectory analysis, on the one hand because the adolescents are not always fully aware of their history and on the other hand because this information is not collected in a structured manner by a centralized organization in Switzerland. Finally, by design, participants were interviewed at varying time points after the beginning of their institutional stay. The MAYSI-2 however is designed to be administered at intake into a juvenile justice facility. Given the time limited nature of the anchoring questions in this screening measure, it can therefore not be excluded that the results have been influenced by the varying time spent already in an institution. We tried to offset this limitation by controlling for time since intake in our analyses.</p>", "<p id=\"Par39\">A second set of limitations concerns the assessment used in the current study. An important point is that many of the tools we used in this study were self-report instruments (MAYSI-2, self-reported delinquency). The use of self-report instruments entails a risk of both overestimation and underestimation. On the other hand, it offers the opportunity to gain more insight into certain aspects (often relating to internalizing mental health) that may have been overlooked when using only third-party assessments. Notably, we used official registered criminal convictions for the outcome variables. However, future research and analyses should include information from multiple sources. Finally, trauma is a broad and multi-faceted concept with often no clear definition leading to an exponential use. We used the traumatic experience scale of the MAYSI-2, which is a very rudimentary screening scale only consisting of a limited number of items. This approach takes little account of the number, duration or effect of a certain (potentially) traumatic experience and is supposed to be a quick screening tool that needs further enhanced clarification and more sophisticated measurement tolls. Nevertheless, it is a short and feasible indicator for possible trauma exposure.</p>" ]
[ "<title>Conclusion</title>", "<p id=\"Par40\">Our results support the approach of placement in residential care institutions based on treatment needs in this Swiss sample. Adolescents’ reason for placement were unrelated to risk of adult criminal conviction when taking into account well documented demographic risk factors (male gender, previous conviction and more time at risk). In addition, there was an effect of trauma histories and mental health needs beyond these static factors, indicating a possible avenue for intervention for all adolescents. Our results thus underscore the importance of assessing trauma and mental health status of all adolescents entering residential or other out-of-home placement and addressing their treatment needs, with a special attention to trauma-informed care. Finally, although it is impossible to make general statements given the differences in legal systems, countries might reflect on whether they want to place adolescents strictly based on the adjudicating court or whether they want to take the (underlying) problems of these youngsters into account. This seems especially pertinent for the high percentage of “cross-over youths”, adolescents who have records in both systems.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Although child welfare youth and juvenile offenders in residential care have different judicial placement reasons, there seems to be overlap in their demographic and psychosocial backgrounds. This could raise the question whether these adolescents should be placed in strictly separated institutions based on their judicial title (civil or criminal law) or together based on their needs. As systematic knowledge on the effects of shared placement of these groups is limited, the aim of the current paper is to examine the demographic, crime-related and psychosocial characteristics of child welfare and juvenile justice youths in shared residential care and subsequently examine its relationship with offending behavior in adulthood.</p>", "<title>Methods</title>", "<p id=\"Par2\">The sample was drawn from the Swiss study for clarification and goal-attainment in youth welfare and juvenile justice institutions (MAZ.) and consisted 354 juveniles (252 child welfare, 102 juvenile justice; 223 boys, 131 girls) between 10 and 18 years. Mental health problems were assessed with the Massachusetts Youth Screening Instrument-Version 2 (MAYSI-2), official adult criminal conviction data up to 10 years later was obtained from the Swiss Federal Office of Statistics. Three sets of logistic regressions were conducted investigating any, violent and non-violent convictions.</p>", "<title>Results</title>", "<p id=\"Par3\">Univariate results showed that that the child welfare sample included more females, more juveniles with the Swiss nationality, and was younger at the time of assessment and at first placement compared to the juvenile justice sample. Furthermore, child welfare youths showed less alcohol/drug use problems and offending behavior than their juvenile justice counterparts. Unadjusted models demonstrated that committing authority predicted adult criminal convictions, but that this distinction disappeared when it was controlled for demographic, crime-related and psychosocial factors. Gender and time at risk were found to be related to adult conviction in all three models. In addition, alcohol/drug use problems were risk factors for general, previous convictions for violent, and traumatic experiences for non-violent convictions in adulthood.</p>", "<title>Conclusions</title>", "<p id=\"Par4\">Our results support the approach of placement in residential care institutions based on treatment needs instead of on judicial title. Special attention should be devoted to trauma informed care and substance use coping. However, more research is needed.</p>", "<title>Keywords</title>" ]
[]
[ "<title>Acknowledgements</title>", "<p>Not applicable.</p>", "<title>Authors’ contributions</title>", "<p>LJ, CB and NS contributed to the manuscript conception and design. Data collection was performed by MS and his team. Analysis were performed by LJ. LJ, MS, DB, AG and CB interpreted the data. LJ and CB wrote the first draft of the manuscript, MS and DB commented on previous versions of the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>The study was funded by the Swiss Ministry of Justice. The costs for the open access publication will be taken over by the University of Basel.</p>", "<title>Availability of data and materials</title>", "<p>The data that support the findings of this study are available from the corresponding author upon request.</p>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par41\">The Ethics Review Committees of Basel, Lausanne (Switzerland) and Ulm (Germany) approved the study.</p>", "<title>Consent for publication</title>", "<p id=\"Par42\">Not applicable.</p>", "<title>Competing interests</title>", "<p id=\"Par43\">The authors declare that they have no competing interest.</p>" ]
[]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Group differences in baseline demographic factors, mental health and history of offending</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Characteristic</th><th align=\"left\">Total (N = 354)</th><th align=\"left\">Welfare Youth (N = 252)</th><th align=\"left\">Juvenile Justice Youth (N = 102)</th><th align=\"left\" colspan=\"2\">Univariate test of difference</th></tr></thead><tbody><tr><td align=\"left\">Demographic factors</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Gender [% male (<italic>n</italic>)]</td><td char=\"(\" align=\"char\">63.0% (223)</td><td char=\"(\" align=\"char\">53.2% (134)</td><td char=\"(\" align=\"char\">87.3% (89)</td><td align=\"left\">χ2(1) = 36.18</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Age mean (SD)</td><td char=\"(\" align=\"char\">16.02 (1.64)</td><td char=\"(\" align=\"char\">15.8 (1.55)</td><td char=\"(\" align=\"char\">16.6 (1.76)</td><td align=\"left\">t(320) = -3.95</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Nationality [% Swiss (n)]</td><td char=\"(\" align=\"char\">83.1% (294)</td><td char=\"(\" align=\"char\">85.7% (216)</td><td char=\"(\" align=\"char\">76.5% (78)</td><td align=\"left\">χ2(1) = 4.41</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> Born in Switzerland [% yes (n)]</td><td char=\"(\" align=\"char\">76.0% (269)</td><td char=\"(\" align=\"char\">77.8% (196)</td><td char=\"(\" align=\"char\">71.6% (73)</td><td align=\"left\">χ2(1) = 1.53</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\">Language region of placement</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\">χ2(2) = 8.81</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> German [% (n)]</td><td char=\"(\" align=\"char\">73.2% (259)</td><td char=\"(\" align=\"char\">72.6% (183)</td><td char=\"(\" align=\"char\">74.5% (76)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> French [% (n)]</td><td char=\"(\" align=\"char\">21.5% (76)</td><td char=\"(\" align=\"char\">19.8% (50)</td><td char=\"(\" align=\"char\">25.5% (26)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Italian [% (n)]</td><td char=\"(\" align=\"char\">5.4% (19)</td><td char=\"(\" align=\"char\">7.5% (19)</td><td char=\"(\" align=\"char\">0.0% (0)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Age at beginning of current placement mean (SD)</td><td char=\"(\" align=\"char\">14.95 (1.74)</td><td char=\"(\" align=\"char\">14.62 (1.72)</td><td char=\"(\" align=\"char\">15.76 (1.51)</td><td align=\"left\">t(350) = -5.89</td><td align=\"left\">***</td></tr><tr><td align=\"left\">Planned duration of current placement mean (SD)</td><td char=\"(\" align=\"char\">27.32 21.4</td><td char=\"(\" align=\"char\">28.05 (22.65)</td><td char=\"(\" align=\"char\">25.51 (17.93)</td><td align=\"left\">t(350) = 1.01</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\">Type of Institution</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\">χ2(3) = 19.32</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Transitional Placement</td><td char=\"(\" align=\"char\">11.9% (42)</td><td char=\"(\" align=\"char\">15.7% (16)</td><td char=\"(\" align=\"char\">10.3% (26)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Group home with school or trade program</td><td char=\"(\" align=\"char\">58.8% (208)</td><td char=\"(\" align=\"char\">53.6% (135)</td><td char=\"(\" align=\"char\">71.6% (73)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Group home without internal educational program</td><td char=\"(\" align=\"char\">25.7% (91)</td><td char=\"(\" align=\"char\">31.7% (80)</td><td char=\"(\" align=\"char\">10.8% (11)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Other</td><td char=\"(\" align=\"char\">3.7% (13)</td><td char=\"(\" align=\"char\">4.4% (11)</td><td char=\"(\" align=\"char\">2.0% (2)</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Institutionalization History</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Previous residential or foster placement [% yes (n)]</td><td char=\"(\" align=\"char\">46.3% (161)</td><td char=\"(\" align=\"char\">45.2% (112)</td><td char=\"(\" align=\"char\">49.0% (49)</td><td align=\"left\">χ2(1) = 0.42</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> Age at first placement mean (SD)</td><td char=\"(\" align=\"char\">13.46 (3.45)</td><td char=\"(\" align=\"char\">12.99 (3.67)</td><td char=\"(\" align=\"char\">14.62 (2.51)</td><td align=\"left\">t(269.86) = -4.81</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Number of previous placements mean (SD)</td><td char=\"(\" align=\"char\">0.97 (1.38)</td><td char=\"(\" align=\"char\">0.93 (1.30)</td><td char=\"(\" align=\"char\">1.09 (1.56)</td><td align=\"left\">t(346) = -0.10</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\">Mental health problems</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Alcohol and drug use mean (SD)</td><td char=\"(\" align=\"char\">2.87 (2.77)</td><td char=\"(\" align=\"char\">2.54 (2.70)</td><td char=\"(\" align=\"char\">3.76 (2.80)</td><td align=\"left\">t(320) = -3.55</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Angry-irritable mean (SD)</td><td char=\"(\" align=\"char\">4.66 (2.68)</td><td char=\"(\" align=\"char\">4.66 (2.61)</td><td char=\"(\" align=\"char\">4.67 (2.87)</td><td align=\"left\">t(320) = -0.02</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> Depressed-anxious mean (SD)</td><td char=\"(\" align=\"char\">3.04 (2.39)</td><td char=\"(\" align=\"char\">3.14 (2.50)</td><td char=\"(\" align=\"char\">2.75 (2.04)</td><td align=\"left\">t(186.61) = 1.46</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> Suicide ideation mean (SD)</td><td char=\"(\" align=\"char\">2.08 (1.70)</td><td char=\"(\" align=\"char\">2.17 (1.76)</td><td char=\"(\" align=\"char\">1.82 (1.51)</td><td align=\"left\">t(172.39) = 2.24</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> More than one elevated MAYSI-2 scale [% yes (n)]</td><td char=\"(\" align=\"char\">80.1% (258)</td><td char=\"(\" align=\"char\">80.4% (189)</td><td char=\"(\" align=\"char\">79.3% (69)</td><td align=\"left\">χ2(1) = 0.50</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\">Violence Exposure and Trauma</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Traumatic Experience [% yes (n)]</td><td char=\"(\" align=\"char\">84.8% (217)</td><td char=\"(\" align=\"char\">84.5% (158)</td><td char=\"(\" align=\"char\">85.5% (59)</td><td align=\"left\">χ2(1) = 0.40</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> Traumatic experiences mean (SD)</td><td char=\"(\" align=\"char\">2.4 (1.47)</td><td char=\"(\" align=\"char\">2.38 (1.42)</td><td char=\"(\" align=\"char\">2.46 (1.61)</td><td align=\"left\">t(320) = -0.42</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> Direct victimization mean (SD)</td><td char=\"(\" align=\"char\">2.04 (1.98)</td><td char=\"(\" align=\"char\">2.11 (2.08)</td><td char=\"(\" align=\"char\">1.85 (1.66)</td><td align=\"left\">t(188.71) = 1.15</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\">Self-reported previous delinquency</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> General delinquency mean (SD)</td><td char=\"(\" align=\"char\">8.02 (7.07)</td><td char=\"(\" align=\"char\">7.04 (6.64)</td><td char=\"(\" align=\"char\">10.65 (7.55)</td><td align=\"left\">t(316) = -4.15</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Violent delinquency mean (SD)</td><td char=\"(\" align=\"char\">1.53 (1.83)</td><td char=\"(\" align=\"char\">1.27 (1.62)</td><td char=\"(\" align=\"char\">2.23 (2.18)</td><td align=\"left\">t(121.19) = -3.74</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Delinquency severity mean (SD)</td><td char=\"(\" align=\"char\">2.50 (1.37)</td><td char=\"(\" align=\"char\">2.30 (1.39)</td><td char=\"(\" align=\"char\">3.07 (1.15)</td><td align=\"left\">t(183.04) = -5.03</td><td align=\"left\">***</td></tr><tr><td align=\"left\">Previous convictions</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Criminal conviction [% yes (n)]</td><td char=\"(\" align=\"char\">47.5% (168)</td><td char=\"(\" align=\"char\">36.9% (93)</td><td char=\"(\" align=\"char\">73.5% (75)</td><td align=\"left\">χ2(1) = 40.75</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Age at first conviction mean (SD)</td><td char=\"(\" align=\"char\">13.93 (1.78)</td><td char=\"(\" align=\"char\">13.91 (1.83)</td><td char=\"(\" align=\"char\">13.95 (1.73)</td><td align=\"left\">t(166) = -0.15</td><td align=\"left\">n.s</td></tr><tr><td align=\"left\"> Violent crime [% yes (n)]</td><td char=\"(\" align=\"char\">17.5% (62)</td><td char=\"(\" align=\"char\">8.7% (22)</td><td char=\"(\" align=\"char\">39.2% (40)</td><td align=\"left\">χ2(1) = 46.71</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Non-violent crime [% yes (n)]</td><td char=\"(\" align=\"char\">44.4% (157)</td><td char=\"(\" align=\"char\">34.5% (87)</td><td char=\"(\" align=\"char\">68.6% (70)</td><td align=\"left\">χ2(1) = 34.22</td><td align=\"left\">***</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Logistic regressions predicting adult criminal conviction</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Parameter Estimates</th><th align=\"left\">OR</th><th align=\"left\" colspan=\"2\">(95% CI)</th><th align=\"left\"><italic>β</italic></th><th align=\"left\"><italic>S.E</italic></th><th align=\"left\" colspan=\"2\">Est./S.E</th></tr><tr><th align=\"left\" colspan=\"8\">Any Adult Criminal Conviction</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"8\">Model 1a</td></tr><tr><td align=\"left\"><p> Committing Authority</p><p> (0 = child welfare; 1 = juvenile justice)</p></td><td char=\".\" align=\"char\">2.66</td><td char=\".\" align=\"char\" colspan=\"2\">(1.47–4.80)</td><td char=\".\" align=\"char\">0.24</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">2.87</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> AIC, BIC, R<sup>2</sup></td><td char=\".\" align=\"char\">16,876.74</td><td char=\".\" align=\"char\" colspan=\"2\"/><td char=\".\" align=\"char\">16,893.47</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.06</td><td align=\"left\">(n.s.)</td></tr><tr><td align=\"left\" colspan=\"8\">Model 1b</td></tr><tr><td align=\"left\"><p>  Committing Authority</p><p>(0 = child welfare; 1 = juvenile justice)</p></td><td char=\".\" align=\"char\">0.93</td><td char=\".\" align=\"char\" colspan=\"2\">(0.50–1.71)</td><td char=\".\" align=\"char\">− 0.02</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">− 0.21</td><td align=\"left\">(n.s.)</td></tr><tr><td align=\"left\"> Gender (0 = female; 1 = male)</td><td char=\".\" align=\"char\">6.34</td><td char=\".\" align=\"char\" colspan=\"2\">(3.62–11.10)</td><td char=\".\" align=\"char\">0.38</td><td char=\".\" align=\"char\">0.06</td><td char=\".\" align=\"char\">6.27</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Age at Beginning of Commitment</td><td char=\".\" align=\"char\">0.89</td><td char=\".\" align=\"char\" colspan=\"2\">(0.68–1.15)</td><td char=\".\" align=\"char\">− 0.09</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">− 0.79</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Traumatic Experiences</td><td char=\".\" align=\"char\">1.23</td><td char=\".\" align=\"char\" colspan=\"2\">(1.00–1.52)</td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">1.57</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Alcohol/Drug Use</td><td char=\".\" align=\"char\">1.13</td><td char=\".\" align=\"char\" colspan=\"2\">(1.03–1.24)</td><td char=\".\" align=\"char\">0.15</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">2.15</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> MAYSI Angry-Irritable</td><td char=\".\" align=\"char\">1.04</td><td char=\".\" align=\"char\" colspan=\"2\">(0.91–1.19)</td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">0.46</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Depressed-Anxious</td><td char=\".\" align=\"char\">1.03</td><td char=\".\" align=\"char\" colspan=\"2\">(0.84–1.27)</td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">0.25</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Suicidal Ideation</td><td char=\".\" align=\"char\">0.98</td><td char=\".\" align=\"char\" colspan=\"2\">(0.82–1.16)</td><td char=\".\" align=\"char\">− 0.02</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">− 0.25</td><td align=\"left\"/></tr><tr><td align=\"left\"> Severity of Previous Delinquency</td><td char=\".\" align=\"char\">0.89</td><td char=\".\" align=\"char\" colspan=\"2\">(0.73–1.10)</td><td char=\".\" align=\"char\">− 0.07</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">− 0.93</td><td align=\"left\"/></tr><tr><td align=\"left\"> Previous Conviction</td><td char=\".\" align=\"char\">1.90</td><td char=\".\" align=\"char\" colspan=\"2\">(1.23–2.96)</td><td char=\".\" align=\"char\">0.14</td><td char=\".\" align=\"char\">0.06</td><td char=\".\" align=\"char\">2.41</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> Time at Risk</td><td char=\".\" align=\"char\">1.03</td><td char=\".\" align=\"char\" colspan=\"2\">(1.01–1.05)</td><td char=\".\" align=\"char\">0.32</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">3.27</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> Time since Intake</td><td char=\".\" align=\"char\">0.98</td><td char=\".\" align=\"char\" colspan=\"2\">(0.96–1.00)</td><td char=\".\" align=\"char\">− 0.12</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">− 1.45</td><td align=\"left\"/></tr><tr><td align=\"left\"> AIC, BIC, R<sup>2</sup></td><td char=\".\" align=\"char\">11,865.95</td><td char=\".\" align=\"char\" colspan=\"2\"/><td char=\".\" align=\"char\">11,905.67</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.40</td><td align=\"left\">***</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"8\">Adult Violent Conviction</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"8\">Model 2a</td></tr><tr><td align=\"left\"><p> Committing Authority</p><p>(0 = child welfare; 1 = juvenile justice)</p></td><td char=\".\" align=\"char\">2.83</td><td char=\".\" align=\"char\">1.45</td><td char=\".\" align=\"char\">5.51</td><td char=\".\" align=\"char\">0.25</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">2.73</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> AIC, BIC</td><td char=\".\" align=\"char\">16,688.45</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">16,705.17</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.06</td><td align=\"left\">(n.s.)</td></tr><tr><td align=\"left\" colspan=\"8\">Model 2b</td></tr><tr><td align=\"left\"><p> Committing Authority</p><p>(0 = child welfare; 1 = juvenile justice)</p></td><td char=\".\" align=\"char\">0.94</td><td char=\".\" align=\"char\">0.51</td><td char=\".\" align=\"char\">1.73</td><td char=\".\" align=\"char\">− 0.01</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">− 0.17</td><td align=\"left\"/></tr><tr><td align=\"left\"> Gender (0 = female; 1 = male)</td><td char=\".\" align=\"char\">4.60</td><td char=\".\" align=\"char\">2.04</td><td char=\".\" align=\"char\">10.39</td><td char=\".\" align=\"char\">0.29</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">3.04</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> Age at Beginning of Commitment</td><td char=\".\" align=\"char\">0.69</td><td char=\".\" align=\"char\">0.47</td><td char=\".\" align=\"char\">1.02</td><td char=\".\" align=\"char\">− 0.25</td><td char=\".\" align=\"char\">0.14</td><td char=\".\" align=\"char\">− 1.80</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Traumatic Experiences</td><td char=\".\" align=\"char\">0.96</td><td char=\".\" align=\"char\">0.72</td><td char=\".\" align=\"char\">1.29</td><td char=\".\" align=\"char\">− 0.02</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">− 0.23</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Alcohol/Drug Use</td><td char=\".\" align=\"char\">1.13</td><td char=\".\" align=\"char\">1.00</td><td char=\".\" align=\"char\">1.27</td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">1.67</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Angry-Irritable</td><td char=\".\" align=\"char\">0.96</td><td char=\".\" align=\"char\">0.81</td><td char=\".\" align=\"char\">1.13</td><td char=\".\" align=\"char\">− 0.05</td><td char=\".\" align=\"char\">0.11</td><td char=\".\" align=\"char\">− 0.44</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Depressed-Anxious</td><td char=\".\" align=\"char\">1.14</td><td char=\".\" align=\"char\">0.92</td><td char=\".\" align=\"char\">1.41</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">1.01</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Suicidal Ideation</td><td char=\".\" align=\"char\">0.95</td><td char=\".\" align=\"char\">0.76</td><td char=\".\" align=\"char\">1.19</td><td char=\".\" align=\"char\">− 0.04</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">− 0.37</td><td align=\"left\"/></tr><tr><td align=\"left\"> Severity of Previous Delinquency</td><td char=\".\" align=\"char\">0.96</td><td char=\".\" align=\"char\">0.71</td><td char=\".\" align=\"char\">1.31</td><td char=\".\" align=\"char\">− 0.02</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">− 0.20</td><td align=\"left\"/></tr><tr><td align=\"left\"> Previous Conviction</td><td char=\".\" align=\"char\">4.32</td><td char=\".\" align=\"char\">1.91</td><td char=\".\" align=\"char\">9.78</td><td char=\".\" align=\"char\">0.29</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">3.29</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> Time at Risk</td><td char=\".\" align=\"char\">1.04</td><td char=\".\" align=\"char\">1.01</td><td char=\".\" align=\"char\">1.06</td><td char=\".\" align=\"char\">0.36</td><td char=\".\" align=\"char\">0.11</td><td char=\".\" align=\"char\">3.34</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> Time since Intake</td><td char=\".\" align=\"char\">0.96</td><td char=\".\" align=\"char\">0.93</td><td char=\".\" align=\"char\">0.99</td><td char=\".\" align=\"char\">− 0.21</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">− 2.38</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> AIC, BIC, R<sup>2</sup></td><td char=\".\" align=\"char\">11,719.86</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">11,759.58</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.48</td><td align=\"left\">***</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"8\">Adult Non-Violent Conviction</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"8\">Model 3a</td></tr><tr><td align=\"left\"><p> Committing Authority</p><p>(0 = child welfare; 1 = juvenile justice)</p></td><td char=\".\" align=\"char\">2.44</td><td char=\".\" align=\"char\">1.27</td><td char=\".\" align=\"char\">4.68</td><td char=\".\" align=\"char\">0.22</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">2.35</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> AIC, BIC, R2</td><td char=\".\" align=\"char\">16,858.54</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">16,875.27</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.05</td><td align=\"left\">(n.s.)</td></tr><tr><td align=\"left\" colspan=\"8\">Model 3b</td></tr><tr><td align=\"left\"><p> Committing Authority</p><p>(0 = child welfare; 1 = juvenile justice)</p></td><td char=\".\" align=\"char\">0.86</td><td char=\".\" align=\"char\">0.43</td><td char=\".\" align=\"char\">1.74</td><td char=\".\" align=\"char\">− 0.03</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">− 0.34</td><td align=\"left\"/></tr><tr><td align=\"left\"> Gender (0 = female; 1 = male)</td><td char=\".\" align=\"char\">7.10</td><td char=\".\" align=\"char\">3.67</td><td char=\".\" align=\"char\">13.73</td><td char=\".\" align=\"char\">0.40</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">5.64</td><td align=\"left\">***</td></tr><tr><td align=\"left\"> Age at Beginning of Commitment</td><td char=\".\" align=\"char\">0.86</td><td char=\".\" align=\"char\">0.65</td><td char=\".\" align=\"char\">1.14</td><td char=\".\" align=\"char\">− 0.11</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">− 0.92</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Traumatic Experiences</td><td char=\".\" align=\"char\">1.36</td><td char=\".\" align=\"char\">1.12</td><td char=\".\" align=\"char\">1.66</td><td char=\".\" align=\"char\">0.19</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">2.35</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> MAYSI Alcohol/Drug Use</td><td char=\".\" align=\"char\">1.09</td><td char=\".\" align=\"char\">0.97</td><td char=\".\" align=\"char\">1.23</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">1.23</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Angry-Irritable</td><td char=\".\" align=\"char\">1.06</td><td char=\".\" align=\"char\">0.92</td><td char=\".\" align=\"char\">1.22</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">0.70</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Depressed-Anxious</td><td char=\".\" align=\"char\">1.00</td><td char=\".\" align=\"char\">0.81</td><td char=\".\" align=\"char\">1.24</td><td char=\".\" align=\"char\">0.00</td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">0.02</td><td align=\"left\"/></tr><tr><td align=\"left\"> MAYSI Suicidal Ideation</td><td char=\".\" align=\"char\">0.97</td><td char=\".\" align=\"char\">0.81</td><td char=\".\" align=\"char\">1.15</td><td char=\".\" align=\"char\">− 0.03</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">− 0.34</td><td align=\"left\"/></tr><tr><td align=\"left\"> Severity of Previous Delinquency</td><td char=\".\" align=\"char\">0.80</td><td char=\".\" align=\"char\">0.65</td><td char=\".\" align=\"char\">0.98</td><td char=\".\" align=\"char\">− 0.13</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">− 1.92</td><td align=\"left\"/></tr><tr><td align=\"left\"> Previous Conviction</td><td char=\".\" align=\"char\">2.04</td><td char=\".\" align=\"char\">1.27</td><td char=\".\" align=\"char\">3.29</td><td char=\".\" align=\"char\">0.15</td><td char=\".\" align=\"char\">0.06</td><td char=\".\" align=\"char\">2.52</td><td align=\"left\">*</td></tr><tr><td align=\"left\"> Time at Risk</td><td char=\".\" align=\"char\">1.03</td><td char=\".\" align=\"char\">1.01</td><td char=\".\" align=\"char\">1.05</td><td char=\".\" align=\"char\">0.32</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">3.26</td><td align=\"left\">**</td></tr><tr><td align=\"left\"> Time since Intake</td><td char=\".\" align=\"char\">0.97</td><td char=\".\" align=\"char\">0.95</td><td char=\".\" align=\"char\">1.00</td><td char=\".\" align=\"char\">− 0.15</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">− 1.84</td><td align=\"left\"/></tr><tr><td align=\"left\"> AIC, BIC, R<sup>2</sup></td><td char=\".\" align=\"char\">11,852.74</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">11,892.46</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.43</td><td align=\"left\">***</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>All p-values from analyses with continuous data are adjusted for multiple comparisons using a Holm-Bonferroni sequential correction. + Mean age among N = 168 with previous conviction</p><p>*** <italic>p</italic> &lt; .001, ** <italic>p</italic> &lt; .01, * <italic>p</italic> &lt; .05</p></table-wrap-foot>", "<table-wrap-foot><p><italic>N</italic> = 354. *** <italic>p</italic> &lt; .001, ** <italic>p</italic> &lt; .01, * <italic>p</italic> &lt; .05 Parameters are standardized; Analyses used cluster-robust standard errors and FIML-Estimation with Montecarlo integration for dichotomous outcomes</p></table-wrap-foot>", "<fn-group><fn id=\"Fn1\"><label>1</label><p id=\"Par34\">The results regarding nationality are difficult to interpret for various reasons. Firstly, (cultural/ethnic) minority groups are defined in the international literature in various ways, e.g., race, ethnicity, nationality, country of birth. Secondly, as far as we know, there are no numbers on nationality among child welfare youths in Switzerland. Numbers from the Swiss Federal Statistical Office show that in 2010 about 21% of young people between the ages of 10 and 18 had a non-Swiss nationality (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.pxweb.bfs.admin.ch/pxweb/de/px-x-0102020000_103/px-x-0102020000_103/px-x-0102020000_103.px\">https://www.pxweb.bfs.admin.ch/pxweb/de/px-x-0102020000_103/px-x-0102020000_103/px-x-0102020000_103.px</ext-link>). As far as convictions and residential placement are concerned, this percentage was 29% (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bfs.admin.ch/bfs/de/home/statistiken/kriminalitaet-strafrecht/strafjustiz/jugend-erwachsenenurteile.assetdetail.8946454.html\">https://www.bfs.admin.ch/bfs/de/home/statistiken/kriminalitaet-strafrecht/strafjustiz/jugend-erwachsenenurteile.assetdetail.8946454.html</ext-link>) and 36% (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bfs.admin.ch/bfs/de/home/statistiken/kriminalitaet-strafrecht/justizvollzug/platzierte-jugendliche.assetdetail.8947222.html\">https://www.bfs.admin.ch/bfs/de/home/statistiken/kriminalitaet-strafrecht/justizvollzug/platzierte-jugendliche.assetdetail.8947222.html</ext-link><underline>)</underline> respectively for this age group in the same year. Our results regarding non-Swiss nationality of the juvenile justice sample are lower than the conviction and residential placement rates of the Swiss Federal Statistical Office. This could be due to the fact that some juveniles with a non-Swiss nationality were unable to complete the questionnaires due to insufficient knowledge of the French, German and Italian language and were therefore excluded from participation. The same could apply to the child welfare sample, which, with 14% of non-Swiss nationals, is underrepresented in relation to the general population.</p></fn><fn id=\"Fn2\"><label>2</label><p id=\"Par37\">It should be taken into account that the concept of trauma-informed care is still under development and is being interpreted in different ways by various authors and agencies.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[{"label": ["2."], "surname": ["Wilson", "Berent", "Donenberg", "Emerson", "Rodriguez", "Sandesara"], "given-names": ["HW", "E", "GR", "EM", "EM", "A"], "article-title": ["Trauma History and PTSD Symptoms in Juvenile Offenders on Probation"], "source": ["Vict Offender"], "year": ["2013"], "volume": ["8"], "issue": ["4"], "fpage": ["465"], "lpage": ["477"], "pub-id": ["10.1080/15564886.2013.835296"]}, {"label": ["4."], "surname": ["Cheng", "Li"], "given-names": ["TC", "QY"], "article-title": ["Adolescent delinquency in child welfare system: a multiple disadvantage model"], "source": ["Children Youth Serv Rev"], "year": ["2017"], "volume": ["73"], "fpage": ["205"], "lpage": ["212"], "pub-id": ["10.1016/j.childyouth.2016.12.018"]}, {"label": ["5."], "surname": ["Lee", "Villagrana"], "given-names": ["SY", "M"], "article-title": ["Differences in risk and protective factors between crossover and non-crossover youth in juvenile justice"], "source": ["Children Youth Serv Rev"], "year": ["2015"], "volume": ["58"], "fpage": ["18"], "lpage": ["27"], "pub-id": ["10.1016/j.childyouth.2015.09.001"]}, {"label": ["6."], "mixed-citation": ["Kuissink D. Advies plaatsing van jeugdigen met strafrechtelijke en met civielrechtelijke titel in gesloten voorzieningen - Placement in secure facilities of juveniles under criminal law and juveniles under civil law. Advisory report for the Dutch Minister for Legal Protection and the Dutch Minister for Health, Welfare and Sport. Council for the Administration of Criminal Justice and Protection of Juveniles; 2018 2018\u201302\u20132. Report No.: 06\u201352872158."]}, {"label": ["7."], "surname": ["Aebersold"], "given-names": ["P"], "source": ["Schweizerisches Jugendstrafrecht"], "year": ["2017"], "publisher-loc": ["Berlin"], "publisher-name": ["St\u00e4mpfli"]}, {"label": ["8."], "mixed-citation": ["Schmid M, K\u00f6lch M, Fegert JM, Schmeck K. Abschlussbericht f\u00fcr den Fachausschuss f\u00fcr die Modellversuche und das Bundesamt f\u00fcr Justiz. Zusammenfassung der wichtigsten Ergebnisse und Erkenntnisse des Modellversuchs Abkl\u00e4rung und Zielerreichung in station\u00e4ren Massnahmen (MAZ.). Basel: Universit\u00e4re Psychiatrische Kliniken Basel; 2013."]}, {"label": ["9."], "surname": ["Averdijk", "Ribeaud", "Eisner"], "given-names": ["M", "D", "M"], "article-title": ["The long-term effects of out-of-home placement in late adolescence: a propensity score matching analysis among Swiss youths"], "source": ["Longitud Life Course"], "year": ["2018"], "volume": ["9"], "issue": ["1"], "fpage": ["30"], "lpage": ["57"]}, {"label": ["10."], "mixed-citation": ["Zatti KB. Das Pflegekinderwesen in der Schweiz. Analyse, Qualit\u00e4tsentwicklung und Professionalisierung. Expertenbericht im Auftrag des Bundesamtes f\u00fcr Justiz. [Foster care in Switzerland. Analysis, development of quality and professionalisation. Expert report on behalf of the Federal Department of Justice]. 2005 2005."]}, {"label": ["11."], "mixed-citation": ["Bundesamt f\u00fcr Justiz. Leitfaden: Neuanerkennung und periodische \u00dcberpr\u00fcfung der Anerkennungsvoraussetzungen. Bundesamt f\u00fcr Justiz BJ; 2017 2017."]}, {"label": ["12."], "surname": ["Gabriel", "Keller", "Bolter", "Martin-Blachais", "S\u00e9raphin"], "given-names": ["T", "S", "F", "M-P", "G"], "article-title": ["Out of home care in France and Switzerland"], "source": ["Psychosocial Intervention"], "year": ["2013"], "volume": ["22"], "issue": ["3"], "fpage": ["215"], "lpage": ["225"], "pub-id": ["10.5093/in2013a25"]}, {"label": ["13."], "mixed-citation": ["Darbellay S, Maillard C, Zoder I. Risikofaktoren f\u00fcr eine Wiederverurteilung straff\u00e4lliger Minderj\u00e4hriger im Erwachsenenalter 1999\u20132015. Neuchatel: Schweizerisches Bundesamt f\u00fcr Statistik; 2018 2018. Report No.: 1710\u20131501\u201305."]}, {"label": ["15."], "surname": ["Bronsard", "Lancon", "Loundou", "Auquier", "Rufo", "Simeoni"], "given-names": ["G", "C", "A", "P", "M", "MC"], "article-title": ["Prevalence rate of DSM mental disorders among adolescents living in residential group homes of the French Child Welfare System"], "source": ["Children Youth Serv Rev"], "year": ["2011"], "volume": ["33"], "issue": ["10"], "fpage": ["1886"], "lpage": ["1890"], "pub-id": ["10.1016/j.childyouth.2011.05.014"]}, {"label": ["17."], "surname": ["Schmid", "Goldbeck", "Nuetzel", "Fegert"], "given-names": ["M", "L", "J", "JM"], "article-title": ["Prevalence of mental disorders among adolescents in German youth welfare institutions"], "source": ["Child Adolesc Psychiatry Mental Health"], "year": ["2008"], "volume": ["2"], "issue": ["1"], "fpage": ["2"], "pub-id": ["10.1186/1753-2000-2-2"]}, {"label": ["19."], "surname": ["Jaritz", "Wiesinger", "Schmid"], "given-names": ["C", "D", "M"], "source": ["Traumatische Lebensereignisse bei Kindern und Jugendlichen in der station\u00e4ren Jugendhilfe : Trauma & Gewalt"], "year": ["2008"], "volume": ["2"], "issue": ["4"], "fpage": ["266"], "lpage": ["277"]}, {"label": ["25."], "surname": ["Ryan", "Testa"], "given-names": ["JP", "MF"], "article-title": ["Child maltreatment and juvenile delinquency: Investigating the role of placement and placement instability"], "source": ["Children and Youth Services Review"], "year": ["2005"], "volume": ["27"], "issue": ["3"], "fpage": ["227"], "lpage": ["249"], "pub-id": ["10.1016/j.childyouth.2004.05.007"]}, {"label": ["26."], "surname": ["Halemba", "Siegel"], "given-names": ["G", "G"], "source": ["Doorways to delinquency: Multi-system involvement of delinquent youth in King County (Seattle, WA)"], "year": ["2011"], "publisher-loc": ["Pittsburgh"], "publisher-name": ["National Center for Juvenile Justice"]}, {"label": ["27."], "surname": ["D\u00f6litzsch", "Fegert", "K\u00fcnster", "K\u00f6lch", "Schmeck", "Schmid"], "given-names": ["C", "JM", "A", "M", "K", "M"], "article-title": ["Mehrfachdiagnosen bei Schweizer Heimjugendlichen"], "source": ["Kindheit und Entwicklung"], "year": ["2014"], "volume": ["23"], "issue": ["3"], "fpage": ["140"], "lpage": ["150"], "pub-id": ["10.1026/0942-5403/a000140"]}, {"label": ["32."], "surname": ["Chen"], "given-names": ["XJ"], "article-title": ["The link between juvenile offending and victimization the influence of risky lifestyles, social bonding, and individual characteristics"], "source": ["Youth Violence Juvenile Justice"], "year": ["2009"], "volume": ["7"], "issue": ["2"], "fpage": ["119"], "lpage": ["135"], "pub-id": ["10.1177/1541204008328799"]}, {"label": ["34."], "mixed-citation": ["Hockenberry S. Juveniles in Residential Placement, 2010. Washington, DC: U.S. Department of Justice, Office of Justice Programs, Office of Juvenile Justice and Delinquency Prevention; 2013."]}, {"label": ["35."], "surname": ["Wibbelink", "Hoeve", "Stams", "Oort"], "given-names": ["CJM", "M", "GJJM", "FJ"], "article-title": ["A meta-analysis of the association between mental disorders and juvenile recidivism"], "source": ["Aggress Violent Beh"], "year": ["2017"], "volume": ["33"], "fpage": ["78"], "lpage": ["90"], "pub-id": ["10.1016/j.avb.2017.01.005"]}, {"label": ["36."], "surname": ["Widom"], "given-names": ["CS"], "article-title": ["Long-term impact of childhood abuse and neglect on crime and violence"], "source": ["Clin Psychol Sci Pract"], "year": ["2017"], "volume": ["24"], "issue": ["2"], "fpage": ["186"], "lpage": ["202"]}, {"label": ["38."], "surname": ["Vaughn", "Shook", "McMillen"], "given-names": ["MG", "JJ", "JC"], "article-title": ["Aging out of foster care and legal involvement: toward a typology of risk"], "source": ["Soc Serv Rev"], "year": ["2008"], "volume": ["82"], "issue": ["3"], "fpage": ["419"], "lpage": ["446"], "pub-id": ["10.1086/592535"]}, {"label": ["39."], "surname": ["Carr", "McAlister", "Mendes", "Snow"], "given-names": ["N", "S", "P", "P"], "article-title": ["The Double-Bind: Looked After Children, Care Leavers and Criminal Justice"], "source": ["Young People Transitioning from Out-of-Home Care: International Research, Policy and Practice"], "year": ["2016"], "publisher-loc": ["London"], "publisher-name": ["Palgrave Macmillan UK"], "fpage": ["3"], "lpage": ["21"]}, {"label": ["40."], "surname": ["Lindquist", "Santavirta"], "given-names": ["MJ", "T"], "article-title": ["Does placing children in foster care increase their adult criminality?"], "source": ["Labour Economics"], "year": ["2014"], "volume": ["31"], "fpage": ["72"], "lpage": ["83"], "pub-id": ["10.1016/j.labeco.2014.10.001"]}, {"label": ["41."], "surname": ["Stewart", "Waterson"], "given-names": ["A", "E"], "source": ["Dennison S"], "year": ["2002"], "publisher-loc": ["Pathways from child maltreatment to juvenile offending"], "publisher-name": ["Australian Institute of Criminology Canberra"], "fpage": ["2002"]}, {"label": ["42."], "surname": ["Vinnerljung", "Sallnas"], "given-names": ["B", "M"], "article-title": ["Into adulthood: a follow-up study of 718 young people who were placed in out-of-home care during their teens"], "source": ["Child Fam Soc Work"], "year": ["2008"], "volume": ["13"], "issue": ["2"], "fpage": ["144"], "lpage": ["155"], "pub-id": ["10.1111/j.1365-2206.2007.00527.x"]}, {"label": ["43."], "surname": ["Friedrich", "Schmid"], "given-names": ["RI", "M"], "article-title": ["Pflegefamilie oder Heim"], "source": ["P\u00e4diatrie"], "year": ["2014"], "volume": ["1"], "issue": ["14"], "fpage": ["25"], "lpage": ["30"]}, {"label": ["46."], "surname": ["Achenbach"], "given-names": ["TM"], "source": ["Manual for the Child Behavior Checklist/4\u201318 and 1991 profile"], "year": ["1991"], "publisher-loc": ["Burlington, VT"], "publisher-name": ["University of Vermont, Department of Psychiatry"]}, {"label": ["47."], "surname": ["Achenbach"], "given-names": ["TM"], "source": ["Manual for the young adult self-report and young adult behavior checklist"], "year": ["1997"], "publisher-loc": ["Vermont"], "publisher-name": ["University of Vermont Department of Psychiatry"]}, {"label": ["48."], "surname": ["Grisso", "Fusco", "Paiva-Salisbury", "Perrauot", "Williams", "Barnum"], "given-names": ["T", "S", "M", "R", "V", "R"], "source": ["The Massachusetts youth screening instrument-version 2 (MAYSI-2): comprehensive research review"], "year": ["2012"], "publisher-loc": ["Worcester, MA"], "publisher-name": ["University of Massachusetts Medical School"]}, {"label": ["49."], "surname": ["Ford", "Chapman", "Pearson", "Borum", "Wolpaw"], "given-names": ["JD", "JF", "G", "R", "JM"], "article-title": ["Psychometric Status and Clinical Utility of the MAYSI-2 with Girls and Boys in Juvenile Detention"], "source": ["J Psychopathol Behav Assess"], "year": ["2007"], "volume": ["30"], "issue": ["2"], "fpage": ["87"], "lpage": ["99"], "pub-id": ["10.1007/s10862-007-9058-9"]}, {"label": ["51."], "mixed-citation": ["Boers K, Reinecke J. Delinquenz im Jugendalter. Erkenntnisse einer M\u00fcnsteraner L\u00e4ngsschnittstudie. Boers K RJ, editor. M\u00fcnster: Waxmann; 2007."]}, {"label": ["52."], "mixed-citation": ["Muth\u00e9n BO, Muth\u00e9n LK. Mplus (Version8.2). [Computer software] Los Angeles, CA: Muthen & Muthen. 2018."]}, {"label": ["53."], "mixed-citation": ["IBM Corporation. IBM SPSS Statistics for Windows. 25.0. ed. Aemonk, NY2017."]}, {"label": ["54."], "mixed-citation": ["Gaetano J. Holm-Bonferroni sequential correction: an EXCEL calculator (1.3)[Microsoft Excel workbook]. 2018."]}, {"label": ["56."], "surname": ["Muth\u00e9n", "Muth\u00e9n"], "given-names": ["BO", "LK"], "source": ["Mplus User\u2019s Guide: Statistical Analysis with Latent Variables: User\u2019s Guide"], "year": ["2018"], "edition": ["7"], "publisher-loc": ["Los Angeles, CA"], "publisher-name": ["Muth\u00e9n & Muth\u00e9n"]}, {"label": ["57."], "mixed-citation": ["Lammers S, Soe-Agnie S, De Haan H, Bakkum G, Pomp E, Nijman H. Middelengebruik en criminaliteit: een overzicht. 2014."]}, {"label": ["58."], "surname": ["Dowden", "Brown"], "given-names": ["C", "SL"], "article-title": ["The role of substance abuse factors in predicting recidivism: A meta-analysis"], "source": ["Psychol Crime Law"], "year": ["2002"], "volume": ["8"], "issue": ["3"], "fpage": ["243"], "lpage": ["264"], "pub-id": ["10.1080/10683160208401818"]}, {"label": ["62."], "mixed-citation": ["Aebi M, Bessler C, Steinhausen HC. A Cumulative Substance Use Score as a Novel Measure to Predict Risk of Criminal Recidivism in Forensic Juvenile Male Outpatients. Child Psychiatry Hum Dev. 2020."]}, {"label": ["63."], "surname": ["Goddard", "Pooley"], "given-names": ["T", "JA"], "article-title": ["The impact of childhood abuse on adult male prisoners: a systematic review"], "source": ["J Police Crim Psychol"], "year": ["2018"], "volume": ["34"], "issue": ["2"], "fpage": ["215"], "lpage": ["230"], "pub-id": ["10.1007/s11896-018-9260-6"]}, {"label": ["64."], "surname": ["Ogloff", "Cutajar", "Mann", "Mullen", "Wei", "Hassan"], "given-names": ["JR", "MC", "E", "P", "FTY", "HAB"], "article-title": ["Child sexual abuse and subsequent offending and victimisation: A 45 year follow-up study"], "source": ["Trends Issues Crime Criminal Justice"], "year": ["2012"], "volume": ["440"], "fpage": ["1"]}, {"label": ["65."], "mixed-citation": ["Kerig PK, Becker SP. 12 Early Abuse and Neglect as Risk Factors for the Development of Criminal and Antisocial Behavior. The Development of Criminal and Antisocial Behavior2015. p. 181\u201399."]}, {"label": ["66."], "surname": ["Aebi"], "given-names": ["M"], "article-title": ["Die Pr\u00e4valenz von psychosozialen Belastungen bei jugendlichen Straft\u00e4tern: eine Metaanalyse"], "source": ["Forensische Psychiatrie, Psychologie, Kriminologie"], "year": ["2019"], "volume": ["13"], "issue": ["2"], "fpage": ["166"], "lpage": ["177"], "pub-id": ["10.1007/s11757-019-00525-8"]}, {"label": ["67."], "surname": ["Hopper", "Bassuk", "Olivet"], "given-names": ["EK", "EL", "J"], "article-title": ["Shelter from the storm: Trauma-informed care in homelessness services settings"], "source": ["Open Health Serv Policy J"], "year": ["2010"], "volume": ["3"], "issue": ["2"], "fpage": ["80"], "lpage": ["100"], "pub-id": ["10.2174/1874924001003020080"]}, {"label": ["69."], "surname": ["Schmid"], "given-names": ["M"], "article-title": ["Psychotherapie von Traumafolgest\u00f6rungen im Kontext der station\u00e4ren Jugendhilfe"], "source": ["Traumatherapie bei Kindern und Jugendlichen"], "year": ["2012"], "volume": ["2"], "fpage": ["404"], "lpage": ["440"]}]
{ "acronym": [], "definition": [] }
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CC BY
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2024-01-13 23:36:46
Child Adolesc Psychiatry Ment Health. 2021 Jan 21; 15:2
oa_package/1a/ba/PMC7819213.tar.gz
PMC7853133
32355286
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[ "<title>Subject terms</title>" ]
[ "<p id=\"Par1\">Dear Editor,</p>", "<p id=\"Par2\">DNA N<sup>6</sup>-methyladenine (6mA), one of the most prevalent epigenetic base modifications in prokaryotes,<sup>##REF##5656625##1##</sup> is recently found in multicellular eukaryotes.<sup>##REF##25936838##2##–##REF##27027282##8##</sup> This nucleobase may have epigenetic roles in regulation of retrotransposons, chromatin organization, and so on.<sup>##REF##25936838##2##–##REF##27027282##8##</sup> However, both our group<sup>##REF##28471639##9##</sup> and Greer’s group<sup>##UREF##0##10##</sup> noticed that eukaryotic DNA is easily contaminated with a minute of bacterial DNA, which carries overwhelmingly abundant 6mA (~2% 6mA/dA).<sup>##REF##5656625##1##</sup> This brings great challenges for accurate detection of DNA 6mA in eukaryotes in terms of both sample pretreatments and analytical technologies.<sup>##REF##28471639##9##,##UREF##0##10##</sup> For example, inconsistent with the report of Wu et al.,<sup>##REF##27027282##8##</sup> Schiffers et al. failed to detect 6mA above background levels in mouse embryonic stem (mES) cells using sensitive ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) analysis.<sup>##REF##28371147##11##</sup> To date, it is of intensive interest to seek conclusive evidence to support the prevalence of this post-replicative adenine modification in mammals.</p>", "<p id=\"Par3\">These issues prompted us to re-investigate DNA 6mA in mammalian cells. To provide robust and reliable data, a contamination-free UHPLC-MS/MS technology, which is being developed in our lab, was used for ultrasensitive and accurate detection of 6mA in mammals. We measured genomic 6mA in four cultured mammalian cell lines. We observed 6mA in three human cell lines, including HEK293T cells (~0.7 6mA per 10<sup>7</sup> dA), human mesenchymal stem cells (~2.0 6mA per 10<sup>7</sup> dA), and human embryonic stem cells (hES cells) (~4.0 6mA per 10<sup>7</sup> dA). We also detected 6mA in mES cells (~7.0 6mA per 10<sup>7</sup> dA) (Supplementary information, Fig. ##SUPPL##0##S1a, b##). As detected by specific PCR analysis, no mycoplasma contamination was observed in all these cultured cells (Supplementary information, Fig. ##SUPPL##0##S1c##). However, the detected level of 6mA is ~10 folds lower than previously reported.<sup>##REF##27027282##8##</sup> This might be associated with incomplete release of 6mA from genomic DNA during enzymatic digestion. By spiking 6mA-absent genomic DNA with synthetic 6mA-containing oligonucleotide, we validated a complete release of 6mA by effective enzymatic digestion (Supplementary information, Fig. ##SUPPL##0##S1d##). On the other hand, the culturing conditions might contribute to the low levels of 6mA in mES cells. Both the study by Schiffers et al. and ours used 2i + LIF, whereas the work by Wu et al.<sup>##REF##27027282##8##</sup> used a 2i-absent culturing medium. Along this line, it was reported that prolonged Mek1/2 suppression impaired the developmental potential of ES cells.<sup>##REF##28746311##12##</sup> The replacement of the Mek1/2 inhibitor (PD0325901) with a Src inhibitor (CGP77675) preserved the epigenetic and genomic integrity as well as the developmental potential of mES cells. Hence, five additional culturing conditions were tested (Supplementary information, Fig. ##SUPPL##0##S1e##). However, similar levels of DNA 6mA were detected (4.0–8.0 6mA per 10<sup>7</sup> dA; Supplementary information, Fig. ##SUPPL##0##S1f##). By treatment of late G1-phase arresting agent L-mimosine (Fig. ##FIG##0##1a##), interestingly, we observed an accumulation of genomic 6mA in both mouse (2.7 6mA per 10<sup>6</sup> dA, Fig. ##FIG##0##1d, e##) and human ES cells (1.9 6mA per 10<sup>6</sup> dA, Supplementary information, Fig. ##SUPPL##0##S2a##). We also observed an increase of 6mA in HEK293T cells (Supplementary information, Fig. ##SUPPL##0##S2b##). By using early G1-phase arresting palbociclib, we observed a moderate increase of 6mA (Supplementary information, Fig. ##SUPPL##0##S2c##). For the first time, we reported the accumulation of 6mA in G1 phase.</p>", "<p id=\"Par4\">Next, we exploited unique stable isotope-labeled deoxyadenosine tracing technology to investigate the 6mA origin. Previously, we showed that [<sup>15</sup>N<sub>5</sub>]-2'-deoxyadenosine ([<sup>15</sup>N<sub>5</sub>]-dA) tracer can be incorporated into genomic DNA in the form of [<sup>15</sup>N<sub>4</sub>]-dA.<sup>##REF##28471639##9##</sup> If there is any methyltransferase-dependent 6mA in any cultured cells, [<sup>15</sup>N<sub>4</sub>]-6mA should be detected. Another advantage of this [<sup>15</sup>N<sub>5</sub>]-dA tracing technology is its capacity of discriminating prototype bacterial mycoplasmas, which mainly carry [<sup>15</sup>N<sub>5</sub>]-6mA but not [<sup>15</sup>N<sub>4</sub>]-6mA, from host cells.<sup>##REF##28471639##9##</sup> As revealed by UHPLC-MS/MS analysis, &gt;67% of genomic dA was labeled in the form of [<sup>15</sup>N<sub>4</sub>]-dA (Fig. ##FIG##0##1b, c##). dG was also efficiently labeled (Supplementary information, Fig. ##SUPPL##0##S3a, b##). These results are consistent with our recent work.<sup>##REF##28471639##9##</sup> Despite efficient labeling of dA, we failed to detect any [<sup>15</sup>N<sub>4</sub>]-6mA in three tested cell lines, including mES cells (Fig. ##FIG##0##1d, e##), hES cells and HEK293T cells (Supplementary information, Fig. ##SUPPL##0##S2##), at all cell cycle phases. Moreover, under six culturing conditions (Supplementary information, Fig. ##SUPPL##0##S1e##), we did not detect any [<sup>15</sup>N<sub>4</sub>]-6mA in the genomes of mES cells. Of note, our assay can detect ten labeled 6mA from one human genome. In contrast, we observed [<sup>15</sup>N<sub>4</sub>]-6mA by transfecting HEK293T cells with a plasmid carrying an <italic>E. coli</italic> 6mA methylase <italic>dam</italic> mutant gene (data not shown), which has an activity of 100 times lower than wild type.</p>", "<p id=\"Par5\">To corroborate our results, we used second stable isotope-labeling reagent [<sup>13</sup>CD<sub>3</sub>] L-methionine. This chemical can be in vivo converted into stable isotope-labeled methyl donor S-adenosyl-L-methionine, which must be utilized by possible 6mA methylases to generate DNA N<sup>6</sup>-methylated adenine. If there was any methyltransferase to act on N<sup>6</sup> atom of dA, we could detect the formed [<sup>13</sup>CD<sub>3</sub>]-6mA. Consistent with the above results, we did not detect any [<sup>13</sup>CD<sub>3</sub>]-6mA in the treated mES cells at all cell cycle phases (Fig. ##FIG##0##1g## and Supplementary information, Fig. ##SUPPL##0##S4b##). In contrast, we did observe [<sup>13</sup>CD<sub>3</sub>]-5mC in mES cells ([<sup>13</sup>CD<sub>3</sub>]-5mC/total 5mC: &gt;50%) (Fig. ##FIG##0##1f## and Supplementary information, Fig. ##SUPPL##0##S4a##).</p>", "<p id=\"Par6\">Collectively, all the above results supported that the observed 6mA is independent of the methylases, proving the origin of methylase-independent 6mA.</p>", "<p id=\"Par7\">In view of a lack of methylase-generated 6mA, we proposed that the observed 6mA is caused by DNA polymerase-dependent incorporation. If so, the tested cells should have an ability to incorporate 6mA into their genomes. Indeed, by treating mES cells with deoxyribonucleoside N<sup>6</sup>-methyldeoxyadenosine (50–800 μM) (Supplementary information, Fig. ##SUPPL##0##S5a, b##), we observed dose-dependent incorporation of 6mA into the genome (0.9–3.5 6mA per 10<sup>6</sup> dA). Similarly, by treatment of ribonucleoside N<sup>6</sup>-methyladenosine (m<sup>6</sup>A, 10–50 μM), we also observed a dose-dependent increase in genomic 6mA (4.2–7.1 6mA per 10<sup>6</sup> dA) (Supplementary information, Fig. ##SUPPL##0##S5c, d##). Moreover, the treatment of N<sup>6</sup>-methyladenine base-containing DNA or RNA fragments also increased the level of genomic 6mA in mES cells (Supplementary information, Fig. ##SUPPL##0##S5e, f##). In contrast, the treatment of N<sup>6</sup>-methyladenine base-absent DNA or RNA fragments could not induce any 6mA increase (Supplementary information, Fig. ##SUPPL##0##S5e, f##).</p>", "<p id=\"Par8\">To further provide direct evidence on the incoporation of 6mA into mammalian genome, we used two stable isotope-labeled N<sup>6</sup>-methyladenines (Supplementary information, Fig. ##SUPPL##0##S6a, b##): 2'-deoxyribonucloside [<sup>15</sup>N<sub>5</sub>]-6mA and ribonucleoside [CD<sub>3</sub>]-m<sup>6</sup>A. Both treatments could induce a dramatic increase in genomic 6mA in the respective labeled forms (Supplementary information, Fig. ##SUPPL##0##S6c–e##).</p>", "<p id=\"Par9\">Now the question is how the genome of mES cells incorporates 6mA. To answer this question, we first explored a routinely used high-fidelity Taq DNA polymerase as a substitute for the template-dependent, high-fidelity replication polymerases in mammalian cells. By replacing 2′-deoxyadenosine triphosphate (dATP) with N<sup>6</sup>-methyl-dATP (N<sup>6</sup>mdATP), we indeed observed the incorporation of 6mA in PCR products (Supplementary information, Fig. ##SUPPL##0##S7a, b##). However, when a mixture of N<sup>6</sup>mdATP and dATP with a ratio of 1:1000 was used for PCR amplification, we could not observe any 6mA incorporation (Supplementary information, Fig. ##SUPPL##0##S7b##). These results suggest that high-fidelity polymerases prefer using dATP rather than N<sup>6</sup>mdATP for DNA synthesis.</p>", "<p id=\"Par10\">Next, we turned our attention to one of the template-independent X family DNA polymerases, Pol λ (lambda). Compared to non-synchronized cells, the mRNA expression of Pol λ increased in late G1 phase mES cells (Supplementary information, Fig. ##SUPPL##0##S8a##). Pol λ knockdown effectively reduced both its mRNA expression and the 6mA abundance in non-synchronized and late G1 phase cells (Supplementary information, Fig. ##SUPPL##0##S8b, c##). We further generated two <italic>Pol λ</italic>-knockout mES cell lines (Supplementary information, Fig. ##SUPPL##0##S8d, e##). We treated the cells with L-mimosine to obtain G1 phase-dominant cells, and observed that the abolishment of <italic>pol λ</italic> reduced the 6mA abundance (Fig. ##FIG##0##1h##, indicated as the unsorted G1). Moreover, we sorted out both sub G1 phase and G1 phase L-mimosine-treated mES cells (Supplementary information, Fig. ##SUPPL##0##S9##), and found that genomic 6mA accumulated at both sub G1 and G1 phases (Fig. ##FIG##0##1h##). The depletion of Pol λ caused a dramatic 6mA decrease in both G1 phase and sub G1 phase. Interestingly, Pol λ depletion also impaired the 6mA incorporation capacity of mES cells as revealed by the treatment of extracellular [CD<sub>3</sub>]-m<sup>6</sup>A (Fig. ##FIG##0##1i##).</p>", "<p id=\"Par11\">Noteworthy, as indicated by an increase in the cell number of apoptosis-related sub G1 phase, L-mimosine treatment also increased apoptosis of pol λ-knockout mES cells (Supplementary information, Fig. ##SUPPL##0##S9a–d##). Since Pol λ participates in non-homologous end joining (NHEJ) repair<sup>##REF##18585102##13##</sup> via its BRCT domain and NHEJ accounts for a large proportion of DNA repair in the G1 phase,<sup>##REF##17363343##14##</sup> Pol λ may incorporate 6mA into the genome through NHEJ repair pathway. This may implicate a potential association of 6mA with NHEJ.</p>", "<p id=\"Par12\">To further investigate the origin of DNA 6mA in mES cells, we knocked out a potential methylase gene <italic>mettl4</italic><sup>##REF##30982744##15##</sup> and a demethylase candidate gene <italic>alkbh1</italic><sup>##REF##27027282##8##</sup> by CRISPR/Cas9 technology. We obtained two <italic>mettl4</italic><sup><italic>−/−</italic></sup> mESC strains (Supplementary information, Fig. ##SUPPL##0##S10a, b##), five <italic>alkbh1</italic><sup><italic>−/−</italic></sup> mESC strains (Supplementary information, Fig. ##SUPPL##0##S11a, b, e##) and one <italic>alkbh1</italic><sup><italic>−/−</italic></sup> HEK293T strain (Supplementary information, Fig. ##SUPPL##0##S11c, d, f##). Evidently, <italic>mettl4</italic> knockout could not reduce  the level of genomic 6mA in mES cells (Supplementary information, Fig. ##SUPPL##0##S10c##). By knockout of <italic>alkbh1</italic> gene in mES cells, interestingly, we could see a 6mA decrease in two strains, but a 6mA increase in three strains (Supplementary information, Fig. ##SUPPL##0##S11g##). By knockout of <italic>alkbh1</italic> gene in HEK293T cells, we only observed a 6mA decrease (Supplementary information, Fig. ##SUPPL##0##S11h##). The observation of contrary trends on genomic 6mA abundance in <italic>alkbh1</italic><sup><italic>−/−</italic></sup> strains should reflect a varying adaption of the mES cells to <italic>alkbh1</italic> knockout rather than elimination of a demethylation function. Of note, even accompanying with the increase in 6mA for three <italic>alkbh1</italic><sup><italic>−/−</italic></sup> strains (Supplementary information, Fig. ##SUPPL##0##S11g##), we did not see any stable isotope-labeled 6mA (data not shown). This observation confirmed the absence of methylase-generated DNA 6mA in cultured mES cells. Moreover, by the treatment of [CD<sub>3</sub>]-m6A, we observed genomic incorporation of [CD<sub>3</sub>]-6mA in both mES cells and HEK293T cells (Supplementary information, Fig. ##SUPPL##0##S11i, j##). However, <italic>Alkbh1</italic> knockout could not increase the level of [CD<sub>3</sub>]-6mA in both cells (Supplementary information, Fig. ##SUPPL##0##S11i, j##). Collectively, these results did not support that Alkbh1 plays a role in the elimination of genomically incorporated 6mA.</p>", "<p id=\"Par13\">In this study, we exploited two stable isotope-labeling strategies for indicating either nucleobase adenine (by ([<sup>15</sup>N<sub>5</sub>]-dA) or N<sup>6</sup>-methyl group (by [<sup>13</sup>CD<sub>3</sub>]-L-methionine) to trace methylase-generated genomic 6mA in mammalian ES cells. Adenine methylation would yield genomic 6mA in a form bearing a stable isotope label. Astonishingly, by both labeling strategies, the detected 6mA is present exclusively in the non-labeled form in both human and murine ES cells. We also found that non-labeled genomic 6mA increases with extracellular N<sup>6</sup>-methyladenine base-containing (deoxy)ribonucleosides and RNA or DNA fragments. By the use of either deoxyribonucleoside [<sup>15</sup>N<sub>5</sub>]-6mA or ribonucleoside [CD<sub>3</sub>]-m<sup>6</sup>A as a stable isotope tracer, strikingly, we observed genomic DNA 6mA predominantly in the stable isotope-labeled form. These data consistently support thepresence of DNA polymerase-dependent incorporation of 6mA and the absence of methylase-generated 6mA at least in the tested cells. DNA polymerase λ is identified as one of major polymerases responsible for 6mA accumulation in late G1 phase. Noteworthy, the incorporated DNA 6mA is not altered by depletion of demethylase candidate Alkbh1 or methylase candidate Mettl4. Overall, our data suggest a new origin of DNA N<sup>6</sup>-methyladenine base in mammals, implicating the complexity of this rare base in the context of genome function.</p>", "<title>Supplementary information</title>", "<p>\n\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p><bold>Supplementary information</bold> accompanies this paper at 10.1038/s41422-020-0317-6.</p>", "<title>Acknowledgements</title>", "<p>This study is supported by the National Natural Science Foundation of China (91743201, E0B70316 and 21527901), the Ministry of Science and Technology of China (2018YFC1005003 and Y9L10301), the Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-DQC017), and the K.C. Wong Education Foundation.</p>", "<title>Author contributions</title>", "<p>H.W. conceived the project; H.W., X.L., W.L. designed the experiments; X.L., W.L., Y.L., S.C., B.L., N.Z., J.M., C.L., J.Z., Y.-R.D., G.J., G.-L.X. acquired and/or analyzed the data; H.W., X.L., W.L. drafted the paper.</p>", "<title>Competing interests</title>", "<p id=\"Par14\">The authors declare no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Genomic incorporation of DNA N<sup>6</sup>-methyladenine and the contribution of DNA polymerase λ.</title><p><bold>a</bold> Flow diagram of tracing DNA 6mA in mES cells by [<sup>15</sup>N<sub>5</sub>]-dA or [<sup>13</sup>CD<sub>3</sub>]-L-methionine. (<bold>b</bold>, <bold>c</bold>) UHPLC-MS/MS chromatograms (<bold>b</bold>) and quantification (<bold>c</bold>) of unlabeled dA, [<sup>15</sup>N<sub>4</sub>]-dA, and [<sup>15</sup>N<sub>5</sub>]-dA in the genome of mES cells. (<bold>d</bold>, <bold>e</bold>) UHPLC-MS/MS chromatograms (<bold>d</bold>) and quantification (<bold>e</bold>) of unlabeled 6mA, [<sup>15</sup>N<sub>4</sub>]-6mA, and [<sup>15</sup>N<sub>5</sub>]-6mA in the genome of mES cells. Note: [<sup>15</sup>N<sub>5</sub>]-dA was used as an initiation tracer and would be converted into [<sup>15</sup>N<sub>4</sub>]-dA in genomic DNA.<sup>##REF##28471639##9##</sup> The distinct cell cycle phases are indicated in (<bold>b–e</bold>). (<bold>f</bold>, <bold>g</bold>) UHPLC-MS/MS quantification of [<sup>13</sup>CD<sub>3</sub>]-5mC (<bold>f</bold>) and [<sup>13</sup>CD<sub>3</sub>]-6mA (<bold>g</bold>) in the genomes of non-synchronized and G1 phase ES cells. [<sup>13</sup>CD<sub>3</sub>]-L-methionine was used for tracing stable isotope-labeled methyl group. <bold>h</bold> UHPLC-MS/MS quantification of genomic 6mA levels in <italic>pol λ</italic><sup><italic>+/+</italic></sup> and <italic>pol λ</italic><sup><italic>−/</italic>−</sup> mES cells. The unsorted cells at G1 phase were directly obtained by L-mimosine treatment. The sorted cells at sub G1 and G1 phases were obtained by flow cytometry sorting of the L-mimosine-treated cells. <bold>i</bold> UHPLC-MS/MS quantification of the labeled genomic 6mA ([CD<sub>3</sub>]-6mA) levels in <italic>pol λ</italic><sup><italic>+/+</italic></sup> and <italic>pol λ</italic><sup>−<italic>/</italic>−</sup> mES cells. The cells were treated with [CD<sub>3</sub>]-m<sup>6</sup>A alone or co-treated with [CD<sub>3</sub>]-m<sup>6</sup>A and L-mimosine. ND, not detected.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>" ]
[ "<fn-group><fn><p>These authors contributed equally: Xiaoling Liu, Weiyi Lai, Yao Li</p></fn><fn><p><bold>Change history</bold></p><p>5/25/2020</p><p>A Correction to this paper has been published: 10.1038/s41422-020-0339-0</p></fn></fn-group>" ]
[ "<graphic xlink:href=\"41422_2020_317_Fig1_HTML\" id=\"d32e531\"/>" ]
[ "<media xlink:href=\"41422_2020_317_MOESM1_ESM.pdf\"><caption><p>Supplementary information, Materials and Figures</p></caption></media>", "<media xlink:href=\"41422_2020_317_MOESM2_ESM.xlsx\"><caption><p>Supplementary information, Tables</p></caption></media>" ]
[{"label": ["10."], "surname": ["O\u2019Brown"], "given-names": ["ZK"], "source": ["BMC Genom."], "year": ["2019"], "volume": ["20"], "fpage": ["445"], "lpage": ["459"], "pub-id": ["10.1186/s12864-019-5754-6"]}]
{ "acronym": [], "definition": [] }
15
CC BY
no
2024-01-13 23:36:43
Cell Res. 2021 Jan 30; 31(1):94-97
oa_package/62/c3/PMC7853133.tar.gz
PMC8318878
32214204
[ "<title>Introduction</title>", "<p id=\"Par2\">Alterations within the hematopoietic system are a normal physiological consequence of the healthy aging process [##REF##23746838##1##]. However, sometimes changes have a pathophysiological impact [##UREF##0##2##], for example, in the development of age-related leukemias, such as acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Though detailed molecular mechanisms of the aging hematopoietic system and dysfunctional hematopoiesis are often unknown, physiological, and pathophysiological mechanisms can be very closely associated. In this context, hematopoietic stem cell (HSC) changes in relation to longevity is a major focus of current research interests, not only because these cells have the potential to become leukemic as a result of genetic alterations, but also because progenitors may reacquire stem-cell characteristics such as self-renewal in the development of leukemic disease. Known hallmarks of aging include epigenetic alterations and mitochondrial dysfunction. Both changes can be influenced by deacetylation processes.</p>", "<p id=\"Par3\">Sirtuins are a family of NAD<sup>+</sup>-dependent protein deacetylases. Seven sirtuins were described in mammals which are associated to many cellular activities, including energy metabolism, stress resistance, maintenance of genomic stability, aging, and tumorigenesis. Since histone acetylation undergoes dynamic changes during differentiation, the sirtuins have been linked to the development and differentiation of embryonic or HSCs [##REF##28994177##3##]. Recently, SIRT2 was established as SIRT2 required for HSC maintenance and regenerative capacity at an old age by repressing the activation of the NLRP3 inflammasome in HSCs cell autonomously [##REF##30673616##4##]. Mohrin et al. described the interaction of the class III histone deacetylase sirtuin 7 (SIRT7) with nuclear respiratory factor 1 (NRF1) in murine HSCs [##REF##25792330##5##–##REF##25857811##7##]. High SIRT7 levels were typically found in young adult HSCs. They inhibited NRF1-mediated transcription initiation of mitochondrial ribosomal proteins. As a result, mitochondrial biogenesis and energy metabolism were low, and HSCs remained quiescent ensuring HSC longevity. Conversely, older HSCs had low SIRT7 levels. NRF1 was active and mitochondrial biogenesis and energy metabolism were increased. This resulted in enhanced cell proliferation—a hallmark of cancer development [##REF##21376230##8##]. In addition to the results of Mohrins et al., Vazquez et al. described accelerated aging and genome instability in SIRT7 knockout mice [##REF##28406750##9##]. Next to the described role in aging and proliferation with tumorigenesis, it plays an important role in ribosome biogenesis, general transcription regulation, metabolic homeostasis, stress response, and genome stability [##REF##28067587##10##]. The first characterization of the SIRT7 gene was performed by Voelter-Mahlknecht et al. [##REF##16525639##11##], who localized the gene on chromosome 17q25.3—a region frequently altered in acute leukemias and lymphomas. The genomic sequence has a length of 6.2 kb with ten exons and one alternative exon 3a. The flanking promoter region is TATA- and CCAAT-boxless and lacks CpG islands. Ensembl Genome Browser searches revealed 21 splice variants in transcription products (ENSG00000187531) with only two protein-coding variants (SIRT7-210 and SIRT7-201). The UniProtKB database indicates three protein-coding isoforms (Q9NRC8; isoform 1 44.9 kDa, isoform 2 20.4 kDa, and isoform 3 35.9 kDa). Detailed information about principles of promoter regulation, potential exon-skipping mechanisms, posttranscriptional or posttranslational modifications, and processing are currently unknown.</p>", "<p id=\"Par4\">The following study aimed to investigate the age dependency of intracellular SIRT7 levels in human hematopoietic cells and the association of low SIRT7 levels to the age-dependent malignant myeloid stem-cell disorders AML and CML. Moreover, the regulation of the SIRT7 promoter by transcription factors CCAAT-enhancer-binding proteins (C/EBP) α, β, and ε, and the effects on the functions and pathomechanisms mediated by SIRT7 were investigated.</p>", "<title>Patients and methods</title>", "<p id=\"Par5\">All chemicals used were of analytical or cell culture grades. All oligonucleotides were purchased from Eurofins Genomics (Ebersberg, Germany).</p>", "<title>Preparation of patient samples</title>", "<p id=\"Par6\">Peripheral blood or bone marrow samples were obtained from healthy donors (<italic>n</italic> = 169), CML (<italic>n</italic> = 78), or AML (<italic>n</italic> = 113) patients and collected in EDTA-tubes. Leukocytes were isolated after erythrocyte lysis according to standard protocols. RNA extraction of leukocytes and cell lines was performed using TRIzol reagent (Invitrogen, Carlsbad, USA) as previously described [##REF##2440339##12##]. Complementary DNA was synthesized with the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, USA) using random hexameric primers. This study was approved by the Jena University Hospital ethics committee (No. 3944-12/12).</p>", "<title>Quantitative real-time PCR (qRT-PCR)</title>", "<p id=\"Par7\">SYBR green‐based analyses were performed using the Mastercycler® ep realplex Real‐time PCR System (Eppendorf, Hamburg, Germany). Primers and conditions are described in Supplementary Table ##SUPPL##0##1##. Housekeeper-gene in human cells or cell lines was β-glucuronidase. In murine cells β-actin was used.</p>", "<title>Exon-specific PCR: analysis of expressed SIRT7-exons</title>", "<p id=\"Par8\">Transcription of all putative exons of the human SIRT7 gene was analyzed by reverse transcription PCR (RT-PCR) as described in Supplementary Fig. ##SUPPL##0##1##.</p>", "<title>Cells and cell culture</title>", "<p id=\"Par9\">Different CML- and AML-cell lines were used as described in Supplementary Table ##SUPPL##0##2##. Intracellular processes were inhibited by FLT3-ITD inhibitor quizartinib (AC220; 20 nM), BCR-ABL inhibitors nilotinib (20–320 nM), imatinib (2000 nM), and dasatinib (10 nM) (all Cayman Chemical, Ann Arbor, USA). THP-1 monocyte cell differentiation was carried out by 5 days phorbol 12-myristate 13-acetate (PMA, 5 ng/ml, Cayman Chemical) incubation according to the protocol of Tsuchiya et al. [##REF##6949641##13##]. For cytomorphological examination cells were Pappenheim stained [##UREF##1##14##] and visualized using an Axio Scope.A1 microscope (Zeiss, Jena, Germany).</p>", "<title>SIRT7-overexpression: production of pseudoviral particles and cell transduction</title>", "<p id=\"Par10\">For the production of retroviral particles plasmids encoding SIRT7 WT (pBABE-SIRT7 WT) or enzymatically inactive SIRT7 (pBABE-SIRT7 H<sub>187</sub>Y) was used [##REF##16618798##15##, ##REF##24210820##16##]. Details are described in the supplementary information.</p>", "<title>Western blot analysis</title>", "<p id=\"Par11\">Western blot analyses were done as described by Kyhse-Andersen [##REF##6530509##17##]. Details and antibodies are described in the supplementary information (antibodies see Supplementary Table ##SUPPL##0##3##).</p>", "<title>Quantification of cell surface molecules by flow cytometry (FACS)</title>", "<p id=\"Par12\">Measurements were done using the FACSCalibur analyzer and Cell QuestTM software (BD Bioscience, Heidelberg, Germany) according to the manufactures protocol (antibodies see Supplementary Table ##SUPPL##0##3##).</p>", "<title>Plasmids</title>", "<p id=\"Par13\">All used plasmids are described in Supplementary Table ##SUPPL##0##4##. Furthermore, generation of hC/EBPα expression plasmids and SIRT7-promoter reporter-gene plasmids are explained in the supplementary information (Supplementary Fig. ##SUPPL##0##2## and Supplementary Table ##SUPPL##0##4##).</p>", "<title>Transfection and luciferase reporter-gene assays</title>", "<p id=\"Par14\">Ba/F3 cells and AR230 cells were transfected using the Nucleofector<sup>TM</sup> I instrument with the Cell Line Nucleofector® Kit V (Lonza, Basal, Switzerland) according to manufacturer’s protocol using the program X-001. To quantify C/EBP-dependent promoter activities, the firefly luciferase-containing pGL3-promoter SIRT7 plasmid and Renilla luciferase-containing pRL-SV40 plasmid were used. Furthermore, different C/EBP-overexpression plasmids were co-transfected (Supplementary Table ##SUPPL##0##4##). Reporter-gene assay measurements were done as described previously [##REF##16750160##18##].</p>", "<title>Chromatin immunoprecipitation (ChIP)</title>", "<p id=\"Par15\">For in vivo evaluation of SIRT7-promoter binding by C/EBPs the SimpleChIP<sup>®</sup> Enzymatic Chromatin IP Kit with magnetic beads (Cell Signaling Technology, Danvers, USA) was used according to manufacturer’s instructions. Antibodies for immunoprecipitation are described in Supplementary Table ##SUPPL##0##3##. Quantification of C/EBP-bound DNA was performed in a StepOnePlus™ Real-Time PCR system (Applied Biosystems) using primers and conditions summarized in Supplementary Table ##SUPPL##0##1##.</p>", "<title>Survival analysis</title>", "<p id=\"Par16\">Survival in SIRT7 high and SIRT7 low-expressing patient groups was analyzed in FLT3-ITD-mutated and FLT3-wild-type AML patients. Detailed information is given in the supplementary information.</p>", "<title>Statistics</title>", "<p id=\"Par17\">Statistical analyses and generation of figures were carried out using the SigmaPlot 13 software. Detailed information is given in the supplementary information and figures.</p>" ]
[ "<title>Patients and methods</title>", "<p id=\"Par5\">All chemicals used were of analytical or cell culture grades. All oligonucleotides were purchased from Eurofins Genomics (Ebersberg, Germany).</p>" ]
[ "<title>Results</title>", "<title>SIRT7 gene expression in leukocytes of healthy people is age dependent</title>", "<p id=\"Par18\">SIRT7 gene expression was measured in leukocytes of healthy people (Fig. ##FIG##0##1##). Four cohorts were distinguished, each containing a 20-year time span. SIRT7-expression decreased significantly in older cohorts. The 80–99-year-old cohort in comparison to the 20–39-year-old cohort showed an expression level of 29.3% of the initial SIRT7-expression.</p>", "<title>CML: SIRT7-expression and SIRT7 protein levels are influenced by BCR-ABL inhibition</title>", "<p id=\"Par19\">SIRT7-expression in leukocytes of CML patients, especially in younger patient cohorts (20–39 and 40–59 years), was significantly lower than in the leukocytes of healthy people (Fig. ##FIG##1##2a##). No differences were measured in elderly people. After BCR-ABL inhibition by tyrosine kinase inhibitors, SIRT7-expression increased in all age cohorts (Fig. ##FIG##1##2b##). SIRT7-expression also increased after BCR-ABL inhibition in CML cell lines (KCL-22, AR230) (Supplementary Fig. ##SUPPL##0##3A## and Fig. ##FIG##1##2c##). Clinical established tyrosine kinase inhibitors (imatinib, nilotinib, and dasatinib) showed the same effects (Supplementary Fig. ##SUPPL##0##3A##). Furthermore, time- and dosage-specific effects in AR230 cell lines depended directly on BCR-ABL activity as nilotinib-resistant AR230 cells showed no SIRT7-expression response to BCR-ABL inhibition by nilotinib (different mechanisms resulted in nilotinib resistance, Supplementary Table ##SUPPL##0##2##). In line with the RNA expression results, protein levels in western blots increased after BCR-ABL inhibition in nilotinib-sensitive AR230 cells. Principal isoform changes of SIRT7 were not seen in exon-specific PCRs or on protein levels (Supplementary Fig. ##SUPPL##0##3B, C##).</p>", "<title>AML: SIRT7-expression linked with clinical treatment response</title>", "<p id=\"Par20\">SIRT7-expression in bone marrow leukocytes of AML patients was also associated with the clinical treatment response (Fig. ##FIG##2##3a##). It increased to 212% (on average) in times of positive treatment effects (decreased percentage of myeloid blasts in bone marrow). In times of disease progress (increased percentage of myeloid blasts in bone marrow) SIRT7-expression decreased down to 51% compared with pretreatment expression. These effects were the same in FLT3-wt and FLT3-ITD mutated leukemias (Supplementary Fig. ##SUPPL##0##4A##). Furthermore, SIRT7-expression differed in pretreatment monitoring too (Supplementary Fig. ##SUPPL##0##4B##). Patients with disease progress had initial higher SIRT7-expression than patients with positive treatments effects. Two clinical case studies illustrated these effects as well (Fig. ##FIG##2##3b##). Target-based FLT3-ITD inhibition by AC220 and positive treatment response resulted in SIRT7-expression increase in case 2. Two other AC220 treated patients were identified in AML patient cohort too. In all cases positive treatment response resulted in SIRT7-expression increase (Supplementary Fig. ##SUPPL##0##4C##).</p>", "<title>AML: low SIRT7-expression associated with poor prognosis in FLT3-ITD mutated and FLT3-wild-type AML patients</title>", "<p id=\"Par21\">Specific genetic alterations in AMLs determine favorable and unfavorable long-time patient prognosis. Especially different internal tandem duplications in FLT3 receptors (FLT3-ITD) with continuous activation of oncogenic signal transduction are associated with an unfavorable prognosis [##UREF##2##19##]. Gene expression data [##REF##18716133##20##] analyzing the expression level of the mRNA encoding SIRT7 revealed that the overall survival probability of FLT3-ITD-mutated patients with a low SIRT7-expression level (<italic>n</italic> = 24) was significantly lower (<italic>p</italic> = 0.011, log-rank test) than the survival probability of FLT3-ITD-mutated patients with a high SIRT7-expression level (<italic>n</italic> = 57) (Supplementary Fig. ##SUPPL##0##5A##). Similarly, high expression of SIRT7 (<italic>n</italic> = 57) also correlated with the overall survival in FLT3-wild-type AML patients and revealed a significantly better survival prognosis (<italic>p</italic> = 0.048) compared with patients with low SIRT7-expression (<italic>n</italic> = 25) (Supplementary Fig. ##SUPPL##0##5B##). Low SIRT7-expression levels were always associated with a poorer survival prognosis. An additional FLT3-ITD mutation reduced survival probability further.</p>", "<title>AML: SIRT7-expression depends on FLT3-ITD mutation</title>", "<p id=\"Par22\">SIRT7-expression measurements in bone marrow leukocytes of AML at diagnosis showed a higher SIRT7-expression in prognostic favorable FLT3-ITD-negative AML samples in comparison to FLT3-ITD-mutated AML samples. Furthermore, differences are highly significant in female subgroup. In male patients, no significant difference was measurable (Fig. ##FIG##2##3c##). The functional linkage of FLT3-ITD mechanism and SIRT7-expression regulation was also seen in cell culture experiments. FLT3-ITD inhibition by AC220 in human FLT3-ITD-mutated MV4-11 cells resulted in an increased SIRT7-expression. SIRT7-expression in FLT3-ITD-negative THP-1 cells was not affected by treatment (Fig. ##FIG##2##3d##). The similar results were found in murine 32D FLT3-WT or -ITD cells (Fig. ##FIG##2##3e##). Furthermore, inhibition of different typical FLT3-ITD mutations [##REF##26487272##21##] (FLT3-ITD E611V, FLT3-ITD G613E, FLT3-ITD 589/99(12), and FLT3-ITD 598/99(22)) in murine Ba/F3 cells by AC220 supported the results generated with the other cell lines (Fig. ##FIG##2##3f##). FLT3-ITD inhibition by AC220 affected cells to increased SIRT7 mRNA-expression. Only in Ba/F3 FLT3-ITD cells AC220 induced higher normalized SIRT7 protein levels on western blots (Supplementary Fig. ##SUPPL##0##4D##), which was accompanied by a SIRT7-isoform shift: the 21 kDa- and 36 kDa-isoforms were suppressed, whereas the predominant 45 kDa isoform increased and a new 42 kDa-variant emerged. Such an isoform change could also be detected indirectly in the exon-specific PCR performed in human FLT3-ITD expressing MV4-11 cells (Supplementary Fig. ##SUPPL##0##4E##). Exon 1–5-specific PCR identified a new 450 bp product upon AC220 treatment, indicating AC220-dependent expression of an exon 1 containing (longer) SIRT7 transcript. This could be responsible for the switch from shorter to longer protein isoforms.</p>", "<title>SIRT7-expression increased in monocyte differentiation</title>", "<p id=\"Par23\">THP-1 cells were differentiated to monocytes by PMA-incubation. Successful differentiation effects were measured by FACS analysis (increase of CD14/40-positive cells) (Fig. ##FIG##3##4a##). Furthermore, typical monocyte differentiation effects were monitored by cytomorphological changes in microscopy. In time of differentiation SIRT7-expression increased (Fig. ##FIG##3##4b##).</p>", "<p id=\"Par24\">Furthermore, overexpression of SIRT7 in THP-1 cells was also associated with differentiation. THP-1 cells were stably transduced with expression constructs encoding wild-type SIRT7 or enzymatically inactive SIRT7 H187Y [##REF##16618798##15##] (Fig. ##FIG##3##4c##). Overexpression of SIRT7 resulted in stimulation of cell differentiation as demonstrated by increase of CD14/40-positive cells and pronounced monocyte-typical morphological changes (Fig. ##FIG##3##4d##). Interestingly, overexpression of inactive SIRT7 H187Y also resulted in partial stimulation of THP-1 differentiation indicating a noncatalytic function of SIRT7 (Fig. ##FIG##3##4d##).</p>", "<title>C/EBPα, -β, and -ε: missing links in FLT3-ITD/SIRT7, BCR-ABL/SIRT7, and monocyte differentiation/SIRT7 relationship</title>", "<p id=\"Par25\">C/EBPα, -β, and -ε proteins regulate many transcriptional mechanisms in hematopoiesis and stem-cell differentiation. C/EBP dysregulation is often associated with hematopoietic stem-cell disorders showing disturbed cell differentiation [##REF##28179278##22##–##REF##12393674##27##]. Therefore, we investigated the influence of C/EBPs on SIRT7-expression. MatInspector database search results revealed 2 putative C/EBP-binding sites in the SIRT7 gene promoter region (C/EBP1: NC_000017.11 (81,921,507–81,921,521); C/EBP2: NC_000017.11 (81,919,160–81,919,174)). The ChIP results confirmed C/EBPα, -β, and -ε binding to both C/EBP-binding sites in the SIRT7-promoter region (Fig. ##FIG##4##5a##). In the THP-1 monocyte differentiation model all C/EBPs revealed increased binding to both binding sites after induction of differentiation. In MV4-11 FLT3-ITD-AML cells C/EBPα binding to C/EBP2 increased after FLT3-ITD inhibition. C/EBPα binding to C/EBP1 and C/EBPβ/ε binding to C/EBP1/2 were unaffected or reduced. In AR230 CML cells C/EBP binding increased after BCR-ABL inhibition by nilotinib (with the exception of C/EBPε-binding to C/EBP2).</p>", "<p id=\"Par26\">BCR-ABL inhibition in AR230 cells (Supplementary Fig. ##SUPPL##0##6A##) or induction of cell differentiation in THP-1 cells (Supplementary Fig. ##SUPPL##0##6B##) resulted in higher C/EBPα, -β, and -ε gene expression (whereas AC220 treatment of THP-1 cells did not; Supplementary Fig. ##SUPPL##0##6C##). FLT3-ITD inhibition in MV4-11 and Ba/F3 FLT3-ITD-positive cells showed somewhat different results (Supplementary Fig. ##SUPPL##0##6C, D##). Here, C/EBPα and -ε expression increased, but C/EBPβ decreased or was unaffected. However, protein isoform analyses in western blots of murine Ba/F3 cells relativized mRNA-expression findings (Supplementary Fig. ##SUPPL##0##6E##). Only the inhibitory C/EBPβ-LIP isoform protein level was reduced in FLT3-ITD expressing Ba/F3 cells. The activating C/EBPβ-LAP1 isoform was not reduced. All in all, there is a relative increase in the activating C/EBPβ isoform (previously described by Haas et al. [##REF##19958352##25##]).</p>", "<p id=\"Par27\">Finally, all C/EBPα and -β isoforms and full-length C/EBPε were overexpressed in Ba/F3 FLT3-wt, Ba/F3 FLT3-ITD 598/599(22)-AML cells, and AR230 CML cells, which were co-transfected with a C/EBP1/2 binding-site containing SIRT7-promoter-luciferase reporter-gene plasmid. In the AML-cell model (Fig. ##FIG##4##5b##), all C/EBPs induced luciferase activity in FLT3-ITD-mutated and wild-type Ba/F3 cells. The strongest effect was measured in case of C/EBPε-overexpression. The weakest inducer was C/EBPβ-LIP. In the CML cell model (Fig. ##FIG##4##5c##), all C/EBPs significantly upregulated luciferase activity in BCR-ABL AR230 cells. The strongest effect again was induced by C/EBPε-overexpression. In general, C/EBP-overexpression reversed the effects of the mutations.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par28\">This study identified an age-dependent decrease of SIRT7 gene expression in healthy leukocytes. SIRT7-expression in humans was age dependent, as observed in the mouse model. Mohrin et al. showed that low SIRT7 gene expression levels were typical in older adult murine HSCs. SIRT7 downregulation was associated with myeloid cell proliferation and reduced longevity [##REF##25792330##5##–##REF##25857811##7##].</p>", "<p id=\"Par29\">Older humans have a higher risk of developing myeloid stem-cell disorders, such as the well-known age-related leukemias AML and CML. It was therefore hypothesized that SIRT7 gene expression might be reduced at the time of disease diagnosis. Conversely, expression should increase as a result of positive treatment response when hematopoiesis reaches normal conditions and pathomechanisms, which lower SIRT7-expression are inhibited. SIRT7 could be a tumor suppressor and a biomarker to evaluate the treatment effects in myeloid stem-cell disorders. To confirm SIRT7 functions as a tumor suppressor additional experiments are needed.</p>", "<p id=\"Par30\">The results presented here confirm this theory. SIRT7-expression levels at the time of CML and AML diagnosis were significantly lower than in healthy young people. Furthermore, expression levels improved after successful targeted treatment. In cases of relapse or disease progress SIRT7-expression levels in AML decreased significantly. Therefore, after further clinical evaluation SIRT7 may serve as a useful biomarker to control the treatment outcome in myeloid stem-cell disorders in correlating with positive and negative treatment responses.</p>", "<p id=\"Par31\">In CML- and FLT3-ITD-mutated AML-cell lines SIRT7 gene expression increase was seen after successful specific treatment. Direct comparison of BCR-ABL inhibition in nilotinib-sensitive and -resistant AR230 CML cells verified direct association to BCR-ABL-specific effects. SIRT7-expression only increased in nilotinib-sensitive cells after BCR-ABL inhibition. Therefore, the detectable effects on SIRT7-expression in patient samples are likely to depend directly on BCR-ABL activity.</p>", "<p id=\"Par32\">Driver mutation dependency was also found in AML. The activity of typical FLT3-ITD mutations affected SIRT7-expression levels in the Ba/F3 cell line model. After inhibition of FLT3-ITD activity by AC220 SIRT7-expression increased.</p>", "<p id=\"Par33\">Interestingly, on the protein level SIRT7-isoform changes were associated with AC220 treatment effects. FLT3-ITD inhibition in MV4-11 cells induced a new unknown 42 kDa isoform of SIRT7. This could be explained by a change in exon usage by, e.g., exon skipping or altered transcription initiation points. Some indications for such mechanisms were detected through exon-specific PCRs of the SIRT7 gene. A new 450 bp PCR-fragment was found in exon 1–5-specific PCR. This points toward versatile transcriptional and maybe translational regulatory processes involved in isoform shifting. The molecular reason is currently not understood.</p>", "<p id=\"Par34\">FLT3-ITD-dependent reduction of SIRT7-expression was also found in bone marrow samples of AML patients at diagnosis. Interestingly, the effect was more dominant in female patients. Influence of female or male hormones on leukemia development and treatment has already been described [##REF##24778052##28##–##REF##25708485##31##]. But detailed mechanisms are unknown. The AC220 effect on SIRT7 was also evident in a case report: AC220 treatment of an AML relapse with FLT3-ITD mutation after allogeneic stem-cell transplantation resulted in increased SIRT7-expression. Therefore, FLT3-ITD-activity is one important regulator of SIRT7 gene expression.</p>", "<p id=\"Par35\">Next to the direct influence of driver mutations on SIRT7-expression, subgroup analyzes of established gene expression data [##REF##18716133##20##] correlated reduced SIRT7 gene expression with poor prognosis in FLT3-ITD-mutated and FLT3-wild-type AML patients. FLT3-ITD mechanisms are one important part in SIRT7-expression regulation. But data revealed an influence in FLT3-wt leukemias too. Therefore, there have to be other unknown mechanisms next to the important FLT3-ITD pathway.</p>", "<p id=\"Par36\">Our results suggest that SIRT7 gene expression is likely regulated by a disease entity-overlapping mechanism. Low SIRT7 levels were always associated with active disease (nonhealthy conditions), in both BCR-ABL- and FLT3-ITD-positive leukemias. In case of a positive treatment response, SIRT7-expression levels increased (healthy condition). In clinical practice a normalized hematopoiesis resulted in better cell maturation and differentiation. The relationship of high SIRT7-expression levels and cell differentiation, especially monocyte differentiation, was investigated.</p>", "<p id=\"Par37\">Monocyte differentiation of THP-1 cells by PMA resulted in increased SIRT7 gene expression. Induced SIRT7-overexpression also affected cell differentiation, as cytomorphological evaluation showed development toward the monocytic phenotype and induction of CD14/40-positive cells. The strong association with cell differentiation points toward SIRT7 having a role as a monocyte cell differentiation factor. Interestingly, overexpression of catalytically inactive SIRT7 H187Y also partially stimulated cell differentiation pointing a noncatalytic function of SIRT7 in this process. A function of enzymatically inactive SIRT7 has been demonstrated earlier [##REF##23750001##32##]. This observation serves as a basis for our further investigations.</p>", "<p id=\"Par38\">One general well-known factor of regulation of gene expression in myeloid stem-cell disorders is the influence of the transcription factor C/EBPα [##REF##28179278##22##]. Functions of C/EBPα can be inhibited by BCR-ABL-induced upregulation of hRNP-E2 with associated C/EBPα mRNA instability in CML and inhibition of C/EBPα dimerization by FLT3-ITD-induced phosphorylation in AML. Furthermore, C/EBPα influences transcriptional control of myeloid cell differentiation [##REF##17934488##23##].</p>", "<p id=\"Par39\">The C/EBP transcription factor family is encoded by six genes, namely C/EBPα, C/EBPβ, C/EBPε, C/EBPγ, C/EBPδ, and C/EBPζ [##REF##12006103##33##]. The most relevant C/EBPs in myeloid cell differentiation are C/EBPα, C/EBPβ, and C/EBPε [##REF##17934488##23##]. C/EBP homo- or heterodimers bind to typical C/EBP <italic>cis</italic>-elements in promoter regions and influence transcriptional activity [##REF##12006103##33##]. Therefore, the roles of C/EBPα, C/EBPβ, and C/EBPε in SIRT7-promoter regulation were investigated. MatInspector database searches revealed two putative C/EBP-binding sites in the SIRT7-promoter region. ChIP experiments proved binding of all investigated C/EBP variants to these sites. In THP-1 cells, monocyte-typic differentiation increased C/EBP binding. Similar results were found in AR230 CML cells after BCR-ABL inhibition. Only C/EBPε binding decreased. In MV4-11 AML cells FLT3-ITD inhibition by AC220 resulted in increased C/EBPα binding. C/EBPβ and C/EBPε binding decreased after inhibition. C/EBP-specific effects were supported by the results of reporter-gene assays when C/EBP-isoforms were overexpressed. All C/EBPs activated the SIRT7 promoter. The strongest inductor was C/EBPε. The weakest inductor was the well-known inhibitory isoform of C/EBPβ called C/EBPβ-LIP. Furthermore, cell-specific increases of C/EBP-expression corresponded with the ChIP results.</p>", "<p id=\"Par40\">These results indicate a positive involvement of C/EBPs in regulation of SIRT7-expression. However, cell-specific differences were observed in ChIP experiments. Overall, C/EBPα activated SIRT7-expression in all cell models.</p>", "<p id=\"Par41\">In contrast to our results Liu et al. characterized C/EBPα as an inhibitor of SIRT7-expression in hepatocellular carcinoma cells [##REF##26704017##34##]. Furthermore, SIRT7 is upregulated in the majority of cancers [##REF##25923013##35##], including colorectal [##REF##24771643##36##], gastric [##REF##25860861##37##], breast [##REF##25973086##38##, ##REF##25922576##39##], bladder [##REF##24396870##40##], ovarian [##REF##25921180##41##], cervical [##REF##25794641##42##], and hepatocellular [##REF##23079745##43##] cancers. High SIRT7-expression is a predictor of poor survival in various cancers. SIRT7 acts in these cases like an oncogene and not like a tumor suppressor. But conversely, Mc Glynn et al. reported a tumor-suppressive effect of SIRT7 in pancreatic cancers [##REF##26121130##44##]. Furthermore, Hubbi et al. described tumor suppressor effects of SIRT7 by negative regulation of HIF1 and HIF2 transcription [##REF##23750001##32##]. All examples show that SIRT7 mediates ambivalent effects in tumor biology. It can act as an oncogene or tumor suppressor in cell-specific ways. Therefore, C/EBPα could inhibit SIRT7-expression in hepatocellular carcinoma cells [##REF##26704017##34##], yet activate SIRT7-expression in hematopoietic cells.</p>", "<p id=\"Par42\">In summary, the intracellular SIRT7-level in human hematopoietic cells was age dependent. The results were consistent with Mohrin’s data [##REF##25792330##5##]. Low SIRT7 levels were associated to age-dependent malignant myeloid stem-cell disorders. Furthermore, successful treatment of CML or AML increased SIRT7 levels. SIRT7 levels increased during hematopoietic cell differentiation. SIRT7-expression was directly influenced by effects of specific driver mutations (BCR-ABL in AML, FLT3-ITD in AML). The activating effects of C/EBP transcription factors (described in detail by Tsukada [##REF##21257317##45##] and Avellino [##REF##28179278##22##]) might explain the relationship between driver mutations effects, cell differentiation, and SIRT7-expression.</p>", "<p id=\"Par43\">Taken together, SIRT7 is an important element in human hematopoietic cell aging and longevity. Furthermore, it is an important tumor suppressor and could serve potentially as a biomarker for monitoring the clinical treatment response in myeloid stem-cell disorders AML and CML.</p>" ]
[]
[ "<p id=\"Par1\">Molecular alterations within the hematopoietic system influence cellular longevity and development of age-related myeloid stem-cell disorders like acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). A reduced SIRT7-expression in aged murine hematopoietic stem cells (HSC) resulted in reduced longevity and increased proliferation. In this study we investigated age-related changes of SIRT7-expression in healthy humans and relevant pathomechanisms in AML and CML. SIRT7-expression in leukocytes of healthy people decreased in an age-dependent manner. Low SIRT7 mRNA levels were also detected in AML and CML patients. With positive treatment response, SIRT7-expression increased, but showed reduction when patients progressed or relapsed. Pharmacologic inhibition of driver mutations in AML (FLT3-ITD) or CML (BCR-ABL) also restored SIRT7 levels in cell lines and patient samples. Furthermore, SIRT7-expression increased with time during PMA-mediated monocyte differentiation of THP-1 cells. SIRT7-overexpression in THP-1 cells resulted in increased expression of differentiation markers. BCR-ABL, FLT3-ITD, and differentiation-associated SIRT7-expression in general were positively regulated by C/EBPα, -β, and -ε binding to two different C/EBP-binding sites within the SIRT7 promoter. SIRT7 is important in human hematopoietic cell aging and longevity. It might act as tumor suppressor and could potentially serve as general biomarker for monitoring treatment response in myeloid stem-cell disorders.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version of this article (10.1038/s41375-020-0803-3) contains supplementary material, which is available to authorized users.</p>", "<title>Acknowledgements</title>", "<p>The authors thank Volker Ast and Rainer König for AML survival analysis (Hans-Knöll-Institute, Jena, Germany). The authors thank Renate Voit (German Cancer Research Center, Heidelberg, Germany) for providing SIRT7-encoding plasmids. The authors gratefully acknowledge José L. Gutiérrez (Universidad de Concepción, Chile), who provided the human C/EBPβ plasmids. The excellent technical assistance of Anja Waldau and Gabriele Eiselt is gratefully acknowledged. We are also grateful for the different cell lines kindly provided by Francois Xavier Mahon (Département d’Hématologie, Bordeaux, France) and Rebekka Grundler (Department of Internal Medicine III, Technical University of Munich, Germany).</p>", "<title>Funding</title>", "<p id=\"Par44\">AK and TE were supported by the Interdisciplinary Center for Clinical Research (IZKF, Universitätsklinikum Jena, Jena, Germany). OH received funding by the DFG graduate school GK2155 ProMoAge. FHH was supported by the Thuringian state program ProExzellenz (RegenerAging—FSU-I-03/14) of the Thuringian Ministry for Research (TMWWDG). Open Access funding enabled and organized by Projekt DEAL.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par45\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Age-dependent SIRT7-expression in blood leukocytes of healthy people.</title><p>The SIRT7-expression decreased significantly depending on age (means ± SEM; significant differences corresponding to the 20–39 years age cohort (*<italic>p</italic> &lt; 0.05); Mann–Whitney rank-sum test).</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>SIRT7 in CML.</title><p><bold>a</bold> SIRT7-expression was depressed in younger age groups with CML at diagnosis. Healthy people (<italic>n</italic>): 20–39 years (50), 40–59 years (39), 60–79 years (32); CML initial diagnosis: 20–39 years (17), 40–59 years (40), 60–79 years (21) (means ± SEM; Mann–Whitney rank-sum test; differences corresponding to 20–39 year age group *<italic>p</italic> &lt; 0.05; differences between healthy people/CML initial diagnosis <sup>#</sup><italic>p</italic> &lt; 0.05). <bold>b</bold> SIRT7-expression doubled age independently after a 12-months tyrosine kinase inhibitor (TKI; included imatinib, nilotinib, and dasatinib) treatment (means ± SEM; Mann–Whitney rank-sum test). <bold>c</bold> BCR-ABL in AR230 nilotinib-sensitive and -resistant cells were treated with nilotinib. SIRT7-expression was measured after 0, 24, 48, 72 h. The increased expression was associated directly with BCR-ABL inhibition, shown by the almost exclusive effect in nilotinib-sensitive AR230 cells (means ± SEM; value normalization to DMSO control; <italic>n</italic> = 3; Student’s <italic>t</italic> test (*<italic>p</italic> &lt; 0.05)).</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>SIRT7 in AML.</title><p><bold>a</bold> SIRT7-expression in AML patients bone marrow depended on treatment response (mean ± SEM; Mann–Whitney rank-sum test (*<italic>p</italic> &lt; 0.05)). <bold>b</bold> Clinical cases: SIRT7-expression in bone marrow corresponding to AML-treatment events in case of FLT3-wt and FLT3-ITD-positive AML is shown. <bold>c</bold> SIRT7-expression was significantly decreased in bone marrow leukocytes of female FLT3-ITD-positive AML patients at first diagnosis compared with wild-type patients (mean ± SEM; Mann–Whitney rank-sum test (*<italic>p</italic> &lt; 0.05)). <bold>d</bold>–<bold>f</bold> MV4-11 (FLT3-ITD-positive) and THP-1 cells (FLT3-wt) (time course), or 32D FLT3-wt- and FLT3-ITD-cells (time course), or Ba/F3 cells (FLT3-wt or different FLT3-ITDs) (24 h) were treated by AC220. FLT3-ITD inhibition resulted in increase of SIRT7-expression (mean ± SEM; value normalization to 0 h sample (<bold>d</bold>, <bold>e</bold>) or DMSO control (<bold>f</bold>); <italic>n</italic> = 3; significant differences corresponding to 0 h (<bold>d</bold>, <bold>e</bold>), FLT3-wt (<bold>f</bold>) (*<italic>p</italic> &lt; 0.05) or between FLT3-wt vs. ITD (<bold>d</bold>, <bold>e</bold>) (<sup>#</sup><italic>p</italic> &lt; 0.05) were identified with Student’s <italic>t</italic> test).</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>SIRT7 in THP-1 monocyte differentiation.</title><p><bold>a</bold> Monocyte differentiation of THP-1 cells was induced by PMA-incubation and measured in FACS by count of CD14/40<sup>+</sup> cells. Monocyte cytomorphological changes were seen microscopically. <bold>b</bold> During PMA induced THP-1 differentiation SIRT7-expression increased. <bold>c</bold> THP-1 cells stably transduced with pBABE-Sirt7-WT (2; SIRT7 active), pBABE-Sirt7-HY (3; SIRT7 inactive), or empty vector (1). Whole cell lysates of THP-1 cells were subjected to SDS-PAGE, blotted to a PVDF membrane, and analyzed with antibodies recognizing SIRT7 and ß-actin as loading control. <bold>d</bold> The overexpression of enzymatically inactive SIRT7 and especially of active SIRT7 in THP-1 cells was associated with increased detection of CD14/40<sup>+</sup> cells. Cells with active SIRT7-overexpression showed more monocytic cytomorphological features than THP-1 wt. Means ± SEM. <italic>n</italic> = 3. Significant differences corresponding to no differentiation (<bold>a</bold>, <bold>b</bold>) or THP-1 wt (<bold>d</bold>) were identified with Student’s <italic>t</italic> test (*<italic>p</italic> &lt; 0.05).</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>C/EBPα, -β, and -ε: missing links in FLT3-ITD/SIRT7, BCR-ABL/SIRT7, and monocyte differentiation/SIRT7 relationships.</title><p><bold>a</bold> Inhibition- or differentiation-dependent binding of C/EBPα, -β, and -ε to putative C/EBP-binding sites (C/EBP1 and C/EBP2) within the human SIRT7 promoter (<bold>a4</bold>) was analyzed by ChIP. Putative C/EBP-binding sites are located near the TSS of human SIRT7-210 and SIRT7-201 isoforms (protein-coding isoforms, derived from Ensembl Genome Browser). <bold>a1</bold> Monocyte differentiation of THP-1 cells was induced by PMA. <bold>a2</bold> FLT3-ITD in MV4-11 cells was inhibited with AC220. <bold>a3</bold> BCR-ABL in AR230 cells was inhibited by nilotinib (mean ± SEM; <italic>n</italic> = 3; Student’s <italic>t</italic> test (*<italic>p</italic> &lt; 0.05); differences corresponding to no differentiation (<bold>a1</bold>) or inhibitor treatment (<bold>a2</bold>, <bold>a3</bold>); no difference mean ratio 1). <bold>b</bold>, <bold>c</bold> C/EBP-isoform dependent SIRT7-promoter activity was analyzed in reporter-gene assays. C/EBPα or -β isoforms and full-length C/EBPε were overexpressed in Ba/F3 cells with FLT3-wt or FLT3-ITD 598/99(22) and in AR230 cells, resulting in increased promoter activities. The activating effect of C/EBPβ<sub>3</sub> (LIP) was lower than that of LAP isoforms in Ba/F3 cells (means ± SEM; <italic>n</italic> = 3; Student’s <italic>t</italic> test (*<italic>p</italic> &lt; 0.05); differences corresponding to empty vector controls).</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>" ]
[ "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Jörg P. Müller, Thomas Ernst</p></fn><fn><p><bold>Change history</bold></p><p>11/16/2021</p><p>A Correction to this paper has been published: 10.1038/s41375-021-01365-4</p></fn></fn-group>" ]
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[ "<media xlink:href=\"41375_2020_803_MOESM1_ESM.pdf\"><caption><p>Supplemental Material</p></caption></media>" ]
[{"label": ["2."], "surname": ["Chung", "Park"], "given-names": ["SS", "CY"], "article-title": ["Aging, hematopoiesis, and the myelodysplastic syndromes"], "source": ["Hematol Am Soc Hematol Educ Progr"], "year": ["2017"], "volume": ["2017"], "fpage": ["73"], "lpage": ["8"]}, {"label": ["14."], "mixed-citation": ["Begemann H, Rastetter J. Staining methods. In: Heilmeyer L, Begemann H. editors. Atlas of clinical haematology. Berlin, Heidelberg: Springer. 1972. p. 9\u201321."]}, {"label": ["19."], "surname": ["Grafone", "Palmisano", "Nicci", "Storti"], "given-names": ["T", "M", "C", "S"], "article-title": ["An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment"], "source": ["Oncol Rev"], "year": ["2012"], "volume": ["6"], "fpage": ["64"], "lpage": ["74"]}]
{ "acronym": [], "definition": [] }
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2024-01-13 23:35:08
Leukemia. 2020 Mar 25; 34(8):2206-2216
oa_package/04/b3/PMC8318878.tar.gz
PMC8318881
32457353
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[]
[ "<title>Conclusions and future challenges</title>", "<p id=\"Par40\">This review compiles current knowledge of CLL-associated alterations in T-cell distribution, phenotypes, and function. Although it is now well established that CLL disease development elicits accumulation of CD4<sup>+</sup> and CD8<sup>+</sup> T-cells, the role of these cells in either supporting the growth of malignant B-cells or controlling disease progression by adaptive immune responses is still under debate. The observed phenotype and functional properties of CD8<sup>+</sup> T-cells in CLL, along with their oligoclonal expansion, suggest an ongoing CLL-reactive adaptive immunity. Failure of tumor control in CLL is likely due to activation-induced exhaustion of T-cells, as described in other cancers. Of note, the exhaustion phenotype of CD8<sup>+</sup> T-cells in CLL was shown to be more severe in lymphoid organs compared with blood, which is likely explained by the tight interactions of T-cells and CLL cells in pseudofollicles in these tissues, leading to immune cell activation. Therefore, future work investigating T-cells in CLL should aim at verifying results derived from blood samples of CLL patients by analyses of cells from lymphoid organs, mouse models, or tissue culture models that mimic the complex lymphoid microenvironment in CLL.</p>", "<p id=\"Par41\">Novel targeted therapies for CLL, such as the inhibition of BTK or PI3Kδ not only impact on the malignant B-cells, but also target other immune cells in the microenvironment of CLL, including T-cells. To overcome adverse events, it is vital that the impact of such inhibitors on immune cell function, particularly T-cell activity, is monitored closely during clinical trials. This will not only increase the safety for patients but also result in a better understanding of the immune compartment under different treatment regimens, which will be essential for the development of rational combination therapies, including immunomodulating agents.</p>", "<p id=\"Par42\">With the availability of novel technologies, such as multiplexed single-cell protein analysis by mass cytometry or single-cell transcriptome analyses, it has become clear that immune cells are much more heterogeneous than initially thought. One example is the stepwise activation and exhaustion process of CD8<sup>+</sup> effector T-cells, where the presence or absence of distinct intermediate cell stages might have profound effects in terms of response to immunotherapies. Therefore, in-depth analyses of T-cells in cancer patients using these novel techniques will be essential to predict outcome and therapy response. In addition, such studies will help to understand processes of immune cell activity and dysfunction and will, therefore, be essential for the identification of novel therapeutic targets. In light of the disappointing treatment results with immune checkpoint inhibitors and CAR T-cell approaches in CLL, it has become clear that our knowledge about the T-cell compartment in this disease is unsatisfactory, and we must strive to deepen our understanding of immune control and escape in CLL in the future.</p>" ]
[ "<p id=\"Par1\">Chronic lymphocytic leukemia (CLL) is a B-cell malignancy, which is associated with profound alterations and defects in the immune system and a prevalent dependency on the microenvironmental niche. An abnormal T-cell compartment in the blood of CLL patients was already reported 40 years ago. Since then, our knowledge of T-cell characteristics in CLL has grown steadily, but the question of whether T-cells act as pro-tumoral bystander cells or possess anti-tumoral activity is still under debate. Increased numbers of CD4<sup>+</sup> T-helper cell subsets are present in the blood of CLL patients, and T-helper cell cytokines have been shown to stimulate CLL cell survival and proliferation in vitro. In line with this, survival and growth of CLL cells in murine xenograft models have been shown to rely on activated CD4<sup>+</sup> T-cells. This led to the hypothesis that T-cells are tumor-supportive in CLL. In recent years, evidence for an enrichment of antigen-experienced CD8<sup>+</sup> T-cells in CLL has accumulated, and these cells have been shown to control leukemia in a CLL mouse model. Based on this, it was suggested that CD8<sup>+</sup> T-cells recognize CLL-specific antigens and exert an anti-leukemia function. As described for other cancer entities, T-cells in CLL express multiple inhibitory receptors, such as PD-1, and lose their functional capacity, leading to an exhaustion phenotype which has been shown to be more severe in T-cells from secondary lymphoid organs compared with peripheral blood. This exhausted phenotype has been suggested to be causative for the poor response of CLL patients to CAR T-cell therapies. In addition, T-cells have been shown to be affected by drugs that are used to treat CLL, which likely impacts therapy response. This review provides an overview of the current knowledge about alterations of T-cells in CLL, including their distribution, function, and exhaustion state in blood and lymphoid organs, and touches also on the topic of how CLL drugs impact on the T-cell compartment and recent results of T-cell-based immunotherapy. We will discuss potential pathological roles of T-cell subsets in CLL and address the question of whether they foster progression or control of disease.</p>", "<title>Subject terms</title>" ]
[ "<title>CD4<sup>+</sup> T-cells</title>", "<p id=\"Par2\">CD4<sup>+</sup> T-cells are a heterogeneous population of cells, broadly divided into conventional CD4<sup>+</sup> (cCD4<sup>+</sup>) T-cells and forkhead box protein P3 (FOXP3<sup>+</sup>) expressing regulatory T-cells (Treg). The identification of cell type-specific cytokines and functions resulted in the definition of three subsets of cCD4<sup>+</sup> T-helper (Th) cells, namely Th1, Th2, and Th17 T-cells [##REF##18362946##1##, ##UREF##0##2##]. More recently, follicular helper T-cells (Tfh) have been described as the most common Th T-cell subset of lymphoid organs [##REF##18173374##3##]. An overview of subset-defining surface markers, key cytokines, and transcriptional regulators is provided in Fig. ##FIG##0##1##.</p>", "<p id=\"Par3\">The majority of CD4<sup>+</sup> T-cells orchestrate the immune responses of surveilling immune cells, e.g., CD8<sup>+</sup> T-cells and B-cells, against Th-specific pathogens [##REF##18362946##1##]. In contrast to the T-helper subsets, Tregs are thought to be immunosuppressive and reduce the activity of effector T-cells [##REF##30804926##4##]. Recent investigations identified cytotoxic CD4<sup>+</sup> T-cells, which kill target cells by granzyme (Gzm)B and perforins in an antigen-specific manner, especially in conditions of chronic viral infections [##REF##28280496##5##].</p>", "<title>CD4<sup>+</sup> T-cells in CLL</title>", "<title>Phenotype of CD4<sup>+</sup> T-cells in CLL</title>", "<p id=\"Par4\">An enrichment of antigen-experienced memory and effector CD4<sup>+</sup> T-cells accompanied by a relative loss of naïve CD4<sup>+</sup> T-cells has been repeatedly observed in chronic lymphocytic leukemia (CLL) [##UREF##1##6##–##REF##29296521##8##]. Moreover, CLL-associated CD4<sup>+</sup> T-cells express higher levels of the inhibitory receptors programmed cell death protein 1 (PD-1) [##UREF##1##6##, ##REF##27927767##7##, ##REF##23247726##9##–##REF##22190592##11##], CD160 [##REF##23247726##9##], and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) [##REF##29296521##8##] as well as human leukocyte antigen (HLA)-DR as a marker of activation [##UREF##1##6##]. Ki-67, a marker for cell proliferation, was detected at higher levels in CLL-derived CD4<sup>+</sup> T-cells in comparison to control T-cells [##REF##27927767##7##]. Hence, CD4<sup>+</sup> T-cells in the blood of CLL patients seem to be more strongly activated than the T-cells from healthy individuals.</p>", "<title>T-helper cell subsets in CLL</title>", "<p id=\"Par5\">As described above, T-helper cell subsets secrete distinct cytokines and exert specific immunological functions. To understand their role in CLL, the distribution and function of these subsets in patient samples need to be determined. Up until now, such attempts have been largely inconclusive, as both the Th1 and Th2 subsets have been reported as being dominant.</p>", "<p id=\"Par6\">In 2005, Görgün and colleagues investigated T-cell function in CLL. Gene expression profiling (GEP) of sorted T-cells from peripheral blood mononuclear cells (PBMCs) of CLL patients suggested that CD4<sup>+</sup> T-cells are skewed toward a Th2 phenotype due to decreased expression of <italic>c-Jun NH2-terminal kinase</italic><italic> (JNK)</italic> and <italic>p38 mitogen-activated protein kinase (MAPK)</italic> pathway-related genes [##REF##15965501##12##]. This is supported by an increased number of interleukin (IL)-4 secreting CD4<sup>+</sup> T-cells in PBMCs of CLL patients in comparison to cells of healthy controls (HC). Studies using the immunocompetent Eµ-TCL1 mouse model of CLL and after adoptive transfer of TCL1 leukemia (TCL1 AT) into BL/6 wild-type (WT) mice revealed more IL-4 secretion of CD4<sup>+</sup> T-cells in comparison to control mice [##REF##12144536##13##, ##REF##25979947##14##]. Similarly, conditioned medium of CLL cells was able to induce secretion of IL-4 in allogeneic mixed lymphocyte reactions (MLR) of healthy individuals, suggesting that soluble factors secreted by CLL cells trigger Th2 T-cell polarization in vitro [##REF##17972945##15##].</p>", "<p id=\"Par7\">In contrast, ex vivo mitogen stimulation of PBMCs using phorbol 12-myristate-13-acetate (PMA)/ionomycin led to higher percentages of interferon (IFN)γ-expressing Th1-like T-cells in CLL patients compared with HC [##REF##12008083##16##, ##UREF##3##17##], which is in line with data from the TCL1 AT mouse model [##UREF##3##17##]. Based on surface marker expression data, increased absolute numbers of both Th1 and Th2 T-cells have been reported in the blood of CLL patients in comparison to HC [##REF##27927767##7##, ##REF##29296521##8##]. The increase of Th1 T-cells was more pronounced in patients with progressive disease [##REF##27927767##7##]. Recently, expression analysis of surface marker proteins and the master transcriptional regulators T-box transcription factor 21 (TBET) and GATA binding protein 3 (GATA3) in patient samples and the TCL1 AT mouse model revealed a more severe accumulation of Th1-like T-cells compared with Th2 cells in CLL [##UREF##3##17##].</p>", "<p id=\"Par8\">Th1-like T-cells were shown to activate autologous CLL cells and induce their proliferation in an antigen- and IFNγ-dependent manner [##REF##23933259##18##, ##UREF##4##19##]. In contrast, <italic>Tbx21</italic><sup><italic>−/−</italic></sup> bone marrow chimeric mice, which showed a profound reduction in IFNγ-producing Th1 T-cells compared with WT controls, showed a comparable CLL progression to control mice [##UREF##3##17##].</p>", "<p id=\"Par9\">Higher numbers and frequencies of IL-17-expressing Th17 T-cells have been reported in the blood of CLL patients compared with HC which is in line with elevated plasma levels of IL-17 in CLL, although numbers of Th17 T-cells were much lower than Th1 and Th2 cells [##REF##22058222##20##, ##REF##24223764##21##].</p>", "<p id=\"Par10\">It was suggested that Tfh T-cells are tumor-supportive in CLL, as IL-21, a cytokine that is highly expressed by these cells, enhanced CLL cell proliferation in vitro [##REF##23710828##22##, ##REF##24014238##23##]. Frequencies of this CD4<sup>+</sup> T-cell subset were found to be higher in blood and particularly lymph nodes of CLL patients [##REF##23710828##22##–##REF##31506282##25##].</p>", "<p id=\"Par11\">In conclusion, an enrichment of T-helper cell subsets in CLL has been reported by several groups. Reports concerning a higher abundance of Th1 or Th2 T-cells are controversial. To date, functional investigations are only available for Th1 T-cells, which showed that reducing their number in a CLL mouse model has no impact on disease progression.</p>", "<title>Regulatory T-cells in CLL</title>", "<p id=\"Par12\">Increased frequencies and numbers of Tregs in the blood of CLL patients compared with HC have been consistently reported [##REF##27927767##7##, ##REF##29296521##8##, ##REF##22190592##11##, ##REF##17657216##26##–##REF##21463298##34##]. This accumulation was shown to be more pronounced in patients’ lymph nodes [##REF##31506282##25##]. Numbers of Tregs in blood positively correlated with malignant B-cell counts, higher Rai and Binet staging, as well as unfavorable genetics, and served as an independent predictor of time to first treatment [##REF##20880586##29##–##REF##30573773##32##, ##REF##22460620##35##, ##REF##23009220##36##], suggesting a negative impact of Tregs on disease progression. Of note, higher secretion of IL-10 and transforming growth factor (TGF)β of CLL-derived Tregs was observed in comparison to control Tregs [##REF##15914560##27##, ##REF##22101351##28##]. Moreover, Tregs of CLL patients exhibited increased expression of markers for activation and immunosuppression, such as cytotoxic T-lymphocyte associated protein 4 (CTLA-4), which was confirmed in Eµ-TCL1 mice [##REF##19332800##37##] as well as the TCL1 AT mouse model [##REF##15914560##27##, ##REF##30573773##32##, ##REF##29439955##38##–##REF##16094420##40##]. Reducing Tregs by 50% using anti-CD25 antibodies in the TCL1 AT model did not affect CLL progression [##REF##30573773##32##]. However, data are lacking in mouse models with a more efficient depletion of Tregs, e.g., <italic>Foxp3</italic><sup><italic>DTR</italic></sup> mice, in which Tregs can be depleted by injection of diphteria toxin [##REF##17136045##41##]. Such a model would help to clarify the role of these cells in CLL development and progression.</p>", "<p id=\"Par13\">In summary, an increase of Tregs in blood of CLL patients has been observed on numerous occasions, but more functional analyses are still required to elucidate the role of Tregs and other T-helper cell subsets in CLL.</p>", "<title>Influence of CD4<sup>+</sup> T-cells on CLL progression</title>", "<p id=\"Par14\">The role of CD4<sup>+</sup> T-cells in CLL, which accumulate in the blood of patients as the disease progresses [##UREF##3##17##, ##REF##2787679##42##, ##REF##21270444##43##], is discussed with contention. Treatment of CLL cells with Th T-cell-derived factors, such as IFNγ [##REF##7678114##44##], IL-21 [##REF##24014238##23##], IL-4 [##REF##24828787##45##], CD40L [##REF##23933259##18##, ##REF##24014238##23##, ##REF##24828787##45##], or their combinations, either reduced apoptosis or induced proliferation of CLL cells in vitro (Table ##TAB##0##1##). In line with this, a pro-leukemic effect has been suggested, as autologous CD4<sup>+</sup> T-cells but not CD8<sup>+</sup> T-cells were indispensable for inducing CLL cell proliferation in patient-derived xenograft (PDX) mouse models (Table ##TAB##0##1##) [##REF##23933259##18##, ##REF##21385850##46##]. Co-culture of CLL cells with autologous CD4<sup>+</sup> T-cells, which were either depleted of PD-1<sup>+</sup>, TIGIT<sup>+</sup>, or PD-1<sup>+</sup> TIGIT<sup>+</sup> double-positive T-cells, revealed a reduced CLL cell viability in all of the depleted co-cultures, suggesting that these T-cell subsets enhance CLL cell survival [##REF##29296521##8##]. Interestingly, TIGIT<sup>+</sup> CD4<sup>+</sup> T-cells produced higher amounts of IFNγ and IL-10 in a TIGIT-dependent manner than their TIGIT<sup>-</sup> counterparts [##REF##29296521##8##], which is in line with the aforementioned beneficial effects of IFNγ on CLL cells in vitro. Correlations of CD4<sup>+</sup> T-cell counts with patients’ progression-free survival (PFS) revealed lower counts to be associated with increased PFS [##REF##31412798##47##]. Accordingly, a higher proportion of PD-1<sup>+</sup> HLA-DR<sup>+</sup> CD4<sup>+</sup> T-cells, which are enriched in CLL patients, was associated with reduced PFS [##UREF##1##6##].</p>", "<p id=\"Par15\">Analyses of the TCL1 AT mouse model of CLL revealed similar alterations of the CD4<sup>+</sup> T-cell compartment as reported for CLL patients. A relative loss of naïve CD4<sup>+</sup> T-cells and an accumulation of antigen-experienced CD4<sup>+</sup> T-cells, as well as a higher expression of the early activation markers CD69 and PD-1, was noted [##UREF##2##10##], suggesting that this model is a useful tool to investigate the role of CD4<sup>+</sup> T-cells in CLL.</p>", "<p id=\"Par16\">In contrast to the pro-tumoral effects of CD4<sup>+</sup> T-cells described above, an anti-tumoral function of CD4<sup>+</sup> T-cells was suggested by experiments using the Eµ-TCL1 mouse model of CLL. TCL1 AT into GK5 mice that lack CD4<sup>+</sup> T-cells resulted in a faster CLL development compared with respective treatment of WT mice [##REF##26522084##48##]. However, CD4<sup>+</sup> T-cell depletion by injecting CD4-specific antibodies into WT mice that had been transplanted with TCL1 leukemia cells induced no difference in CLL development [##REF##30267008##49##]. These controversial results might be explained by differences in the depletion efficacy of CD4<sup>+</sup> T-cells in GK5 mice versus antibody-treated mice, which might also result in a difference in the degree of simultaneous depletion of other CD4-expressing cell types, such as dendritic cells, which has been observed in GK5 mice [##UREF##6##50##]. In support of a role for CD4<sup>+</sup> T-cells in leukemia control, we have recently shown that these cells are able to control CLL progression in the absence of CD8<sup>+</sup> T-cells in the TCL1 AT mouse model in an <italic>Eomesodermin</italic> (<italic>Eomes)</italic>- and <italic>Il10rb</italic>-dependent manner [##UREF##2##10##]. However, it remains unclear whether cytotoxic CD4<sup>+</sup> T-cells are involved in CLL control in immunocompetent mice with functional CD8<sup>+</sup> T-cells, and whether such cells are of relevance in CLL patients.</p>", "<p id=\"Par17\">In conclusion, in vitro as well as xenograft experiments suggest that CD4<sup>+</sup> T-cells enhance CLL cell survival and proliferation, which is supported by correlations of CD4<sup>+</sup> T-cell counts and clinical outcome. Nevertheless, results of the Eµ-TCL1 mouse model suggest a more diverse role of CD4<sup>+</sup> T-cells within a complex immune microenvironment. As this mouse model might be limited in its ability to reflect the situation in CLL patients, the use of humanized PDX mouse models containing all major immune cell subsets of human origin might help to clarify whether CD4<sup>+</sup> T-cells have a pro- or anti-tumoral role in CLL.</p>", "<title>CD8<sup>+</sup> T-cells</title>", "<title>CD8<sup>+</sup> T-cell subsets and their role in immune responses</title>", "<p id=\"Par18\">CD8<sup>+</sup> T-cells are pivotal for adaptive immunity, for example, in response to a viral infection [##REF##21867926##51##]. Activation of CD8<sup>+</sup> T-cells requires multiple signals that lead to their clonal expansion and differentiation into effector cells. These cells are functionally and phenotypically heterogeneous, and include short-lived effector T-cells (SLEC) that undergo apoptosis once an acute infection is cleared [##REF##21867926##51##], but also effector cells that survive after antigen clearance and form a memory population [##REF##29236683##52##]. CD8<sup>+</sup> T-cells target infected cells by secretion of perforins, which permeabilize the cell membrane of target cells and enable the diffusion of granzymes into target cells, which cause cell death [##REF##23377437##53##]. In addition, CD8<sup>+</sup> T-cells can induce clearance of target cells by induction of apoptosis via FAS ligand (FASL) [##REF##21517838##54##].</p>", "<p id=\"Par19\">Long-lived memory CD8<sup>+</sup> T-cells develop during acute infections from effector cells to provide a fast immunological response upon repeated infections [##REF##29236683##52##]. Effector memory T-cells (T<sub>EM</sub>), which express CD45RO and are negative for C-C motif chemokine receptor 7 (CCR7) in humans, reside in non-lymphoid tissues and retain cytolytic activity [##REF##15032595##55##]. A fraction of T<sub>EM</sub> expresses CD45RA and is therefore defined as T<sub>EMRA</sub>. These cells contain the highest amounts of perforins [##REF##15032595##55##]. In contrast, CD45RO<sup>+</sup>, CCR7<sup>+</sup> central memory (T<sub>CM</sub>) cells home to secondary lymphoid organs and are not capable of immediate effector function [##REF##15032595##55##]. A further subset of memory T-cells, CD103<sup>+</sup> tissue-resident memory T-cells (T<sub>RM</sub>) has been recently identified [##REF##25526304##56##]. T<sub>RM</sub> cells reside in non-lymphoid tissues, such as the skin. They exert first responses after infections in the tissues but do not recirculate [##REF##25526304##56##].</p>", "<title>CD8<sup>+</sup> T-cells in conditions of chronic antigen persistence</title>", "<p id=\"Par20\">During chronic infections and in cancer, the persistence of antigens leads to a gradual loss of CD8<sup>+</sup> T-cell function, a process called T-cell exhaustion [##REF##21739672##57##]. Continuous antigen-driven activation of CD8<sup>+</sup> T-cells causes the upregulation of multiple inhibitory receptors, such as PD-1, CD244 (2B4), lymphocyte activation gene 3 (LAG-3), and CD160 [##REF##21739672##57##]. In addition to enhanced expression of these inhibitory receptors, exhausted T-cells gradually lose their proliferative potential and the ability to produce cytokines such as IL-2, tumor necrosis factor-alpha (TNFα), and IFNγ [##REF##21739672##57##]. These functional changes are associated with a distinct epigenetic profile that differs from that of effector and naïve T-cells, and cannot be reversed by antibody-mediated immune checkpoint blockade targeting PD-1 [##REF##27789795##58##, ##REF##27789799##59##]. PD-1 blockade has been shown to induce proliferation and reactivation of a precursor exhausted CD8<sup>+</sup> T-cell subset with features of self-renewal and the ability to differentiate into terminally exhausted T-cells [##REF##31591533##60##].</p>", "<p id=\"Par21\">Besides in chronic viral infections, for example with human immunodeficiency virus (HIV), CD8<sup>+</sup> T-cell exhaustion has been described in several cancer entities, including non-small cell lung carcinoma (NSCLC) and melanoma [##REF##16921384##61##–##REF##20819923##63##].</p>", "<title>CD8<sup>+</sup> T-cells in CLL</title>", "<p id=\"Par22\">Elevated T-cell numbers in the blood of CLL patients were described as long as 40 years ago [##REF##2787679##42##, ##REF##21270444##43##, ##UREF##7##64##, ##REF##6218347##65##] with the observation of the decrease of the CD4 : CD8 T-cell ratio coming shortly after [##UREF##3##17##, ##REF##2787679##42##, ##REF##30267008##49##, ##REF##6982129##66##–##REF##22397719##70##]. An inversion of the CD4 : CD8 T-cell ratio with values below 1 was found to be associated with shorter time to first treatment (TTFT), shorter overall survival (OS) [##UREF##1##6##, ##REF##27302925##71##], and shorter PFS independently of other prognostic markers [##UREF##1##6##, ##REF##22190592##11##]. Using a ratio of CD8<sup>+</sup> T-cells per monoclonal, malignant B-cell in CLL blood samples, higher relative numbers of CD8<sup>+</sup> T-cells correlated with better OS [##REF##20846097##72##], suggesting that CD8<sup>+</sup> T-cells might participate in leukemia control.</p>", "<p id=\"Par23\">In addition to the altered numbers of CD8<sup>+</sup> T-cells, detailed descriptions of their phenotype and leukemia-associated changes in blood of CLL patients are also available. The CD8<sup>+</sup> T-cells of CLL patients were found to be enriched in antigen-experienced effector memory and effector cells [##UREF##1##6##–##REF##23247726##9##, ##REF##22190592##11##, ##REF##25979947##14##, ##REF##21270444##43##, ##REF##30267008##49##, ##REF##22397719##70##, ##REF##12689926##73##–##UREF##9##75##] and were shown to express markers of T-cell activation such as HLA-DR and CD69 more strongly than cells of healthy individuals [##UREF##1##6##, ##REF##2787679##42##, ##REF##30267008##49##]. Unsupervised computational analysis of the expression levels of 29 proteins analyzed by flow cytometry showed that blood-derived CD8<sup>+</sup> T-cells from CLL patients are phenotypically distinct from those of healthy donors [##REF##30906665##76##].</p>", "<p id=\"Par24\">In line with the concept of T-cell exhaustion as an activation-induced dysfunction, several reports have independently demonstrated increased expression of exhaustion markers. Initiated by Motta et al. [##REF##16094420##40##], and followed by Nunes et al. [##REF##22190592##11##] and Riches et al. [##REF##23247726##9##], higher expression of PD-1 [##REF##23247726##9##, ##REF##22190592##11##, ##REF##30267008##49##, ##REF##27302925##71##, ##REF##23300177##74##, ##REF##26066608##77##, ##REF##28306177##78##], CD160 [##REF##23247726##9##], CD244 [##REF##23247726##9##], T-cell immunoglobulin mucin 3 (TIM-3) [##REF##28306177##78##], killer cell lectin-like receptor G1 (KLRG1) [##UREF##9##75##] and CTLA-4 [##REF##27927767##7##, ##REF##16094420##40##] was reported on CD8<sup>+</sup> T-cells in the blood of CLL patients. Counts of PD-1<sup>+</sup> CD8<sup>+</sup> T-cells were found to positively correlate with CLL burden [##REF##27927767##7##, ##REF##30267008##49##]. These results were recapitulated in the Eµ-TCL1 and TCL1 AT mouse models of CLL, implying that these models are useful to investigate CD8<sup>+</sup> T-cell immunity in CLL [##REF##25979947##14##, ##UREF##3##17##, ##REF##29439955##38##, ##REF##30221064##39##, ##REF##30267008##49##, ##REF##21606964##79##, ##REF##31519123##80##].</p>", "<title>Function or dysfunction of CD8<sup>+</sup> T-cells in CLL</title>", "<p id=\"Par25\">Although detailed phenotypic characterizations of CD8<sup>+</sup> T-cells in CLL are available, investigations of their role during development and progression of CLL are largely missing. The idea of defective or diminished leukemia control by CD8<sup>+</sup> T-cells was supported by the reduced proliferative capacity and target cell lysis observed in patient blood-derived CD8<sup>+</sup> T-cells after T-cell receptor (TCR) engagement ex vivo [##REF##23247726##9##]. Moreover, gene expression analysis of patient-derived CD8<sup>+</sup> T-cells revealed deregulation of actin polymerization, vesicle formation and trafficking, and cytotoxicity-associated pathways, in comparison to control T-cells [##REF##15965501##12##]. Further, CD4<sup>+</sup> and CD8<sup>+</sup> T-cells of CLL patients were found to form impaired immunological synapses with CLL cells. Super-antigen pulsed CLL cells from untreated patients formed less conjugates with autologous T-cells, and a lower F-actin accumulation at the immune synapse was detected in comparison to experiments with healthy donor B- and T-cells [##REF##18551193##81##], which was also seen in a study using T-cells from Eµ-TCL1 mice [##REF##19332800##37##]. Impaired immune synapse formation was further observed when healthy donor-derived T-cells were co-cultured with CLL cells for 48 h, suggesting that this defect is induced by the malignant CLL cells. Blocking inhibitory ligands, such as CD200, programmed cell death 1 ligand 1 (PD-L1), or CD276, with neutralizing antibodies in CLL co-cultures improved this defect of immune synapse formation [##REF##22547582##82##].</p>", "<p id=\"Par26\">In the recent years, several reports suggested an active anti-leukemia immunity by CD8<sup>+</sup> T-cells, as TCR analyses of CLL blood samples or spleens of TCL1 AT mice revealed an enrichment of clonally expanded CD8<sup>+</sup> T-cells [##REF##30267008##49##, ##REF##27904140##83##–##UREF##10##85##] with the major clonotypes persisting for about 2 years in patients [##REF##27904140##83##]. This concept is further supported by an analysis of HLA ligandomes, in which CLL-specific antigens, which cause spontaneous, autologous T-cell activation, were identified [##REF##25548167##86##]. In addition, we recently reported that CD8<sup>+</sup> T-cells are able to control CLL progression in the TCL1 AT model which leads to longer survival of leukemic mice in the presence of these immune cells [##REF##30267008##49##]. This is in line with a recent study that analyzed CLL development in Eµ-TCL1 mice crossed with mice that harbor a B-cell specific knockout of interferon regulatory factor 4 (IRF4) [##REF##31537531##87##]. Lack of IRF4 in malignant B-cells resulted in faster development of CLL, which was associated with a downregulation of major histocompatibility complex (MHC) molecules on CLL cells and reduced activation of T-cells, suggesting an abrogation of T-cell-mediated leukemia control in these mice [##REF##31537531##87##].</p>", "<title>Exhaustion of CD8<sup>+</sup> T-cells in CLL</title>", "<p id=\"Par27\">As mentioned above, a hallmark of T-cell exhaustion is enhanced expression of inhibitory receptors, such as PD-1, along with decreased cytokine production after ex vivo stimulation [##REF##26205583##88##]. Although CLL patient blood-derived CD8<sup>+</sup> T-cells have been shown to express PD-1 and other inhibitory receptors, accompanied by defective immune synapse formation [##REF##23247726##9##], increased cytokine production by these cells has also been described (Fig. ##FIG##1##2##) [##REF##23247726##9##, ##REF##30267008##49##]. This has resulted in the conclusion that CD8<sup>+</sup> T-cells in CLL are “pseudoexhausted” [##REF##23247726##9##]. Building on these findings, a comparative analysis of CD8<sup>+</sup> T-cells of paired blood and lymph node samples of CLL patients showed an accumulation of PD-1<sup>+</sup> CD8<sup>+</sup> T-cells harboring all key features of exhaustion in lymph nodes but not blood samples, namely the expression of several inhibitory receptors and reduced cytokine production (Fig. ##FIG##2##3##) [##REF##31506282##25##, ##REF##30267008##49##]. The presence of such exhausted T-cells was confirmed in spleen samples but to a much lower extent in blood of TCL1 AT mice [##REF##30267008##49##], suggesting that T-cell exhaustion in CLL is a phenomenon that happens in lymphoid tissues rather than blood.</p>", "<p id=\"Par28\">In conclusion, emerging data clearly emphasize that CD8<sup>+</sup> T-cells in CLL likely recognize tumor-specific antigens, but fail to control disease due to their functional exhaustion, a phenotype that is more pronounced in lymphoid organs compared with the blood of CLL patients.</p>", "<title>Impact of novel kinase inhibitors on T-cells</title>", "<title>PI3Kδ inhibitors impair T-cell function in CLL</title>", "<p id=\"Par29\">Inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) by idelalisib has proven to be a clinically efficient treatment option for CLL [##REF##26472751##89##–##REF##24450857##91##]. However, immune-related adverse events were observed during this treatment, such as autoimmune-mediated elevation of liver enzymes associated with an infiltration of lymphocytes in the liver, as well as a decrease in Tregs [##REF##27247136##92##], and increased rates of infections [##REF##26472751##89##–##REF##24450857##91##, ##REF##25726955##93##, ##REF##29764250##94##]. As PI3Kδ is expressed by all leukocytes [##REF##12669022##95##], T-cells are also affected during treatment with PI3Kδ inhibitors which contributes to such adverse events [##REF##30573773##32##, ##REF##30457982##96##, ##UREF##11##97##]. Idelalisib treatment of CLL patients resulted in reduced numbers, proliferation, and suppressive function of Tregs, which was confirmed in the TCL1 AT mouse model [##REF##30573773##32##, ##REF##27247136##92##, ##REF##30457982##96##, ##UREF##11##97##]. Ex vivo analysis of T-cells from idelalisib-treated patients before and during treatment revealed a reduced expression of activation markers in Tregs in treated samples [##UREF##11##97##]. Reduced Treg activity by PI3Kδ inhibition was shown to unleash CD8<sup>+</sup> T-cell activity, leading to better CD8<sup>+</sup> T-cell-mediated control of solid tumors [##REF##24919154##98##]. In contrast, PI3Kδ inhibitor treatment of leukemic TCL1 AT mice resulted in reduced differentiation of CD8<sup>+</sup> T-cells towards antigen-experienced effector cells and diminished effector function [##REF##30573773##32##], which was likely caused by diminished TCR signaling by PI3Kδ inhibition [##REF##30573773##32##, ##UREF##11##97##].</p>", "<p id=\"Par30\">In conclusion, clinically approved PI3Kδ inhibitors can cause a potentially beneficial reduction of immunosuppressive functions of Tregs. However, these drugs were also shown to reduce CD8<sup>+</sup> T-cell effector capacity. Therefore, the imbalance of regulatory and cytotoxic T-cell subsets likely explains autoimmune events as well as increased infection rates in idelalisib-treated CLL patients.</p>", "<title>Ibrutinib inhibits TCR signaling in CLL</title>", "<p id=\"Par31\">Ibrutinib, an irreversible Bruton’s tyrosine kinase (BTK) inhibitor commonly used for the treatment of CLL, was shown to inhibit interleukin-2-inducible T-cell kinase (ITK), which is important for the activity of T-cells [##REF##23886836##99##], as reviewed recently by Mhibik et al. [##UREF##12##100##]. Analysis of T-cells derived from ibrutinib-treated CLL patients revealed an occupancy of ITK by ibrutinib of up to 55% [##REF##23886836##99##]. Furthermore, a decline in total T-cell numbers as well as CD4<sup>+</sup> and CD8<sup>+</sup> T-cell numbers in peripheral blood was reported during treatment with ibrutinib [##UREF##13##101##, ##REF##26660519##102##]. In contrast, Long et al. noted an increase in T-cell numbers in a cohort of 18 CLL patients treated with ibrutinib [##REF##28714866##103##]. Besides its impact on T-cell numbers, ibrutinib was shown to reduce the activation, proliferation, and cytokine production of T-cells upon treatment ex vivo [##UREF##14##104##] and in the TCL1 AT mouse model of CLL [##UREF##15##105##]. Moreover, ibrutinib but not acalabrutinib, a covalent BTK inhibitor with lower affinity for ITK, reduced TCR signaling of murine T-cells in vitro [##UREF##15##105##]. Other recent data have further shown that CLL patients gained a broader TCR repertoire diversity after 1 year of treatment with ibrutinib compared with pre-treatment samples [##UREF##13##101##].</p>", "<p id=\"Par32\">As ibrutinib leads to an efficient reduction of CLL cells in almost all affected tissues, the described alterations within the microenvironment of ibrutinib-treated patients might be due to a diminished impact of the malignant cells on their surroundings. Therefore, it still remains to be elucidated whether ibrutinib-mediated alterations of the T-cell compartment in CLL patients are due to a direct inhibition of ITK in T-cells, or indirectly due to the reduction of CLL tumor burden by ibrutinib and a subsequent normalization of the inflammatory milieu [##UREF##13##101##–##REF##28714866##103##, ##REF##26813675##106##–##REF##28123879##109##].</p>", "<p id=\"Par33\">In summary, ibrutinib blocks ITK, a kinase that regulates T-cell activation after TCR engagement, which could explain reduced numbers of T-cells with a more diverse TCR repertoire in ibrutinib-treated CLL patients. However, ongoing studies with acalabrutinib and other novel and more specific BTK inhibitors will ultimately clarify whether the reported effects are due to ITK inhibition or normalization of the inflammatory microenvironment in ibrutinib-treated CLL patients.</p>", "<title>Immunotherapeutic approaches involving T-cells</title>", "<title>Immune checkpoint blockade</title>", "<p id=\"Par34\">Immune checkpoint blockade has changed the treatment strategies of many different cancer entities. The disruption of the interaction of PD-1 and its ligands and the blocking of CTLA-4 are the best-studied examples.</p>", "<p id=\"Par35\">In CLL, initial pre-clinical investigations were encouraging. Using a blocking antibody against PD-L1 in the TCL1 AT mouse model of CLL achieved a substantial reduction of leukemia burden and a reversion of the exhausted T-cell phenotype [##REF##25800048##110##]. Comparable results were obtained combining antibody therapies directed against PD-1 and LAG-3 [##REF##29439955##38##]. Of note, single-agent treatment with αPD-1 antibodies did not reduce leukemia development in this study [##REF##29439955##38##]. In line with this, treatment of 16 relapsed CLL patients with a humanized αPD-1 antibody also did not result in a response in any of the CLL patients [##REF##28424162##111##]. However, four out of nine patients harboring Richter transformation showed a response to this treatment (<ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT02332980\">NCT02332980</ext-link>) [##REF##28424162##111##]. As these results were disappointing, combination therapies of kinase inhibitors and checkpoint blockade are currently under investigation (<ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT02329847\">NCT02329847</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT02332980\">NCT02332980</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT02362035\">NCT02362035</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT02420912\">NCT02420912</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT03514017\">NCT03514017</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT03153202\">NCT03153202</ext-link>, and <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT03283137\">NCT03283137</ext-link>) and first results indicate response rates of 61% [##REF##30642819##112##]. This agrees with encouraging results in the TCL1 AT model, where combination of ibrutinib with either αPD-1 or αPD-L1 considerably improved leukemia control and CD8<sup>+</sup> T-cell effector function, with more pronounced effects in the ibrutinib/αPD-L1 combination [##UREF##15##105##]. Of interest, this combination led to an almost complete eradication of CLL cells in the bone marrow, which was not achieved with ibrutinib or checkpoint blockade monotherapies, suggesting that the combination of ibrutinib with αPD-L1 might be able to eradicate CLL cells more efficiently also in lymphoid tissues [##UREF##15##105##].</p>", "<p id=\"Par36\">In conclusion, more investigations are needed to explore the potential of immune checkpoint blockade in CLL and to develop rational combination approaches to overcome T-cell dysfunction and immune escape in this disease.</p>", "<title>Chimeric antigen receptor (CAR) cell therapies</title>", "<p id=\"Par37\">Increasing the specificity of immune cells against cancer cells by genetic introduction of a CAR has boosted immunotherapeutic approaches in B-cell leukemia and lymphoma. Thus far, two products, tisagenlecleucel and axicabtagen-ciloleucel, have been approved by the Food and Drug Administration (FDA). About 60% of CLL patients treated within clinical trials with autologous CAR T-cells directed against the B-cell antigen CD19, showed a response [##REF##26333935##113##, ##UREF##16##114##]. Complete remissions were seen in about 29% [##REF##26333935##113##], and 21% [##UREF##16##114##] of CLL patients treated with CAR T-cells, which is considerably lower than in B-cell acute lymphoblastic leukemia patients. Estimated median progression-free survival of CD19 CAR T-cell treated CLL patients was reported as 7 months [##REF##26333935##113##] or 8.5 months [##UREF##16##114##]. During the treatment with CAR T-cells, a high rate of patients suffered of side effects such as cytokine release syndrome [##REF##26333935##113##, ##UREF##16##114##].</p>", "<p id=\"Par38\">Major limitations of the CAR T-cell approach were recently overcome using cord blood-derived, CAR-transduced natural killer (NK)-cells (<ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03690193?term=NCT03056339\">NCT03056339</ext-link>) [##REF##32023374##115##]. The advantage of this approach is that no full HLA match between donor and host cells is required, which circumvents the necessity of using autologous cells for CAR transduction, thereby shortening and simplifying the generation of CAR cell products [##REF##32023374##115##]. CAR NK-cell treatment induced remission in four of five patients with CLL, including patients harboring Richter transformation [##REF##32023374##115##].</p>", "<p id=\"Par39\">In summary, more therapeutic options that are based on T-cells and other immune cells, are currently being developed for CLL which have the potential to complement the small molecule-based treatment paradigms of CLL.</p>" ]
[ "<title>Acknowledgements</title>", "<p>The authors would like to thank Dr. Selcen Öztürk, Laura Llaó Cid, and Dr. Emma Philipps for their critical revision of the manuscript. Graphs were created with BioRender.com.</p>", "<title>Author contributions</title>", "<p>PMR and MS reviewed the literature, prepared the figures, wrote, and revised the manuscript.</p>", "<title>Funding</title>", "<p>Open Access funding enabled and organized by Projekt DEAL.</p>", "<title>Compliance with ethical standards</title>", "<title>Conflict of interest</title>", "<p id=\"Par43\">The authors declare that they have no conflict of interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>CD4<sup>+</sup> T-cell subset diversity and distribution in CLL.</title><p>CD4<sup>+</sup> T-cell subsets are defined by the expression of distinct surface markers (CXCR3 (2, 3), CCR4 (2), CCR6 (2), CXCR5 (3), PD-1 (3) and CD25 (4)), cytokines (IFNγ (1–3), IL-4 (1–3), IL-17 (1–3), IL-21 (1, 3) and IL-10 (1, 4)), and transcription factors (TBET (3), GATA3 (3), RORγT (3), BCL6 (3) and FOXP3(4)). In comparison to healthy controls (HC), CD4<sup>+</sup> T-cell subsets are more abundant in the blood of CLL patients.</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Comparison of CD8<sup>+</sup> T-cells from blood of CLL patients and healthy controls.</title><p>Blood-derived CD8<sup>+</sup> T-cells in CLL harbor a higher expression of inhibitory receptors PD-1, CD244, and CD160, and of effector molecules granzyme B (GzmB), TNFα, IFNγ and IL-2 compared with respective cells of healthy controls (HC). Overview of differential marker expression of T-cell activation, inhibitory receptors, cytokines and cytotoxic molecules as well as other T-cell functions of CD8<sup>+</sup> T-cells from HC (left) versus CLL patients (right). cCTLA-4: cytoplasmatic expression of CTLA-4; sCTLA-4: surface expression of CTLA-4.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>T-cell exhaustion phenotype of CD8<sup>+</sup> T-cells in blood versus lymph nodes of CLL patients.</title><p>Comparison of blood (left) and lymph node-derived (right) CD8<sup>+</sup> T-cells of CLL patients. CD8<sup>+</sup> T-cell exhaustion is more severe in lymph nodes compared with blood of CLL patients, with increased expression of PD-1 and CD69, as well as reduced production of effector molecules. Overview of differential marker expression of T-cell activation, inhibitory receptors, cytokines and cytotoxic molecules, cell distribution and other CD8<sup>+</sup> T-cell functions of blood (PB, left) versus lymph nodes of CLL patients (LN, right) or spleens of CLL mouse models. T<sub>EMRA</sub>: effector memory RA T-cells; <sup>#</sup> results derived from the TCL1 AT mouse model of CLL.</p></caption></fig>" ]
[ "<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Function of CD4<sup>+</sup> T-cell subsets in CLL.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>CD4<sup>+</sup> T-cell subset</th><th>Functional data - in vitro</th><th>Functional data - in vivo</th></tr></thead><tbody><tr><td>Th1</td><td><p>- Activation and proliferation of CLL cells [##REF##23933259##18##, ##UREF##4##19##].</p><p>- rIFNγ inhibits CLL cell apoptosis [##REF##7678114##44##].</p></td><td><p>- Activation and proliferation of CLL cells in a PDX xenograft model [##REF##23933259##18##].</p><p>- Depletion does not affect CLL progression in TCL1 AT mice [##UREF##3##17##].</p></td></tr><tr><td>Th2</td><td><p>- rIL-4 induces CLL cell proliferation cooperatively with rIL-21 [##REF##23710828##22##].</p><p>- rIL-4 rescues CLL cells from apoptosis [##REF##24828787##45##].</p></td><td>-</td></tr><tr><td>Th17</td><td> -</td><td>-</td></tr><tr><td>Tfh</td><td><p>- rIL-21 induces CLL cell proliferation cooperatively with rIL-4 [##REF##23710828##22##].</p><p>- rIL-21 induces CLL cell proliferation cooperatively with rCD40L stimulation [##REF##24014238##23##].</p></td><td>-</td></tr><tr><td>Treg</td><td> -</td><td>- Depletion by 50% does not affect CLL progression in TCL1 AT mice [##REF##30573773##32##].</td></tr></tbody></table></table-wrap>" ]
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[ "<table-wrap-foot><p>Overview of published functional data of CD4<sup>+</sup> T-cell subsets and their derived cytokines in vitro and the impact of Th T-cell subsets on CLL progression in vivo.</p></table-wrap-foot>", "<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p><bold>Change history</bold></p><p>11/16/2021</p><p>A Correction to this paper has been published: 10.1038/s41375-021-01362-7</p></fn></fn-group>" ]
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[{"label": ["2."], "surname": ["Appay", "van Lier", "Sallusto", "Roederer"], "given-names": ["V", "RA", "F", "M"], "article-title": ["Phenotype and function of human T lymphocyte subsets: consensus and issues"], "source": ["Cytom Part A: J Int Soc Anal Cytol"], "year": ["2008"], "volume": ["73"], "fpage": ["975"], "lpage": ["83"]}, {"label": ["6."], "surname": ["Elston", "Fegan", "Hills", "Hashimdeen", "Walsby", "Henley"], "given-names": ["L", "C", "R", "SS", "E", "P"], "article-title": ["Increased frequency of CD4(+) PD-1(+) HLA-DR(+) T cells is associated with disease progression in CLL"], "source": ["British J. Haematol"], "year": ["2020"], "volume": ["188"], "fpage": ["872"], "lpage": ["80"]}, {"label": ["10."], "mixed-citation": ["Roessner PM, LLa\u00f3 Cid L, Lupar E, Roider T, Bordas M, Schifflers C, et al. EOMES and IL-10 regulate anti-tumor activity of PD-1+ CD4+ T-cells in B-cell Non-Hodgkin lymphoma. 2020. 10.1101/2020.03.09.983098."]}, {"label": ["17."], "surname": ["Roessner", "Hanna", "Ozturk", "Schulz", "Llao Cid", "Yazdanparast"], "given-names": ["PM", "BS", "S", "R", "L", "H"], "article-title": ["TBET-expressing Th1 CD4(+) T cells accumulate in chronic lymphocytic leukaemia without affecting disease progression in Emicro-TCL1 mice"], "source": ["British J. Haematol"], "year": ["2020"], "volume": ["189"], "fpage": ["133"], "lpage": ["45"]}, {"label": ["19."], "surname": ["Burgler", "Gimeno", "Parente-Ribes", "Wang", "Os", "Devereux"], "given-names": ["S", "A", "A", "D", "A", "S"], "article-title": ["Chronic lymphocytic leukemia cells express CD38 in response to Th1 cell-derived IFN-gamma by a T-bet-dependent mechanism"], "source": ["J Immunol (Baltim, Md: 1950)"], "year": ["2015"], "volume": ["194"], "fpage": ["827"], "lpage": ["35"]}, {"label": ["24."], "surname": ["Cha", "Zang", "Guo", "Rechlic", "Olasnova", "Gu"], "given-names": ["Z", "Y", "H", "JR", "LM", "H"], "article-title": ["Association of peripheral CD4+ CXCR5+ T cells with chronic lymphocytic leukemia"], "source": ["Tumour Biol: J Int Soc Oncodev Biol Med"], "year": ["2013"], "volume": ["34"], "fpage": ["3579"], "lpage": ["85."]}, {"label": ["50."], "surname": ["Zhan", "Brown", "Deliyannis", "Seah", "Wijburg", "Price"], "given-names": ["YF", "LE", "G", "S", "OL", "J"], "article-title": ["Responses against complex antigens in various models of CD4 T-cell deficiency - Surprises from an anti-CD4 antibody transgenic mouse"], "source": ["Immunologic Res."], "year": ["2004"], "volume": ["30"], "fpage": ["1"], "lpage": ["14"]}, {"label": ["64."], "surname": ["Catovsky", "Miliani", "Okos", "Galton"], "given-names": ["D", "E", "A", "DA"], "article-title": ["Clinical significance of T-cells in chronic lymphocytic leukaemia"], "source": ["Lancet (Lond, Engl)"], "year": ["1974"], "volume": ["2"], "fpage": ["751"], "lpage": ["2"]}, {"label": ["67."], "surname": ["Platsoucas", "Galinski", "Kempin", "Reich", "Clarkson", "Good"], "given-names": ["CD", "M", "S", "L", "B", "RA"], "article-title": ["Abnormal T lymphocyte subpopulations in patients with B cell chronic lymphocytic leukemia: an analysis by monoclonal antibodies"], "source": ["J Immunol (Baltim, Md: 1950)"], "year": ["1982"], "volume": ["129"], "fpage": ["2305"], "lpage": ["12"]}, {"label": ["75."], "surname": ["Gothert", "Eisele", "Klein-Hitpass", "Weber", "Zesewitz", "Sellmann"], "given-names": ["JR", "L", "L", "S", "ML", "L"], "article-title": ["Expanded CD8+ T cells of murine and human CLL are driven into a senescent KLRG1+ effector memory phenotype"], "source": ["Cancer Immunol, Immunotherapy: CII"], "year": ["2013"], "volume": ["62"], "fpage": ["1697"], "lpage": ["709."]}, {"label": ["85."], "surname": ["Serrano", "Monteiro", "Allen", "Kolitz", "Schulman", "Lichtman"], "given-names": ["D", "J", "SL", "J", "P", "SM"], "article-title": ["Clonal expansion within the CD4+CD57+ and CD8+CD57+ T cell subsets in chronic lymphocytic leukemia"], "source": ["J Immunol (Baltim, Md: 1950)"], "year": ["1997"], "volume": ["158"], "fpage": ["1482"], "lpage": ["9"]}, {"label": ["97."], "surname": ["Chellappa", "Kushekhar", "Munthe", "Tjonnfjord", "Aandahl", "Okkenhaug"], "given-names": ["S", "K", "LA", "GE", "EM", "K"], "article-title": ["The PI3K p110delta isoform inhibitor idelalisib preferentially inhibits human regulatory T cell function"], "source": ["J Immunol (Baltim, Md: 1950)"], "year": ["2019"], "volume": ["202"], "fpage": ["1397"], "lpage": ["405"]}, {"label": ["100."], "mixed-citation": ["Mhibik M, Wiestner A, Sun C. Harnessing the Effects of BTKi on T Cells for Effective Immunotherapy against CLL. Int J Mol Sci. 2019;21."]}, {"label": ["101."], "surname": ["Yin", "Sivina", "Robins", "Yusko", "Vignali", "O\u2019Brien"], "given-names": ["Q", "M", "H", "E", "M", "S"], "article-title": ["Ibrutinib therapy increases T cell repertoire diversity in patients with chronic lymphocytic leukemia"], "source": ["J Immunol (Baltim, Md: 1950)"], "year": ["2017"], "volume": ["198"], "fpage": ["1740"], "lpage": ["7"]}, {"label": ["104."], "surname": ["Hofland", "de Weerdt", "Ter Burg", "de Boer", "Tannheimer", "Tonino"], "given-names": ["T", "I", "H", "R", "S", "SH"], "article-title": ["Dissection of the effects of JAK and BTK inhibitors on the functionality of healthy and malignant lymphocytes"], "source": ["J Immunol (Baltim, Md: 1950)"], "year": ["2019"], "volume": ["203"], "fpage": ["2100"], "lpage": ["9"]}, {"label": ["105."], "mixed-citation": ["Hanna BS, Yazdanparast H, Demerdash Y, Roessner PM, Schulz R, Lichter P, et al. Combining ibrutinib and checkpoint blockade improves CD8+ T-cell function and control of chronic lymphocytic leukemia in Em-TCL1 mice. Haematologica. 2020. 10.3324/haematol.2019.238154."]}, {"label": ["114."], "surname": ["Turtle", "Hay", "Hanafi", "Li", "Cherian", "Chen"], "given-names": ["CJ", "KA", "LA", "D", "S", "X"], "article-title": ["Durable molecular remissions in chronic lymphocytic leukemia treated with CD19-specific chimeric antigen receptor-modified T cells after failure of ibrutinib"], "source": ["J Clin Oncol: Off J Am Soc Clin Oncol"], "year": ["2017"], "volume": ["35"], "fpage": ["3010"], "lpage": ["20."]}]
{ "acronym": [], "definition": [] }
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2024-01-13 23:35:09
Leukemia. 2020 May 26; 34(8):2012-2024
oa_package/21/30/PMC8318881.tar.gz
PMC8421345
34489451
[ "<title>Introduction</title>", "<p id=\"Par3\">Naïve T cells (Tn) are mature post-thymic T cells that reside in secondary lymphoid organs/tissues, awaiting encounter with their cognate antigen. In mice, Tn cells express L-selectin (CD62L) and CCR7 but not the memory/activation markers CD44, CD25, or CD49d<sup>##REF##12047742##1##</sup>. Adequate numbers and diversity of Tn cells are essential for immune defense against newly encountered pathogens<sup>##REF##15040585##2##</sup>. T cell homeostasis maintains the numbers and diversity of the Tn pool<sup>##REF##19935802##3##</sup>. Seminal findings of Suhr, Sprent, and colleagues have shown that Tn cells require tonic, subthreshold T cell receptor (TCR) stimulation by self-peptide:MHC (s-pMHC), followed by binding of lymph node stroma-produced IL-7 to the IL-7 receptor (IL-7R), for survival and maintenance, as well as for homeostatic proliferation<sup>##REF##21739670##4##</sup>. These and almost all other studies on Tn homeostasis were done in inbred mice housed under specific pathogen-free (SPF) or even germ-free or antigen-free<sup>##REF##15749843##5##</sup> conditions, in the almost complete absence of infection and inflammation. A recent landmark study<sup>##REF##27096360##6##</sup> compared immune homeostasis in wild-caught mice, mice from pet stores (PS), and inbred, laboratory SPF mice. Authors found that SPF mice exhibited T cell subset distribution and activation patterns similar to that of human neonates, whereas the same parameters in wild-caught, pet-store, or inbred SPF mice co-housed with PS mice, faithfully approximated the distribution of T cell subsets (with low levels of naïve and high levels of memory cells), T cell subset activation and responses to infection seen in adult humans.</p>", "<p id=\"Par4\">We previously described a subset of phenotypically naive human CD8<sup>+</sup> T cells with memory characteristics<sup>##REF##27270402##7##</sup> that rapidly secreted multiple cytokines in response to persistent infections. In our hands, no similar population could be found in SPF mice. Given the powerful impact of non-SPF conditions on the murine immune system, we here investigated the phenotype and function of CD8<sup>+</sup> Tn cells in C57BL/6 mice following viral or bacterial mono-infection, as well as following co-housing exposure to pet-store animals (CH in the text).</p>", "<p id=\"Par5\">Here we show that infections and inflammation have a long-lasting antigen-nonspecific (bystander) effect upon CD8<sup>+</sup> Tn cell homeostasis, dominated by an expansion of CD8<sup>+</sup> Tn cells expressing a memory marker Ly6C. In CH animals, the expansion of CD8<sup>+</sup> Tn Ly6C+ is driven by type I interferons (IFN-I) and dependent upon tonic TCR signaling. Compared to their Ly6C<sup>−</sup> counterparts, Ly6C<sup>+</sup>CD8<sup>+</sup> Tn cells are preferentially home to lymph nodes and display enhanced homeostatic and effector properties. Overall, our results demonstrate a novel and powerful role of infection-mediated IFN-I in CD8<sup>+</sup> Tn homeostasis.</p>" ]
[ "<title>Methods</title>", "<title>Ethics statement</title>", "<p id=\"Par28\">Mouse studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Protocols were approved by the Institutional Animal Care and Use Committee at the University of Arizona (IACUC protocol 08-102, PHS Assurance No. A3248-01).</p>", "<title>Mice</title>", "<p id=\"Par29\">Female C57/BL6 mice and OT-I TCR transgenic mice were obtained from Jackson Laboratories (Bar Harbor, ME). IFNAR-KO mice (IFN-αβR-) were bred in our colony from the nucleus obtained from Jackson Laboratories. Adult C57BL/6 (B6) mice were infected with 1000 plaque-forming units of the West Nile virus strain 385-99<sup>##REF##19901080##8##</sup> via footpad injection.</p>", "<p id=\"Par30\">For co-housing, we purchased female outbred pet-store mice from two local vendors, individually tagged them, and co-housed them for two weeks to maximize microbial exchange in large (rat) cages. We then co-housed them in large cages with young female C57/BL6 mice (Jackson Laboratories) separated by a perforated barrier. All mice were bled before co-housing (baseline) and then at 30 and 60 days, followed by the sacrifice of certain animals on post day 60. We have selected a 60 day period as the first harvest point since this is a conventionally accepted time for any acute immune response to entering the memory phase steady state<sup>##REF##8600537##41##</sup>.</p>", "<title>Flow cytometry and cytokine measurements</title>", "<p id=\"Par31\">Blood was collected into heparin-coated tubes and lysed hypnotically. Splenocytes were mechanically dissociated through a 40 μm plastic mesh to prepare a single-cell suspension. Cells were stained with surface antibodies, and then fixed and permeabilized using the FoxP3 Fix/Perm kit (eBioscience, San Diego, CA). Following antibodies were used anti: Ly6C(HK1.4), CD3(17A2), CD8a(53-6.7), CD44(IM7), CD4 (GK1.5), CD5 (53-7.3), CD62L(MEL-14), CD25(PC6.1), SCA1(D7), CD69(H1.2F3), CXCR3 (CXCR#173) (Biolegend, San Diego, CA), Eomes(Dan11mag), Phospho-ZAP70/Syk(n3kobu5) (Invitrogen, Carlsbad, CA). Cytokines were measured with bead-based flow cytometric immunoassay (Legendplex, Biolegend). Serum levels of IFN-I were measured using a bioassay as previously described<sup>##REF##19901080##8##</sup>. Type I IFN standards (National Institute of Allergy and Infectious Diseases international standard) and mouse serum were serially diluted twofold in complete media, 10% FBS, Pen/Strep, and DME. IFN-responsive L929 cells were plated at 5 × 10<sup>4</sup> cells/well, incubated overnight with the serum, and media containing 5 PFU VSV Indiana was added to each well, except for control wells. Twenty-four hours later, media was aspirated, the plate washed twice with PBS, and the monolayer fixed with 5% formaldehyde, incubated for 10 min, and stained with 0.05% crystal violet for 10 min. Washed monolayers were allowed to dry, and 100% methanol was used to elute the dye. Absorbance is measured at 595 nm on an ELISA plate reader (Invitrogen).</p>", "<p id=\"Par32\">Samples were acquired using a BD LSR Fortessa cytometer with FACS DIVA v 8.0.1. software (BD Biosciences) and analyzed by FlowJo software (Tree Star, Ashland, OR) with a minimum of 20000 CD8<sup>+</sup> T cells collected per sample. For MHC-I blocking anti-H-2Kb/H-2Db antibody (clone 28-8-6, Biolegend) was used. Phosphorylation of Erk and ZAP-70 was measured using Phosflow fixation and permeabilization solutions (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions.</p>", "<p id=\"Par33\">Flow cytometry files from SPF and WNV infected mice were uploaded to Cytobank, a cloud-based computational platform. Dead cells were excluded by manual gating and CD3+ events were input into the FLOWSOM clustering algorithm.</p>", "<title>Cell separation by MACS enrichment and in vitro stimulation of naïve T cells</title>", "<p id=\"Par34\">Naïve splenic CD8<sup>+</sup> T cells were magnetically enriched using an AutoMACS pro (Miltenyi Biotec) using the CD8a T cell isolation kit supplemented with anti-CD44-biotin (eBioscience). The cells were cultured at 2 × 10<sup>5</sup> cells/ml in RPMI-1640 with L-glutamine (Lonza, Basel, Switzerland) + 10% FCS 1:1 with α-CD3/α-CD28 immobilized on beads (Miltenyi), 10 U/ml rmIL-2 (eBioscience). The purity of isolated naïve T cells was assessed by flow cytometry and was &gt;95% in all experiments. Lymph node (LN) stromal cells were obtained by dissociating lymph nodes in Liberase TL enzyme mixture (Roche, Basel, Switzerland) using GentleMACS tissue dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). When lymph nodes were fully digested lymph node stromal cells were negatively enriched using anti CD45 and anti Ter119 magnetic beads (Miltenyi Biotec). Stromal cells were passaged 5–10 times in alpha-modified MEM (Sigmaaldrich, St.Louis, Mo) with 20% FCS. Naïve T cells were cultured with lymph node stromal cells in 1:10 ratio in 96 well-round plates.</p>", "<title>Adoptive transfer of naïve T cells</title>", "<p id=\"Par35\">Adoptive transfers of naïve CD8<sup>+</sup> T cells were performed as previously described<sup>##REF##22862959##42##</sup>. Briefly, naïve CD8<sup>+</sup> T cells from TCR transgenic OT-1 mice were sorted by Ly6C expression on the FACS Aria III sorter (BD Biosciences) based on the phenotype of CD8<sup>+</sup>C62L<sup>+</sup>CD44<sup>lo.</sup> CD45.1<sup>+</sup>OT-1 Ly6C<sup>+</sup> or Ly6C<sup>−</sup> cells were transferred into adult congenic B6 recipients; 24 h later, recipient mice were infected with 1–3 × 10<sup>4</sup> CFU of Lm expressing or not expressing the immunodominant epitope of ovalbumin (SIINFEKL, OVA)<sup>##UREF##7##43##</sup> (Lm-OVA) intravenously, in 100 µl PBS. Analysis was performed on days 5 and 8 after infection.</p>", "<title>RNAseq analysis</title>", "<p id=\"Par36\">2 × 10<sup>5</sup> CD8<sup>+</sup>CD62L<sup>+</sup>CD44<sup>LO</sup>Ly6C<sup>+</sup>and Ly6C<sup>−</sup> naïve T cells were isolated from 4 SPF and 4 cohoused mice using a CD8a<sup>+</sup> T cell isolation kit (Miltenyi Biotec) followed by FACS Aria III sorting (BD Biosciences). Raw sequence reads were first demultiplexed using the SMART-Seq DE3 Demultiplexer software (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.takarabio.com/products/next-generation-sequencing/rna-seq/ultra-low-input-rna-seq/smart-seq-de3-demultiplexer\">www.takarabio.com/products/next-generation-sequencing/rna-seq/ultra-low-input-rna-seq/smart-seq-de3-demultiplexer</ext-link>) and read 1 file were used. Sequencing reads were evaluated using FastQC (v0.11.3, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.bioinformatics.babraham.ac.uk/projects/fastqc/\">www.bioinformatics.babraham.ac.uk/projects/fastqc/</ext-link>) and preprocessed for quality using Trim Galore (v0.4.0, Phred score threshold of 20 and minimum length of 50 bp, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\">www.bioinformatics.babraham.ac.uk/projects/trim_galore/</ext-link>). Quality reads were then uniquely mapped to the mouse reference genome (GRCm38) using Tophat2 (v2.1.1). Raw read counts for each sample were obtained by mapping reads at the gene level using HTSeq-count tool from the Python package HTSeq. DESeq2 R package (v1.8.2) was then used to perform differential expression and statistical analysis. Functional enrichment of the differentially expressed genes relative to a background gene list (from all filtered expressed genes) was performed using clusterProfiler R package.</p>", "<title>Statistical analysis</title>", "<p id=\"Par37\">SPSS and Graph Pad Prism were used for statistical analysis. Depending on data distribution, differences were calculated by Student’s <italic>t</italic>-test, Mann Whitney U-test, one-way ANOVA, or Kruskal Wallis test with SIdak post hoc correction. For all statistical differences *<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001.</p>", "<title>Reporting summary</title>", "<p id=\"Par38\">Further information on research design is available in the ##SUPPL##2##Nature Research Reporting Summary## linked to this article.</p>" ]
[ "<title>Results</title>", "<title>Unbiased clustering identifies upregulation of Ly6C on the surface of CD8<sup>+</sup> Tn cells following WNV infection</title>", "<p id=\"Par6\">To examine the impact of infection upon the maintenance of Tn cells, we infected C57BL/6 (B6) mice with the West Nile virus strain 385-99<sup>##REF##19901080##8##</sup>. Flow cytometric analysis was performed on day 7 (d7, peak of the immune response) in an unbiased manner to reveal potential changes in the circulating T cell pool, using a 15-marker flow cytometric panel, including commonly used memory and activation markers (Table ##SUPPL##0##S1##). We have used the Flowsom visualization algorithm<sup>##UREF##0##9##</sup>, an unsupervised technique for clustering and dimensionality reduction, to visualize changes among CD8<sup>+</sup>T cell subsets following WNV infection. FlowSOM software visualizes flow cytometric data as a minimal spanning tree result very similar to more commonly used SPADE software but with greater computing speed. Flowsom identified the standard subsets of CD8<sup>+</sup>T cells, including the effector/effector memory ((EM) CD44<sup>hi</sup>CD62L<sup>−</sup>), central memory ((CM) CD44<sup>hi</sup>CD62L<sup>+</sup>CD49d<sup>+</sup>), virtual memory ((VM) CD44<sup>hi</sup>CD62L<sup>+</sup>D49d<sup>−</sup>) and naïve ((N) CD44<sup>lo</sup>CD62L<sup>+</sup>CD49d<sup>−</sup>) cells (Fig. ##FIG##0##1##). However, the naïve T cells were divided into two distinct subsets, divided by the expression of Ly6C, with the Ly6C<sup>+</sup> cells unexpectedly and markedly increased in WNV-infected mice (Fig. ##FIG##0##1##, arrow). Therefore, unbiased clustering identified the upregulation of Ly6C on CD8<sup>+</sup>Tn cells as one of the most pronounced changes in circulating T cells during WNV infection.</p>", "<title>Ly6C is upregulated on phenotypically naïve CD8<sup>+</sup> T cells transiently during acute viral and bacterial infection and permanently under ‘non-SPF’ conditions</title>", "<p id=\"Par7\">Next, we analyzed the kinetics of Ly6C upregulation. Flow cytometric strategy for identifying Ly6C<sup>+</sup>CD8<sup>+</sup> Tn cells is shown in Fig. ##FIG##1##2A##, (depicting day 7 post-infection with WNV) and was used for all analysis, unless otherwise indicated. WNV-infected mice displayed more than the twofold higher expansion of the Ly6C<sup>+</sup>CD8<sup>+</sup> Tn cells by day 3 p.i. (Fig. ##FIG##1##2B##) compared to day 0 of infection. However, the upregulation of Ly6C on CD8<sup>+</sup> Tn was transient. Ly6C expression declined by d16 and returned to baseline by d28 p.i. Interestingly, virtually all CXCR3<sup>+</sup>CD8<sup>+</sup> Tn cells were contained within the Ly6C<sup>+</sup> subpopulation (Fig. ##FIG##1##2A##), making them phenotypically similar to human T<sub>MNP</sub> cells which displayed higher CXCR3 expression<sup>##REF##27270402##7##</sup>. The proportion of double-positive Ly6C<sup>+</sup>CXCR3<sup>+</sup> cells was also higher on d7 post-WNV infection (Fig. ##SUPPL##0##S1A##) but this increase was lower than the ~2 fold increase in the total Ly6C<sup>+</sup> population. To examine whether the Ly6C upregulation on CD8<sup>+</sup> Tn cells was specific to viral infections, we repeated our experiments using the bacterium <italic>Listeria monocytogenes</italic> (Lm). In Lm-infected mice we found similar transient increased expression of Ly6C on CD8<sup>+</sup> Tn cells, peaking on day 3 but returning to baseline somewhat earlier than during the WNV infection (Fig. ##FIG##1##2C##).</p>", "<p id=\"Par8\">We next measured Ly6C expression longitudinally in mice co-housed (CH) with pet store mice. As previously described<sup>##REF##27096360##6##</sup>, we found that cohousing of laboratory mice with PS mice resulted in transmission of multiple pathogens (Table ##SUPPL##0##S2##). To examine the systemic effects of these infections, we performed measurements of multiple cytokines in secondary lymphoid tissue homogenates on day 60 after cohousing. Multiple cytokines were elevated as depicted in the heatmap in Fig. ##FIG##1##2D##. These results indicate that transmission of multiple pathogens to B6 mice resulted in prolonged stimulation of the immune system, evidenced by the elevation of multiple cytokines even after 60 days of continuous cohousing. Under such conditions, upregulation of Ly6C on CD8<sup>+</sup> Tn was permanent and did not return to baseline even after 300 days following CH (Fig. ##FIG##1##2E##). Female mice were used for all cohousing experiments and their basal Ly6C expression on CD8<sup>+</sup> Tn cells was lower than their SPF male counterparts (Fig. ##SUPPL##0##S1B##). Only a slight upregulation of Ly6C was observed on CD4 T cells in CH mice (Fig. ##SUPPL##0##S1C##). Similarly, the number of double-positive Ly6C<sup>+</sup>CXCR3<sup>+</sup> naïve CD8<sup>+</sup>s was also increased long term (Fig. ##SUPPL##0##S1D##). Thus, the long-lasting increase in Ly6C expression in ‘non-SPF’ mice might be maintained by persistent/chronic infections and/or by continuous exchange of pathogens between infected individuals, reflective of a situation found in the naturally-dwelling mammalian communities. While transient bystander effects of inflammatory cytokines on memory T cells have previously been reported<sup>##REF##8658169##10##,##REF##16616476##11##</sup> our results indicate that chronic infections and inflammation can powerfully remodel even the Tn pool over long periods of time.</p>", "<title>Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells display rapid and enhanced effector function in vitro</title>", "<p id=\"Par9\">Previously we have shown, in human subjects, an increase in CD8<sup>+</sup> T memory cells with naïve phenotype (T<sub>MNP</sub>) which were specific for persistent microbial pathogens<sup>##REF##27270402##7##</sup>. Such cells exhibited signs of basal activation and displayed rapid effector function upon polyclonal stimulation. To investigate whether the Ly6C<sup>+</sup>CD8<sup>+</sup> Tn cells in mice cells exhibit enhanced functional response, we sorted Ly6C ± CD3<sup>+</sup>CD8<sup>+</sup>CD62L<sup>+</sup>CD44<sup>lo</sup> cells from secondary lymphoid tissues of SPF and CH mice (Fig. ##FIG##2##3A##, complete gating strategy in Fig. ##SUPPL##0##S7A##) and stimulated them with α-CD3/α-CD28 beads and phorbol-myristate acetate (PMA) and the calcium ionophore ionomycin. Following 24 h of stimulation with α-CD3/α-CD28 beads, GzB expression was two-fold higher in Ly6C<sup>+</sup> over Ly6C<sup>−</sup> cells from CH mice while both cell types from SPF mice produced very little GzB (Fig. ##FIG##2##3B##, left panel). After 36 h, Ly6C<sup>+</sup> cells from both groups of mice produced more GzB than their Ly6C<sup>−</sup> counterpart although the difference was now less than two-fold (Fig. ##FIG##2##3B##, right panel). As expected, of Tn cells short (6 h) PMA/ionomycin stimulation resulted in low IFN-γ production (~1%); nonetheless, twice as many Ly6C<sup>+</sup> cells produced IFN-γ in cells from both SPF and CH mice (Fig. ##FIG##2##3D##). Production of TNF-α was much higher, again with the same pattern of increased production by Ly6C<sup>+</sup> cells in both groups of mice (Fig. ##FIG##2##3E##). This suggested that Ly6C<sup>+</sup> cells may be at a higher basal activation state even in SPF mice, prompting us to examine phosphorylation of the Erk kinase, which integrates several signaling pathways in T cells<sup>##REF##21205892##12##</sup>. We found that pErk levels at basal state and after 20 min of polyclonal stimulation with α-CD3/α-CD28 beads, showed a small but significant increase in Ly6C<sup>+</sup> cells (Fig. ##FIG##2##3F##).</p>", "<p id=\"Par10\">This suggested that Ly6C<sup>+</sup> cells might have been receiving increased TCR and/or cytokine signals, leading us to measure levels of phosphorylated ZAP-70/SYK. Upon TCR engagement, phosphorylation of ZAP-70 Y319 by Lck is necessary for T cell receptor (TCR)-dependent association of ZAP-70 with Lck and downstream signaling<sup>##UREF##1##13##</sup>. Ly6C<sup>+</sup> cells at both the basal and activated states expressed similarly small, but reproducibly and significantly higher levels of pZAP-70/SYK (Fig. ##FIG##2##3G##), further supporting the possibility that these cells are receiving TCR-mediated signals. Given that overall numbers of Ly6C<sup>+</sup> cells in CH, mice were twofold higher, these results indicated that in CH mice most of the CD8<sup>+</sup> Tn pool was in a state of higher basal activation, affording them higher production of effector molecules.</p>", "<title>Microbial colonization increases Ly6C expression on naïve T cells in a bystander manner</title>", "<p id=\"Par11\">Since both Ly6C and CXCR3 have been reported to be expressed primarily on effector and memory T cells<sup>##REF##25769612##14##</sup> it was possible that our Ly6C<sup>+</sup> population was contaminated by recently activated effector T (Te) cells. We examined the expression of multiple memory and activation markers such as CD49d, CD25, KLRG1, and CD69 on phenotypically naïve CD8<sup>+</sup> T cells from cohoused mice. All of these markers are upregulated upon activation via TCR, with CD69 exhibiting the earliest upregulation<sup>##REF##7804122##15##</sup>. We found that effector/memory CD44<sup>hi</sup> cells in CH mice expressed KLRG1, CD49d, and CD69. By contrast, CD8<sup>+</sup> Tn cells failed to express any of these markers regardless of Ly6C expression (Fig. ##FIG##3##4A##), implying that this population contains very few cells recently stimulated by antigen. Next, using fluorescence-minus-one control, we divided the CD44<sup>lo</sup> CD62L<sup>hi</sup> cells into the CD44 negative and CD44 intermediate subpopulations and compared their Ly6C expression to that on CD44<sup>hi</sup>CD62L<sup>hi</sup> Tcm cells. CD44<sup>lo</sup>CD62L<sup>hi</sup> cells doubled their expression of Ly6C upon cohousing (Fig. ##FIG##3##4B##). While CD44<sup>int</sup>CD62L<sup>hi</sup> cells contained a higher fraction of Ly6C<sup>+</sup> cells at baseline, cohousing also upregulated their expression of Ly6C; no such upregulation was seen on CD44<sup>hi</sup> CM cells. One could argue that the fraction of CD8<sup>+</sup>CD44<sup>int</sup>CD62L<sup>hi</sup> cells, commonly included in the Tn gate in the field, as well as in most of our experiments, represent recently activated memory-like cells that could obscure our results. However, the above results argue against that, because these cells did not exhibit upregulation of antigen-induced activation markers (Fig. ##FIG##3##4A##) and, while these cells expressed a higher frequency of Ly6C<sup>+</sup>, they were still able to upregulate it further in response to cohousing (Fig. ##FIG##3##4B##).</p>", "<p id=\"Par12\">Previously we have shown in a parabiosis model that naïve CD44<sup>lo</sup>CD62L<sup>hi</sup> cells freely circulate and equilibrate between parabiont host<sup>##UREF##2##16##</sup>. Thus, it was not surprising that Ly6C upregulation on Tn CD8<sup>+</sup>s was detected at similar levels in blood, spleen, and lymph nodes (Fig. ##FIG##3##4C##). To further confirm that increase in Ly6C expression is of bystander type and not a result of TCR cross-reactivity, we treated SPF mice with poly(I:C), an innate immune agonist known to induce multiple inflammatory cytokines<sup>##REF##25900439##17##</sup>. Treated mice upregulated Ly6C on Tn (Fig. ##SUPPL##0##S2A##) to a similar extent previously observed in WNV infection. To confirm that Ly6C upregulation is not dependent on cognate antigen we have transferred CFSE labeled Tn CD8<sup>+</sup>s from OT-1 mice into cohoused mice. After 14 days transferred cells have largely remained CD44<sup>lo</sup>CD62L<sup>hi</sup> and have not diluted out CFSE; however, &gt;70% of the undivided cells upregulated Ly6C (Fig. ##FIG##3##4D##). Jointly, these results confirm that Ly6C upregulation is a bystander effect of inflammation on CD8<sup>+</sup> Tn cells and not a result of antigenic stimulation or contamination with T effector or memory cells.</p>", "<p id=\"Par13\">To examine in detail the differentiation status of Ly6C<sup>+</sup>CD8<sup>+</sup> Tn cells, we performed extensive FCM analysis of multiple surface proteins (chemokines, chemokine receptors, adhesion molecules, cytokine receptors, and costimulatory molecules) involved in T cell homeostasis and maturation. One pronounced change observed on all CD8<sup>+</sup> Tn cells in co-housed mice (CH) was the upregulation of stem cell antigen 1 (SCA-1) (Fig. ##FIG##3##4E##). SCA-1, which also belongs to the Ly-6 superfamily, is expressed on memory T cells and gets upregulated on almost all T cells during viral infection<sup>##REF##19384870##18##</sup>. However, SCA-1 levels were increased in CD8<sup>+</sup> Tn cells in CH mice regardless of the Ly6C expression. Another surface marker, CD5 was highly expressed on Ly6C<sup>+</sup> cells from both SPF and CH mice (Fig. ##FIG##3##4E##). CD5 is a negative regulator of T cell receptor (TCR)-mediated signaling<sup>##REF##9858516##19##</sup> and its expression on T cells correlate with TCR affinity so that clonotypes with higher affinity for self p-MHC exhibit higher CD5. Increased levels of CD5 on Ly6C<sup>+</sup> cells together with increased pZAP-70/SYK (Fig. ##FIG##2##3D##) suggested that tonic TCR signals may play a role in the maintenance of this population. We further measured intracellular levels of multiple molecules involved in T cell homeostasis and activation/memory maintenance. Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells from both CH and SPF mice exhibited significantly higher expression of Eomesodermin (Eomes) (Fig. ##FIG##3##4F##) and BCL-2 (Fig. ##FIG##3##4G##), as well as slightly higher levels of IRF-4 and MCL-1 (Fig. ##SUPPL##0##S2B##) with no expression/difference in Tbet, BCL-6 and BLIMP-1 levels (Fig. ##SUPPL##0##S2C##). Eomes is a key transcription factor involved in the generation of effector and memory T cells<sup>##REF##16273099##20##</sup> and in the maintenance of the memory T cell pool<sup>##REF##20935204##21##</sup>. BCL-2 is an anti-apoptotic factor that is positively regulated by homeostatic cytokines such as IL-7<sup>##REF##11062503##22##</sup> and the expression of which correlates with prolonged cell survival. We conclude that CD8<sup>+</sup> Tn cells from cohoused mice are phenotypically and functionally altered. Their dominant phenotype, as observed by flow cytometry was Ly-6C<sup>+</sup>CD62L<sup>hi</sup>CD44<sup>lo</sup> Eomes<sup>hi</sup>BCL-2<sup>hi</sup>CD5<sup>hi</sup>SCA1<sup>hi</sup>.</p>", "<title>RNA-seq reveals Ly6C<sup>+</sup> cells as a transcriptionally distinct subset of CD8<sup>+</sup> Tn cells</title>", "<p id=\"Par14\">We next performed RNA-seq on CD8<sup>+</sup> Tn cells from both SPF and CH mice sorted by Ly6C expression to define Ly6C-dependent transcriptomic differences. When analyzed independently, principal component analysis (PCA) of global gene expression of Ly6C<sup>+</sup> vs. Ly6C<sup>−</sup> Tn cells yielded robust clustering of samples in SPF mice (51% of the variance, PC1), with markedly more modest clustering in CH mice (36% of the variance, PC1). When analyzed together, global gene expression of all samples affirmed a strong transcriptional difference between SPF and CH mice, associated with the first principal component (PC1), accounting for 41% of the total variance (Fig. ##FIG##4##5A##). Differential gene expression analysis between the Ly6C<sup>+</sup> and Ly6C<sup>−</sup> cells identified 154 (SPF) and 46 (CH) significantly [false discovery rate (FDR) &lt; 0.05] downregulated genes, and 220 (SPF) and 50 (CH) upregulated genes (Fig. ##FIG##4##5B##). Not surprisingly, <italic>Ly6C1</italic> and <italic>Ly6C2</italic> exhibited the most significant upregulation, and the results confirmed our previous observations made at the protein level, such as the increased expression of Eomes and CXCR3 mRNA in Ly6C<sup>+</sup> CD8 Tn cells (Fig. ##FIG##4##5B–D##, S##SUPPL##0##3A##). Fifty of the 374 significantly differentially expressed genes from SPF mice overlapped significantly (<italic>P</italic> &lt; 2.2e−16, Fisher’s exact test) with those 96 genes from CH mice (Fig. ##FIG##4##5C##). Gene ontology (GO) and KEGG enrichment analysis identified multiple immune-specific pathways (<italic>q</italic> &lt; 0.1) including “chemokine signaling pathway” and “viral protein interaction with cytokine and cytokine receptor” (Fig. ##SUPPL##0##S3B, C##). Despite the differences between SPF and CH mice (Fig. ##FIG##4##5A##), hierarchical clustering of the 50 common gene expression matrix showed that the Ly6C<sup>+</sup> cell cluster in both SPF and CH mice (dark green) was separate from their Ly6C<sup>−</sup> counterpart (light green) (Fig. ##FIG##4##5D##). These data support a model whereby, at the genome-wide scale, a distinct subset of immune function-related genes is transcribed to convey enhanced effector function to the Ly6C<sup>+</sup> CD8 Tn cells in both SPF or CH mice, consistent with data from Fig. ##FIG##2##3##.</p>", "<title>Ly6C upregulation is mediated by IFN-I and involves tonic TCR recognition of pMHC</title>", "<p id=\"Par15\">Given that Ly6C expression on CD8<sup>+</sup> Tn cells is induced in a bystander manner, a mode of action consistent with the influence of a soluble mediator, we have examined multiple inflammatory and homeostatic cytokines for their ability to upregulate Ly6C<sup>+</sup> on CD8<sup>+</sup> Tn cells. We have cultured CD8<sup>+</sup> Tn cells for 24 h with 100 ng/ml of each cytokine. Only IFN-I (both IFN-α and IFN-β), we're able to induce Ly6C expression (Fig. ##FIG##5##6A##). This was consistent with Ly6C<sup>+</sup> cells expressing high levels of Eomes, as Eomes expression in CD8<sup>+</sup> T cells has been shown to be regulated by IFN-I<sup>##REF##25953241##23##</sup>. Recently it has been reported that IL-27 can induce moderate Ly6C expression on Tn cells after 72 h after culture<sup>##UREF##3##24##</sup>, yet we observed no such effect after 24 h. To confirm the role of IFN-I in vivo, we have measured Ly6C expression in mice lacking IFN-I receptor (Ifnar1<sup>−/−</sup> mice). Ly6C<sup>+</sup> population was almost entirely missing in Ifnar1<sup>−/−</sup> mice as only ~5% CD8<sup>+</sup> Tn cells expressed Ly6C (Fig. ##FIG##5##6B##). While Ifnar1<sup>−/−</sup> mice are known to be highly susceptible to most viral diseases, they have been shown to be resistant to Listeria infection<sup>##UREF##4##25##</sup>. Therefore, to assess the ability of CD8<sup>+</sup> Tn cells to upregulate Ly6C during infection in the absence of IFN-I signaling we have infected Ifnar1<sup>−/−</sup> mice. We found that while Ifnar1<sup>−/−</sup> mice were able to slightly upregulate Ly6C expression on CD8<sup>+</sup> Tn cells on d3 post-infection, the fraction of Ly6C<sup>+</sup> cells in these mice was at least threefold lower than in Lm-infected wt mice (Fig. ##FIG##5##6B##). Therefore, we conclude that IFN-I is the main and necessary soluble signal for induction of Ly6C expression on Tn cells. We then measured levels of type I IFNs in serum of SPF and CH mice and observed long term (6 mo) upregulation of type I IFNs in continuously CH mice (Fig. ##SUPPL##0##S4A##) explaining the previously observed long term upregulation of Ly6C in these animals (Fig. ##FIG##1##2F##).</p>", "<p id=\"Par16\">In light of the critical role of IFN-I, the increased pZAP-70/SYK and CD5 levels could signify an additional role for TCR-mediated signals in the upregulation of Ly6C. Specifically, IFN-I is a well-known inducer of MHC molecules, potentially providing an increase in TCR ligands for homeostatic, tonic TCR stimulation on CD8<sup>+</sup> Tn cells. To investigate the potential role of sp-MHC affinity and tonic TCR signals in the upregulation of Ly6C, we sorted the top and bottom 20% of CD8<sup>+</sup> Tn cells by CD5 expression (Fig. ##FIG##5##6C##, left panel). Previously it was reported that CD5<sup>hi</sup> Tn cells had some similarities to memory CD8<sup>+</sup> T cells<sup>##REF##25419629##26##</sup>, including higher CD44, CXCR3, and Eomes expression. Here we tested the ability of CD5<sup>hi</sup> and CD5<sup>lo</sup> cells to upregulate Ly6C in response to IFN-I. We found that most of the CD5<sup>hi</sup> cells upregulated Ly6C robustly, whereas very few CD5<sup>lo</sup> cells did (Fig. ##FIG##5##6C##) when stimulated with low (10 ng/ml) IFN-α. By contrast, both populations were able to upregulate SCA-1 (Fig. ##FIG##5##6C##, right panel). With higher IFN-α concentrations of 100 ng/ml, a subset of ~40% of CD5lo cells was also able to upregulate Ly6C, which was still less compared to &gt;80% of the CD5hi cells (Fig. ##FIG##5##6D##). This result strongly suggested that tonic TCR signaling may be necessary as an additional signal to upregulate Ly6C following IFN-I stimulation. Because in our experiment CD8<sup>+</sup> Tn cells were cultured alone, we inferred that this signal could only be coming from neighboring T cells. To address this issue, we stimulated highly purified CD8<sup>+</sup> Tn cells with IFN-α in the presence of an MHC-I blocking antibody. Under such conditions, the IFN-α mediated upregulation of Ly6C was completely abrogated (Fig. ##FIG##5##6E##, left), while the expression of SCA-1 was significantly reduced (Fig. ##FIG##5##6E##, right). Therefore, in addition to IFN-I signaling, tonic MHC-dependent TCR signaling was necessary to induce Ly6C on CD8<sup>+</sup> Tn cells. To further investigate this relationship in vivo, we have treated Nur77<sup>GFP</sup> transgenic reporter mice with 0.5 µg/mouse IFN-α. Nur77 mice express GFP from the immediate early gene Nr4a1 (Nur77) locus which is upregulated by TCR stimulation, but not by inflammatory stimuli<sup>##UREF##5##27##</sup>. Upon treatment with IFN-α, Ly6C was upregulated on CD8<sup>+</sup> Tn cells (Fig. ##FIG##5##6F##, left) to a similar extent previously observed in WNV infection (Fig. ##FIG##1##2A##). The Nur77<sup>GFP</sup> signal was increased on all CD8<sup>+</sup> Tn cells by IFN-α treatment (Fig. ##FIG##5##6F##, right) but MFI was higher on Ly6C<sup>+</sup> cells (Fig. ##FIG##5##6G##, left). This increase in GFP signal was, as expected, much lower than the one elicited by stimulation of the same cells by cognate antigen (Nur77 OT-1 stimulated with SIINFEKL peptide, Fig. ##FIG##5##6G##, right), consistent with differences in signal intensities for antigenic vs. tonic subthreshold stimulation and in agreement with a previous report showing a slight increase of Nur77 expression on CD5hi cells<sup>##REF##25419629##26##</sup>. This validated our in vitro findings and confirmed that tonic TCR signaling and IFN-I signaling jointly upregulate Ly6C. Because the Ly6C<sup>+</sup> population is scarce in Ifnar1<sup>−/−</sup> mice, we have measured CD5 levels on CD8<sup>+</sup> Tn cells in these mice and found that compared to WT or IFN-α-treated mice, Ifnar1<sup>−/−</sup> mice exhibited significantly reduced CD5 levels (Fig. ##FIG##5##6H##). This implied that tonic TCR signaling is significantly reduced in the absence of type I interferon signaling directly to the T cells. We next measured MHC-I levels on LN stromal cells from wt and IFN-α treated mice. These stromal cells are critically involved in the homeostatic maintenance of Tn cells in the T cell zones of LN<sup>##REF##17893676##28##</sup>. MHC-I levels were significantly increased by IFN-α treatment of wt mice (Fig. ##FIG##5##6H##). No such increase of MHC-I expression was seen in CD11c+ dendritic cells or T cells (Fig. ##SUPPL##0##S4B##). Based on this we hypothesize that LN stromal cells are the cell type mediating tonic TCR signals in vivo, however, a formal confirmation of this hypothesis requires further investigation outside of the scope of this study.</p>", "<title>Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells are preferentially home to lymph nodes and show improved homeostatic properties</title>", "<p id=\"Par17\">To investigate homeostatic properties of Ly6C<sup>+</sup> cells, we transferred equal numbers of CD8 Tn Ly6C<sup>+</sup> and Ly6C<sup>−</sup> cells, differentially labeled with Cell Trace Violet (CTV) or CFSE, into lymphopenic RAG-1<sup>−/−</sup> mice, a situation that leads to vigorous homeostatic proliferation of Tn cells, driven by reduced competition for signals from self-MHC-I<sup>##REF##10557316##29##</sup> and IL-7<sup>##REF##11447288##30##</sup>. Under such conditions Ly6C<sup>+</sup> cells proliferated more than their Ly6C<sup>−</sup> counterparts and this was more pronounced in the LN (Fig. ##FIG##6##7A##, complete gating strategy in Fig. ##SUPPL##0##S7B##) where the number of undivided cells was lower than in the spleen. This suggested that Ly6C<sup>+</sup> cells might be exhibiting increased reactivity to IL-7. Indeed, we found that the IL-7R (CD127) exhibited significantly higher expression on Ly6C<sup>+</sup> cells (Fig. ##FIG##6##7B##). Next, we assessed the proliferation of Ly6C-separated CD8<sup>+</sup> Tn cell subsets when stimulated with homeostatic cytokines IL-7 and IL-15. Ly6C<sup>+</sup>, but not Ly6C<sup>−</sup>, subset proliferated in response to IL-7 (Fig. ##FIG##6##7C##, left panel), While neither cell type proliferated in response to IL-15 (Fig. ##FIG##6##7C##, middle panel), the addition of IL-15 did potentiate the proliferative effect of IL-7 specifically in Ly6C<sup>+</sup> cells (Fig. ##FIG##6##7C##, right panel). This suggested that Ly6C<sup>+</sup> T cells begin to acquire functional responsiveness to IL-15. To determine if Ly6C<sup>+</sup> cells are also more reactive to IL-7 in vivo, we treated mice with IL-7/Anti-IL-7Ab complexes, to increase the in vivo half-life and potency of IL-7. Ly6C<sup>+</sup> cells displayed higher reactivity to IL-7c in vivo, as measured by increased expression of the division-associated antigen Ki-67 (Fig. ##FIG##6##7D##).</p>", "<p id=\"Par18\">Ly6C was previously reported to assist in the LN homing of CM T cells<sup>##REF##21308682##31##</sup>. To assess if this holds for CD8<sup>+</sup> Tn cells, we transferred equal numbers of magnetically enriched Tn CD8<sup>+</sup>s from CD45.1<sup>+</sup> control mice (CTV labeled), from IFN-α treated mice (not labeled) and IFN-α treated mice blocked with anti-Ly6C Ab (clone HK1.4; CFSE-labeled). Tn from IFN-α treated mice exhibited higher expression of Ly6C (60.6%) compared to control cells (19.6%). Forty-eight hours after transfer, we identified CD45.1+ donor cells in spleens and LN of recipients and divided them into three populations based on CFSE and CTV. Proportions of all three populations were equal in recipient spleens (Fig. ##FIG##6##7E##, left panel), but differed in their migration to the LN, where there was an increased proportion of IFN-α treated cells which expressed a higher level of Ly6C, and this increase was completely abrogated by Ly6C blockade (Fig. ##FIG##6##7E##). These results demonstrate that Ly6C supports the LN homing of Tn cells as well.</p>", "<p id=\"Par19\">Since Ly6C<sup>+</sup> cells are BCL-2<sup>hi</sup> and Eomes<sup>hi</sup> and induced by IFN-I, we examined the relationship between Ly6C<sup>+</sup> phenotype and survival. We incubated magnetically enriched total Tn CD8<sup>+</sup>s with 100 ng/ml IFN-α either alone or in the presence of CD45<sup>−</sup> LN stroma. IFN-α increased survival of Tn CD8<sup>+</sup> cells in vitro (Fig. ##FIG##7##8A##, left panel). Increased survival was even more pronounced in coculture with LN stromal cells, where more than half of IFN-α cells survived &gt;7 days (Fig. ##FIG##7##8A##, right panel). Because LN stromal cells increase survival of Tn cells through the production of homeostatic factors such as IL-7 and CCL21<sup>##REF##17893676##28##</sup>, this suggests that IFN-α prolongs Tn survival both directly and in combination with other homeostatic factors. To confirm the role of IFN-I we stimulated Tn CD8<sup>+</sup>s from Ifnar1<sup>−/−</sup> and wt mice cocultured with LN stroma from Ifnar1<sup>−/−</sup> mice. As expected, IFN-α had no effect on the survival of Ifnar1<sup>−/−</sup> T cells (Fig. ##FIG##7##8B##, left panel). On the other hand, survival of wt T cells cocultured with Ifnar1<sup>−/−</sup> stroma (Fig. ##FIG##7##8B##, right panel) was increased by IFN-α, similarly to wt stroma. This confirms that type I IFN has a direct homeostatic effect on Tn cells. Simultaneously, we have measured the expression of Ly6C on surviving wt cells cultured with LN stroma with or without IFN-α. CD8<sup>+</sup> Tn cells in some wells were labeled with CFSE to determine whether there was any cell proliferation under this condition. By day 7 most (&gt;90%) surviving IFN-α-stimulated cells expressed Ly6C (Fig. ##FIG##7##8C##), and that was not caused by increased proliferation as CFSE remained undiluted (not shown)., We conclude that this is a consequence of increased survival and accumulation of Ly6C<sup>+</sup> cells. At the same time, surviving Tn cells cultured with stroma alone were gradually losing Ly6C expression in the absence of type I IFN signaling (Fig. ##FIG##7##8C##). Overall, these results show that IFN-I preferentially increased the survival of Ly6C<sup>+</sup> Tn CD8<sup>+</sup> cells. Given the preferential LN homing of Ly6C<sup>+</sup> cells and the powerful in vitro homeostatic effect of type I IFNs, we have determined absolute numbers of Ly6C+/− subsets of Tn CD8<sup>+</sup>s in LN of SPF and CH mice. As previously reported, CH mice display a decrease in the proportion of cells with the naïve CD44<sup>lo</sup>CD62L<sup>hi</sup> phenotype, due to an increase in numbers of memory and effector cells (Fig. ##SUPPL##0##S5A##). However, because LNs of CH mice exhibited greatly increased overall cellularity (Fig. ##SUPPL##0##S5B##), they actually exhibit increased absolute numbers of CD8<sup>+</sup> Tn cells, which was significant only for the Ly6C<sup>+</sup> population (Fig. ##SUPPL##0##S5C##). Because IFN-I is the main signal for induction of Ly6C on Tn CD8<sup>+</sup> cells, we conclude that this accumulation of Ly6C<sup>+</sup> Tn is at least partly if not mostly driven by IFN-I signaling.</p>", "<title>Ly6C<sup>+</sup> naïve CD8<sup>+</sup> T cells show enhanced effector function against low-affinity peptide ligands</title>", "<p id=\"Par20\">In vitro polyclonal stimulation of CD8<sup>+</sup> Tn cells sorted by Ly6C expression showed that Ly6C<sup>+</sup> cells produce higher levels of effector molecules, especially GzB. To determine if Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells exhibit improved effector function in vivo, we infected B6 SPF mice with Listeria-OVA genetically engineered to express the native immunodominant CD8<sup>+</sup> ovalbumin peptide SIINFEKL (N4) or its two altered peptide ligands (APLs) for which the OT-I TCR exhibits decreasing sensitivity: Y3 &gt; Q4<sup>##REF##19182777##32##</sup> (Fig. ##FIG##8##9A##). Twenty-four hours later we transferred 3 × 10<sup>4</sup> sorted Ly6C<sup>+</sup> or Ly6C<sup>−</sup> cells from OT-1 mice. On day 5 we measured the number of GzB-producing donor cells in the spleen and liver, as well as host bacterial burdens. Mice infected with Listeria expressing native ligand N4 which received Ly6C<sup>+</sup> cells exhibited a decreased number of GzB<sup>+</sup> cells in the spleen but this was not associated with a lower bacterial burden (Fig. ##FIG##8##9B, C##). On the other hand, mice infected with Lm expressing lower affinity APL Y3 which received Ly6C<sup>+</sup> cells displayed a higher number of GzB producing cells and lower bacterial burden (Fig. ##FIG##8##9B, C##). A similar trend was observed with the lowest affinity APL Q4—the increase in GzB<sup>+</sup> cells in the Ly6C<sup>+</sup> group was not statistically significant (Fig. ##FIG##8##9B##), but the decrease in the bacterial burden was significant (Fig. ##FIG##8##9C##).</p>", "<p id=\"Par21\">These results indicate that in vivo effector function of Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells might be particularly improved for low-affinity clones. To further investigate this, we have treated OT-1 transgenic mice with poly-I:C, isolated Tn CD8s and stimulated them with APL in vitro. As expected, the majority of OT-1 Tn cells upregulated Ly6C (Fig. ##SUPPL##0##S5A##). After stimulation with the original agonist N4 (SIINFEKL) peptide, poly-I:C pretreated OT-1 cells exhibited higher production of GzB especially at early time points (24 h and 36 h) while at 72 h untreated cells have almost caught up with GzB production (Fig. ##FIG##8##9D-E##). A similar trend was observed with all 3 peptides of decreasing affinity (Fig. ##FIG##8##9F##), indicating that type I interferon exposure is particularly important for rapid acquisition of effector function. Increased IRF4 and Eomes (Fig. ##FIG##8##9G##) levels, (both transcription factors known to regulate CD8 effector function), pointed to a potential mechanism of how preexposure to type I interferons may increase effector function. Finally, we examined how the poly-I:C pretreatment affects in vivo function by performing transfers (scheme in Fig. ##SUPPL##0##S5B##, left panel) of Tn CD8<sup>+</sup>s from untreated and poly-I:C-treated into Lm-OVA APL infected mice. In this in vivo setting, similar to experiments performed with Ly6C<sup>+</sup> sorted CD8<sup>+</sup> Tn OT-1 cells in vitro, the increased GzB production was evident for the low-affinity APL Q4 (Fig. ##SUPPL##0##S5C##, left panel). Paralleling that observation, functional reduction of bacterial burden (Fig. ##SUPPL##0##S5C##, right) was significant and evident for that same low-affinity ligand, providing evidence of functional importance of the increased Ly6C expression on CD8<sup>+</sup> Tn cells in the face of low-affinity microbial pathogen variants.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par22\">In this work, we uncover a profound impact of bystander infection and inflammation on the CD8<sup>+</sup> Tn cell pool in mice housed under ‘non SPF conditions’. These conditions induce an expansion of a Ly6C<sup>+</sup> subpopulation of CD8<sup>+</sup> Tn cells which are Eomes<sup>hi</sup>BCL-2<sup>hi</sup>CD5<sup>hi</sup>SCA1<sup>hi</sup> and exhibit rapid effector function upon TCR stimulation. The Human Ly-6/uPAR protein family has 20 members, yet has no known human homolog of Ly6C<sup>##REF##23186997##33##</sup>. The exact function of Ly6C is unknown but it has been reported that Ly6C assists LN homing of central Tm cells<sup>##REF##21308682##31##</sup>. Our transfer experiments support that this is the case for CD8<sup>+</sup> Tn cells and that increasing Ly6C expression leads to superior LN homing.</p>", "<p id=\"Par23\">Our in vitro data shows that IFN-I can selectively and specifically upregulate Ly6C on CD8<sup>+</sup> Tn cells, whereas CD8<sup>+</sup> Tn cells deficient for the IFN-I receptor expressed very little Ly6C. That allowed us to identify IFN-I as the main signals that upregulate Ly6C in a bystander manner on CD8<sup>+</sup> Tn cells. While this upregulation was transient in viral and bacterial monoinfections, it was permanent under ‘non-SPF’ conditions.</p>", "<p id=\"Par24\">IFN-I are known for their role as signal 3 cytokines, involved in directing effector immunity, as well as T cell memory formation<sup>##REF##16129706##34##,##UREF##6##35##</sup>. Homeostatic roles of IFN-I on T cells are much less studied<sup>##REF##20627800##36##</sup>, with the exception that it has been shown that they induce bystander proliferation of memory T cells<sup>##REF##8658169##10##</sup> and subsequent attrition<sup>##REF##11390598##37##</sup> during viral infection. Even before antigen presentation, exposure to IFN-I sensitizes T cells to rapidly exert effector functions, such as IFN-production, on contact with their cognate antigen<sup>##REF##20592282##38##</sup>. Our results show that IFN sensitization of CD8<sup>+</sup> Tn cells programs such cells to express Ly6C and exhibit rapid and enhanced production of effector molecules. This was especially pronounced for low-affinity clones, consistent with previous data<sup>##REF##26999026##39##</sup>. This effect is likely mediated by IFN-I induction of the Eomes transcription factor, which is necessary for the expression of effector molecules and memory formation<sup>##REF##20935204##21##</sup>.</p>", "<p id=\"Par25\">Further clues into the biology of Ly6C<sup>+</sup> cells were provided by their high expression of CD5 and BCL-2. CD5 is a negative regulator of TCR signaling and its expression is considered a measure of the strength of encounter with self-ligand<sup>##REF##25419629##26##</sup>. CD5<sup>lo</sup> cells were not able to upregulate Ly6C when stimulated with IFN-I. Moreover, blocking of MHCI molecules abrogated Ly6C upregulation. This showed that tonic TCR signaling is a necessary cofactor for IFN-I induced Ly6C expression. It is well established that Tn homeostasis depends on weak, tonic signaling from the TCR:spMHC interaction<sup>##REF##19100699##40##</sup> followed by IL-7 signaling<sup>##REF##11447288##30##</sup>. IL-7-mediated survival is regulated by the transcription factors BCL-2 and MCL-1. Therefore, increased BCL-2 and MCL-1 expression on Ly6C<sup>+</sup> Tn cells suggested increased IL-7 reactivity. Indeed, Ly6C<sup>+</sup> cells expressed higher levels of the IL-7R and proliferated more when stimulated with IL-7 in vitro. Consistent with that, Ly6C<sup>+</sup> cells also proliferated more vigorously in the lymphopenic RAG-KO environment, especially in the LN.</p>", "<p id=\"Par26\">In addition, IFN-I directly increased the survival of CD8<sup>+</sup> Tn cells in culture, with the Ly6C<sup>+</sup> cells surviving the longest. Furthermore, in vivo treatment of Nur77-driven GFP reporter mice<sup>##UREF##5##27##</sup> with type I IFNs resulted in low but reproducible increased GFP expression, providing evidence for increased homeostatic TCR signaling. Thus, our results point to IFN-I as the new homeostatic factor, which maintains the Tn Ly6C<sup>+</sup> subpopulation characterized by increased lymph node homing, tonic TCR signaling, and IL-7 reactivity.</p>", "<p id=\"Par27\">Lastly, we show that Ly6C<sup>+</sup>CD8<sup>+</sup> Tn cells exhibit increased effector function particularly towards low-affinity APLs in vivo. This suggests that Ly6C<sup>+</sup> cells might be recruited in the response at a higher rate when ligands are of lower affinity, and this may result in their increased lymph node or tissue homing. Contrary to in vivo data where effector function was improved only with low-affinity ligands our in vitro data showed increased effector function with all three APLS. The reason for this discrepancy could be immediate exposure of transferred cells to host type I IFNs which is difficult to circumvent as IFNARKO Tn CD8<sup>+</sup>s do not express Ly6C and cytokine knockouts do not exist. These findings might have practical implications for adoptive T cell therapy of tumors, which will require further investigation.</p>" ]
[]
[ "<p id=\"Par1\">Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide–MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5<sup>hi</sup>CD8<sup>+</sup> cells, which in the case of polyinfection translates into a numerical CD8<sup>+</sup> Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C<sup>+</sup> Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C<sup>+</sup>CD8<sup>+ </sup>Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.</p>", "<p id=\"Par2\">Infections induce activation of naïve T cells for protective immunity, but insights for this host-pathogen crosstalk are still missing. Here the authors show that infection-induced type I interferon (IFN-I) signaling promote the differentiation, expansion and functional maturation of naïve CD8 T cells, particularly for low affinity clones, to enhance anti-microbial immunity.</p>", "<title>Subject terms</title>" ]
[ "<title>Supplementary information</title>", "<p>\n\n\n\n</p>", "<title>Source data</title>", "<p>\n\n</p>" ]
[ "<title>Supplementary information</title>", "<p>The online version contains supplementary material available at 10.1038/s41467-021-25645-w.</p>", "<title>Acknowledgements</title>", "<p>The authors would like to thank University of Arizona Flow Cytometry core facility personnel for help with cell sorting. We would also like to thank all the members of the JNZ lab for constructive discussion. Supported by USPHS awards AG020719 and AG048021 and the Elizabeth Bowman Endowed Chair in Medical Sciences to J.N-Z.</p>", "<title>Author contributions</title>", "<p>M.J., M.S., D.B. and J.N.-Z. designed experiments. M.J., C.P.C., J.L.U. and S.C. performed experiments. M.J., C.P.C., D.B., J.L.U. and S.C. analyzed the data. M.J. and J.N.-Z. wrote the manuscript. J.N.-Z. administered the project.</p>", "<title>Data availability</title>", "<p>The data and materials that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper. The RNA-seq data discussed in Fig. ##FIG##4##5## of this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159076\">GSE159076</ext-link>. The flow cytometric data used for unbiased clustering in Fig. ##FIG##0##1## has been deposited to flow repository.org and is accessible through accession number <ext-link ext-link-type=\"uri\" xlink:href=\"https://flowrepository.org/id/FR-FCM-Z4AS\">FR-FCM-Z4AS</ext-link>. <xref ref-type=\"sec\" rid=\"Sec22\">Source data</xref> are provided with this paper.</p>", "<title>Competing interests</title>", "<p id=\"Par39\">J.N.Ž. is on the scientific advisory board and receives research funding from Young Blood, Inc. The remaining authors declare no competing interests.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Unbiased clustering identifies upregulation of Ly6C on the surface of CD8<sup>+</sup> Tn cells following WNV infection.</title><p>C57BL/6 mice (<italic>N</italic> = 9) were infected with 1000 PFU WNV virus by footpad injection. WNV infected and uninfected control mice (<italic>N</italic> = 9) were bled retro-orbitally at days 7 post-infection and stained with a 15 marker flow cytometry panel. FCS files were uploaded in Cytobank, cloud-based clustering software, CD3<sup>+</sup> live cells were gated prior to clustering. Flowsom visualization algorithm identified the standard subsets of CD8<sup>+</sup>T cells effector/effector memory ((EM) CD44<sup>hi</sup>CD62L<sup>−</sup>), central memory ((CM) CD44<sup>hi</sup>CD62L<sup>+</sup>CD49d<sup>+</sup>), virtual memory ((VM) CD44<sup>hi</sup>CD62L<sup>+</sup>CD49d<sup>−</sup>) and naïve ((N) CD44<sup>lo</sup>CD62L<sup>+</sup>CD49d<sup>−</sup>). Tn was divided into two subsets based on Ly6C expression, Ly6C<sup>+</sup> subset (indicated by red arrow) was enlarged at d7 of WNV infection. Data is from one experiment (with <italic>N</italic> = 9 mice per group).</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Naïve CD8<sup>+</sup> T cells upregulate Ly6C transiently during acute viral and bacterial infection and permanently under ‘non-SPF’ conditions.</title><p><bold>A</bold> Flow cytometry gating strategy to analyze the expression of Ly6C and CXC3 on naïve (CD44<sup>lo</sup>CD62L<sup>hi</sup>) CD8<sup>+</sup> T cells Briefly, live CD3<sup>+</sup>CD8<sup>+</sup> cells were gated with the standard phenotypic definition for Tn cells (CD62L<sup>hi</sup>CD44l<sup>o</sup>) and then divided into the positive and negative population by Ly6C expression. <bold>B</bold> C57BL/6 mice were infected with 1000 PFU WNV virus by footpad injection. Mice were bled retro-orbitally at days 3, 5, 16, 20, and 28 post-infection. Blood was hypotonicially lysed and surface stained with antibodies for flow cytometric analysis. (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, **<italic>p</italic> = 0.0049, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001) <bold>C</bold> Mice were inoculated intravenously (i.v.) with 10<sup>4</sup> CFU Listeria monocytogenes. Mice were bled retro-orbitally at days 3, 5, 8, and 16 p.i. The proportion of Ly6C<sup>+</sup> positive CD44loCD62Lhi cells were analyzed by flow cytometry (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>D</bold> Expression of various inflammatory cytokines was measured in homogenates of secondary lymphoid tissue of cohoused and SPF control mice by Legendplex flow cytometry multiplex immunoassay (<italic>n</italic> = 5 mice per group) (<bold>E</bold>) cohoused mice were bled periodically and proportion of Ly6C<sup>+</sup> positive naïve CD8<sup>+</sup>s was measured by flow cytometry (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, **<italic>p</italic> = 0.0022, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>A</bold>–<bold>E</bold> Data are representative of two independent experiments (with <italic>n</italic> = 5 mice per group) (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>B</bold>–<bold>E</bold> unpaired one-way ANOVA with Dunett’s correction for multiple comparisons.</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells display rapid and enhanced effector function in vitro.</title><p><bold>A</bold> CD8 T cells were enriched by magnetic sorting from secondary lymphoid tissues (spleen and pool of brachial, inguinal, and cervical lymph nodes) of SPF and CH mice. Ly6C<sup>+</sup> and Ly6C<sup>−</sup> CD44<sup>lo</sup>CD62L<sup>hi</sup> cells were then sorted by flow cytometric sorting. Forty thousand sorted cells were plated in round-bottom plates and simulated α-CD3/α-CD28 beads in 1:1 cell to bead ratio for 24 h or 36 h and with phorbol-myristate acetate (PMA) and ionomycin for 6 h in presence of brefeldin A. <bold>B</bold> Expression of GzB was measured on live cells by intracellular flow cytometric staining following 24 h (left panel, <italic>n</italic> = 5 mice per group, data presented as mean ± sd, left to right **<italic>p</italic> = 0.0021, **<italic>p</italic> = 0.0055) or 36 h stimulation with α-CD3/α-CD28 beads (right panel, <italic>n</italic> = 4 mice per group, data presented as mean ± sd) (<bold>C</bold>) IFN-γ expression was measured on live cells by intracellular flow cytometric staining (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, left to right *<italic>p</italic> = 0.0184, *<italic>p</italic> = 0.0479), as well as expression of (<bold>D</bold>) TNF-α. (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, *<italic>p</italic> = 0.0131, **<italic>p</italic> = 0.0055). <bold>E</bold> Phosphorylation of Erk (p-Erk) was measured by phosflow on unstimulated sorted cells or stimulated for 20 min with an α-CD3/α-CD28 bead (<italic>n</italic> = 6 mice, data presented as mean ± sd, ****<italic>p</italic> &lt; 0.0001), as well as (<bold>F</bold>) phosphorylation of ZAP-70 Y319 (<italic>n</italic> = 6 mice, data presented as mean ± sd, **<italic>p</italic> = 0.0014, ***<italic>p</italic> &lt; 0.001). <bold>A</bold>, <bold>C</bold>, <bold>D</bold> Data are representative of two experiments (with <italic>n</italic> = 5 mice per group). <bold>E</bold>, <bold>F</bold> Data are representative of two experiments (with <italic>n</italic> = 6 mice) (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>A</bold>, <bold>B</bold>, <bold>D</bold>, <bold>E</bold>, <bold>F</bold> one-way ANOVA with Sidak post-hoc correction for multiple comparisons: C-one way Kruskal-Wallis test with Dunn’s correction for multiple comparisons.</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Microbial colonization increases Ly6C expression on CD8<sup>+</sup> Tn cells in a bystander manner.</title><p><bold>A</bold> Female C57BL/6 mice were cohoused with outbred, wild-type pet shop mice in large rat cages, separated by a perforated barrier. After &gt;60 days of cohousing expression of Cd49d, CD69, KLRG1, CD27, and CD25 was analyzed by flow cytometry on subpopulations of Tn CD8<sup>+</sup> T cells from lymph nodes of CH mice and SPF controls (Color scheme: red-Tn Ly6C<sup>−</sup> from SPF mice; blue-Tn Ly6C<sup>+</sup> from SPF mice; orange-Tn Ly6C<sup>−</sup> from CH mice; light green-Tn Ly6C<sup>+</sup> from CH mice; dark green-CD44<sup>hi</sup> from SPF mice; brown-CD44<sup>hi</sup> from CH mice). <bold>B</bold> Upregulation of Ly6C was upregulated on central memory (CM) cytotoxic T cells (CD8<sup>+</sup>CD44<sup>hi</sup>CD62L<sup>hi</sup>), CD44<sup>neg</sup>CD62L<sup>hi</sup> and CD44<sup>int</sup>CD62L<sup>hi</sup> cells (<italic>n</italic> = 4 SPF mice, <italic>n</italic> = 5 CH mice per group, data presented as mean ± sd, ***<italic>p</italic> &lt; 0.001). <bold>C</bold> Ly6C was upregulated on Tn CD8<sup>+</sup> cells in the blood, lymph nodes, and spleen (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, ****<italic>p</italic> &lt; 0.0001). <bold>D</bold> Tn CD8<sup>+</sup> cells were magnetically enriched<sup>,</sup> CFSE labeled and transferred into CH mice for 14 days (<italic>n</italic> = 7 ctrl mice, <italic>n</italic> = 8 CH mice, data presented as mean ± sd, ***<italic>p</italic> = 0.0003). <bold>E</bold> Expression of CD5 and SCA-1 on Ly6C ± CD8<sup>+</sup> Tn from SPF and CH (Color scheme<sup>:</sup> red-Tn Ly6C<sup>−</sup> from SPF mice; blue-Tn Ly6C<sup>+</sup> from SPF mice; orange-Tn Ly6C<sup>−</sup> from CH mice; light green-Tn Ly6C<sup>+</sup> from CH mice). <bold>F</bold> Ly6C ± CD8<sup>+</sup> Tn from SPF and CH (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, left to right *<italic>p</italic> = 0.0481, *<italic>p</italic> = 0.0336) were stained intracellularly for expression of Eomes and (<bold>G</bold>) BCL-2 (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, *<italic>p</italic> = 0.0168). <bold>A</bold>, <bold>B</bold>, <bold>C</bold>, <bold>E</bold>, <bold>F</bold>, and <bold>G</bold> data are representative of two experiments (with <italic>n</italic> = 5 mice per group). <bold>D</bold> Data are pooled from two experiments (total <italic>N</italic> = 7 mice per group (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>D</bold> Two-tailed Mann-Whitney U-test; <bold>A</bold>, <bold>B</bold>, <bold>C</bold>, <bold>E</bold>, <bold>F</bold>, and <bold>G</bold>) one-way ANOVA, with Sidak post hoc correction.</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Transcriptional distinction between Ly6C<sup>+</sup> and Ly6C<sup>−</sup> CD8<sup>+</sup> Tn cells.</title><p><bold>A</bold> PCA shows differentially clustered Ly6C<sup>+</sup> and Ly6C<sup>−</sup> CD8 Tn cells in SPF or co-housed (CH) mice, or both combined (Color scheme: blue-Tn Ly6C<sup>−</sup> from SPF mice; green-Tn Ly6C<sup>+</sup> from SPF mice; red-Tn Ly6C<sup>−</sup> from CH mice; orange-Tn Ly6C<sup>+</sup> from CH mice). <bold>B</bold> Volcano plot comparing gene expression changes between Ly6C<sup>+</sup> vs. Ly6C<sup>−</sup> cells in SPF (left panel) and CH (right panel) mice, where the vertical axis is logarithmically transformed due to very small FDR values of <italic>Ly6C1</italic> and <italic>Ly6C2</italic> genes. Counts of significantly (FDR &lt; 0.05) downregulated and upregulated genes (colored orange), separated by a slash (/), are shown in the top strip label. Genes with log2FoldChange &gt; 3.5 and FDR &lt; 0.0001 are indicated. <bold>C</bold> Venn diagram displaying the significant overlap (<italic>P</italic> &lt; 2.2E−16, Fisher’s exact test) between significantly regulated genes in SPF with CH mice. <bold>D</bold> Expression matrix of these 50 genes. Data was normalized through a regularized logarithm transformation (rlog) implemented in DESeq2. Ly6C<sup>−</sup> (light green) and Ly6C<sup>+</sup> (dark green) cell clusters are highlighted<sup>.</sup> Data is from one experiment with <italic>n</italic> = 4 mice/group.</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Ly6C upregulation is a direct consequence of IFN-I and tonic TCR signaling.</title><p><bold>A</bold> CD62L<sup>hi</sup>CD44<sup>lo</sup> CD8<sup>+</sup> T cells were magnetically enriched from pooled secondary lymphoid tissues (spleen, brachial and inguinal lymph nodes) to &gt;95% purity. Cells were then cultured in RPMI media + 10% FCS and 100 ng/ml of each cytokine TNF-α, IFN-γ, IL-12, IL-18, IL-7, IL-15, IL-6, IFN-α, and IFN-β. Twenty-four hours later expression of Ly6C was measured (<italic>n</italic> = 6 mice, data presented as mean ± sd, <italic>p</italic> = 0.0237) <bold>B</bold> Ifnar1<sup>−/−</sup> mice were infected i.v. with 10<sup>4</sup> CFU Lm. Expression of Ly6C on Tn CD8<sup>+</sup> cells was measured in Lm infected Ifnar1<sup>−/−</sup> mice (72 h p.i.), uninfected Ifnar1<sup>−/−</sup> mice, and wt Lm infected mice (<italic>n</italic> = 7 mice per group, data presented as mean ± sd, ****<italic>p</italic> &lt; 0.0001). <bold>C</bold> Top and bottom 20% of CD8<sup>+</sup> Tn cells by CD5 MFI were FACS sorted (Color scheme: red-CD5lo; blue-CD5hi) and (<bold>D</bold>) cultured in RPMI media + 10% FCS and 10 or 100 ng/ml IFN-α. After 24 h expression of Ly6C and SCA-1 were measured by flow cytometry (<italic>n</italic> = 6 mice per group, data presented as mean ± sd, ****<italic>p</italic> &lt; 0.0001). <bold>E</bold> CD8<sup>+</sup> Tn cells were magnetically enriched to &gt;95% purity and stimulated with 100 ng/ml IFN-α.± MHCI blocking antibody (clone 28-8-6) for 24 h (<italic>n</italic> = 4 mice, mean ± sd). <bold>F</bold> Nur77<sup>GFP</sup> mice (<italic>n</italic> = 5) were treated with 0.75 µg/mouse of IFN-α. 48 h post-treatment mice were bled retroorbitally and expression of GFP and Ly6C was measured on CD8<sup>+</sup> Tn cells (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, ****<italic>p</italic> &lt; 0.0001). <bold>G</bold> Left panel expression of GFP was compared between Ly6C<sup>+</sup> and Ly6C<sup>−</sup> subpopulations of Tn CD8<sup>+</sup> cells (<italic>n</italic> = 10 mice, data presented as individual dots and paired <italic>t-</italic>test lines, *<italic>p</italic> = 0.0363, ****<italic>p</italic> &lt; 0.0001); right panel GFP histograms of Ly6C ± Tn CD8<sup>+</sup> cells were overlayed with GFP histogram of antigen (SIINFEKL peptide, 10<sup>−7 </sup>M) stimulated OT-1 Nur77<sup>GFP</sup> (Color scheme:red-Ly6C<sup>−</sup>; blue-Ly6C<sup>+</sup>, orange-antigen (SIINFEKL peptide) specific). <bold>H</bold> Ifnar1<sup>−/−</sup> mice, wt mice, and IFN-α treated mice were bled and CD5 MFI was measured on CD8 <sup>+ </sup>Tn cells<sup>.</sup> (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, left to right *<italic>p</italic> = 0.0362, *<italic>p</italic> = 0.0480). <bold>A</bold>, <bold>B</bold> Data are pooled from two experiments, <bold>C</bold>–<bold>G</bold> Data are representative of two independent experiments (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). A one-way Kruskal-Wallis test with Dunn’s correction for multiple comparisons, <bold>B</bold>, <bold>D</bold>, <bold>E</bold>, <bold>H</bold> one-way ANOVA with Sidak post hoc correction, <bold>F</bold>, <bold>G</bold> two-tailed Student’s <italic>t</italic>-test.</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig. 7</label><caption><title>Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells preferentially home to lymph nodes and show improved homeostatic properties.</title><p><bold>A</bold> CD8<sup>+</sup> T cells were magnetically enriched isolated LN and spleen of C57BL/6 mice (<italic>N</italic> = 6), Ly6C<sup>+</sup> and Ly6C<sup>−</sup> Tn cells were sorted by FACS. Ly6C<sup>+</sup> cells were labeled with CFSE and Ly6C<sup>−</sup> with CTV and 4 × 10<sup>5</sup> cells were adoptively transferred into RAG-1<sup>−/−</sup> hosts Four days later, the proliferation of donor cells was analyzed by FACS (Color schemed: blue-undivided cells; red-divided cells) in the LN and spleen of host mice (<italic>n</italic> = 4 recipient mice, data presented as mean ± sd, *<italic>p</italic> = 0.001, ***<italic>p</italic> &lt; 0.001). <bold>B</bold> MFI of IL-7R (CD127) was measured on CD8<sup>+</sup> Tn cells from C57/BL6 mice (<italic>n</italic> = 15 mice, data presented as mean ± sd). <bold>C</bold> CD62L<sup>hi</sup>CD44<sup>lo</sup> CD8<sup>+</sup> T cells were magnetically enriched from pooled secondary lymphoid tissues (<italic>n</italic> = 5 mice) and FACS sorted by Ly6C expression. Ly6C ± cells were labeled with CFSE and stimulated with 500 ng/ml rIL-7, 100 ng/mL rIL-15, or both. 7 days letter proliferation was measured on live cells (Color scheme: red-Ly6C<sup>−</sup> cells; blue-Ly6C<sup>+</sup> cells). <bold>D</bold> C57BL/6 mice were treated with IL-7/Anti-IL-7Ab (M25) complexes (1.5 µg/mouse every 48 h). On day 6 after the start of treatment mice were bled retroorbitally and expression of Ki67 was measured on CD8<sup>+</sup> Tn cells (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, *<italic>p</italic> = 0.02, ****<italic>p</italic> &lt; 0.0001). <bold>E</bold> We transferred 2 × 10<sup>6</sup> of magnetically enriched Tn CD8<sup>+</sup>s from CD45.1 control mice (CTV labeled), from IFN-α treated mice (48 h prior to harvest; not labeled) and IFN-α treated mice blocked with anti-Ly6C Ab (clone HK1.4; CFSE-labeled) to 5 recipient C57BL/6 mice. 20 min later spleen and LN pools (inguinal, brachial, cervical) were harvested and the proportion of three transferred cell populations was measured (<italic>n</italic> = 10 recipient mice, data presented as mean ± sd). <bold>A</bold>, <bold>C</bold>, <bold>D</bold> data are representative of two independent experiments. <bold>B</bold>, <bold>E</bold> data are pooled from two experiments (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>A</bold>, <bold>D</bold>, <bold>E</bold>, one-way ANOVA, with Sidak post hoc correction; B paired two-tailed Student’s test.</p></caption></fig>", "<fig id=\"Fig8\"><label>Fig. 8</label><caption><title>Type I interferons enhance survival of preferentially Ly6C<sup>+</sup> CD8<sup>+</sup> Tn cells in vitro.</title><p><bold>A</bold> We magnetically enriched CD8<sup>+</sup> Tn cells to &gt;95% purity. 3 × 10<sup>5</sup> cells were cultured in round bottom 96 well plates either alone or in the presence CD45- LN stroma in RPMI media + 10% FCS and 0.05 mM beta-mercaptoethanol ±100 in ng/ml IFN-α. Cell viability was assessed by flow cytometry on days 1, 3, 5, and 7 of culture (<italic>n</italic> = 8 mice, data presented as mean ± sd). <bold>B</bold> viability of wt CD8<sup>+</sup> Tn cells cocultured with Ifnar1<sup>−/−</sup> stroma and vice versa Ifnar1<sup>−/−</sup> CD8<sup>+</sup> Tn cells stimulated by 100 ng/ml IFN-α was measured on days 1, 3, 5, and 7 (<italic>n</italic> = 8 mice, data presented as mean ± sd). <bold>C</bold> expression of Ly6C on live CD8<sup>+</sup> Tn wt cells cultured with LN stroma with or without IFN-α (<italic>n</italic> = 8 mice, data presented as mean ± sd). Data are pooled from two experiments, <italic>n</italic> = 8, (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>A</bold>, <bold>B</bold>, <bold>C</bold> For the survival of cultured T cells cultured with or without stroma, statistical significance was calculated with a paired two-tailed Student’s <italic>t</italic>-test between corresponding time points.</p></caption></fig>", "<fig id=\"Fig9\"><label>Fig. 9</label><caption><title>Ly6C<sup>+</sup> naïve CD8<sup>+</sup> T cells show enhanced effector function against low-affinity alternative peptide ligands.</title><p><bold>A</bold> Mice were inoculated i.v. with ~10<sup>4</sup> CFU Lm genetically engineered to express the native immunodominant CD8<sup>+</sup> ovalbumin peptide SIINFEKL (N4) or its two altered peptide ligands (APLs) Y3 and Q4. Twenty-four hours later we transferred 3 × 10<sup>4</sup> sorted Ly6C± cells from OT-1 mice into total 6 groups of mice (<italic>n</italic> = 5 mice per group) (transfer scheme). <bold>B</bold> On day 5 post-transfer the number of GzB-producing donor cells in the spleen and liver (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, *<italic>p</italic> = 0.0372, **<italic>p</italic> = 0.0029). <bold>C</bold> Host bacterial burdens in the liver were measured for every Lm APL administered (<italic>n</italic> = 5 mice per group, data presented as mean ± sd, *<italic>p</italic> = 0.0159, **<italic>p</italic> = 0.0079). <bold>D</bold>, <bold>E</bold> Tn CD8<sup>+</sup>s were magnetically enriched from control and poly(I:C) treated mice (Color scheme: red-control mice; blue-poly(I:C) treated mice) and stimulated in vitro with 10<sup>−7</sup> M SIINFEKL peptide and Y3 and Q4 APLs. Production of GzB was measured at 24 h (<italic>n</italic> = 5 mice), 36 and 72 h of stimulation (<italic>n</italic> = 6 mice, data presented as mean ± sd, *<italic>p</italic> = 0.0022, ****<italic>p</italic> &lt; 0.0001) <bold>F</bold> At 36 h GzB expression was higher in cells from mice pretreated with poly(I:C) when stimulated with any of the three peptides (<italic>n</italic> = 6 mice, data presented as mean ± sd). <bold>G</bold> At the same time point expression of transcription factors Eomes and IRF4 was increased in poly(I:C) pretreated cells (<italic>n</italic> = 5 mice, data presented as mean ± sd, *<italic>p</italic> = 0.0491, **<italic>p</italic> = 0.0031, ****<italic>p</italic> &lt; 0.0001). <bold>A</bold>–<bold>G</bold> Data are representative of two independent experiments (*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001, ****<italic>p</italic> &lt; 0.0001). <bold>B</bold>\n<italic>p</italic> values calculated by unpaired Students <italic>t-</italic>test between mice groups receiving same APL, <bold>C</bold> Mann-Whitney U test between mice groups receiving same APL <bold>E</bold>, <bold>F</bold>, <bold>G</bold> Students <italic>t-</italic>test.</p></caption></fig>" ]
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[ "<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"MOESM4\"></supplementary-material>" ]
[ "<fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Stephen Jameson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p><bold>Change history</bold></p><p>1/11/2024</p><p>A Correction to this paper has been published: 10.1038/s41467-024-44731-3</p></fn></fn-group>" ]
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[ "<media xlink:href=\"41467_2021_25645_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>", "<media xlink:href=\"41467_2021_25645_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>", "<media xlink:href=\"41467_2021_25645_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media>", "<media xlink:href=\"41467_2021_25645_MOESM4_ESM.xlsx\"><caption><p>Source Data</p></caption></media>" ]
[{"label": ["9."], "surname": ["Van Gassen"], "given-names": ["S"], "article-title": ["FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data"], "source": ["Cytom. Part A"], "year": ["2015"], "volume": ["87"], "fpage": ["636"], "lpage": ["645"], "pub-id": ["10.1002/cyto.a.22625"]}, {"label": ["13."], "mixed-citation": ["Smith-Garvin, J. T cell activation. "], "italic": ["Annu. Rev."]}, {"label": ["16."], "surname": ["Davies", "Thompson", "Pulko", "Torres", "Nikolich-\u017dugich"], "given-names": ["JS", "HL", "V", "JP", "J"], "article-title": ["Role of cell-intrinsic and environmental age-related changes in altered maintenance of murine T cells in lymphoid organs"], "source": ["J. Gerontol."], "year": ["2018"], "volume": ["73"], "fpage": ["1018"], "lpage": ["1026"], "pub-id": ["10.1093/gerona/glx102"]}, {"label": ["24."], "mixed-citation": ["Delong, J. H. et al. Cytokine- and TCR-mediated regulation of T cell expression of Ly6C and Sca-1. 10.4049/jimmunol.1701154 (2019)."]}, {"label": ["25."], "surname": ["Connell"], "given-names": ["RMO"], "article-title": ["Type I interferon production enhances susceptibility to listeria monocytogenes infection"], "source": ["J. Exp. Med."], "year": ["2004"], "volume": ["200"], "fpage": ["4"], "lpage": ["12"], "pub-id": ["10.1084/jem.20040712"]}, {"label": ["27."], "mixed-citation": ["Moran, A. E. et al. T cell receptor signal strength in T reg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. "], "italic": ["J. Exp. Med."], "bold": ["208"]}, {"label": ["35."], "mixed-citation": ["Curtsinger, J. M., Valenzuela, J. O., Agarwal, P., Lins, D. & Mescher, M. F. Cutting Edge: Type I IFNs provide a third signal to CD8. 8\u201313, 10.4049/jimmunol.174.8.4465 (2020)."]}, {"label": ["43."], "surname": ["Hogquist"], "given-names": ["KA"], "article-title": ["T cell receptor antagonist peptides induce positive selection"], "source": ["Cell"], "year": ["2017"], "volume": ["76"], "fpage": ["17"], "lpage": ["27"], "pub-id": ["10.1016/0092-8674(94)90169-4"]}]
{ "acronym": [], "definition": [] }
43
CC BY
no
2024-01-13 23:35:10
Nat Commun. 2021 Sep 6; 12:5303
oa_package/7d/d8/PMC8421345.tar.gz
PMC9827708
36624511
[ "<title>Introduction</title>", "<p id=\"Par49\">In contrast to the central nervous system, peripheral nerves can regenerate following crush injury or even complete transection. Schwann cells (SCs) serve as a regenerative cell type in adult nerves and their plasticity plays an essential role in reverting to a repair competent state following nerve injury [##REF##32697392##1##]. Briefly, Wallerian degeneration will happen at the distal nerve stump after nerve injury and SCs create a permissive environment by accelerating clearance of axonal and myelin debris. After differentiation, SCs proliferate and extend longitudinally to form Büngner bands which can guide axon regrowth and rebuild the connection from injury site to reinnervate target organs. Moreover, SCs secrete several signaling molecules, such as pro-inflammatory factors, cytokines, growth factors, neurotrophic factors and extracellular matrix molecules, to recruit macrophages, support neuronal survival and accelerate axonal regeneration. Finally, remyelinate regenerated axons and repel infiltrated macrophages to complete the process of peripheral nerve repair [##REF##33766800##2##, ##REF##35431954##3##].</p>", "<p id=\"Par50\">However, the regenerative ability of peripheral nerve often fails to transform to satisfactory functional outcome resulting in loss of sensory, motor and autonomic functions. Besides the slow rate of regeneration and chronical denervation, lack of specificity including disordered axonal regeneration and incorrect reconnection with target organs, is also one of important reasons for insufficient and unsatisfactory functional recovery [##REF##22609046##4##]. Vascularization is crucial for nerve regeneration and angiogenesis precedes neurogenesis while sharing the same structural principles and molecular mechanisms as those responsible for nervous system wiring [##REF##17950371##5##, ##REF##32209756##6##]. In the early stage of peripheral nerve regeneration, endothelial cells (ECs) form micro-vessels rapidly to provide oxygen and nutrient supply which is regulated by the vascular endothelial growth factor (VEGF) [##REF##26864683##7##]. Zhou et al. proved that microvascular ECs could engulf myelin debris and promote macrophage recruitment and fibrosis after neural injury [##REF##30664769##8##]. Cattin et al. first reported that blood vessels direct the migrating cords of SCs and disrupting the organization of the newly formed blood vessels in vivo could compromise SCs directionality resulting in defective nerve repair [##REF##26279190##9##]. In addition, Muangsanit et al. suggested that engineered neural tissue which contain aligned networks of ECs tube-like structures could provide both enhanced vascularization and direct support for regenerating axons [##REF##33766800##2##]. Our previous study showed that Radix Astragalus polysaccharide enhanced functional recovery by accelerating angiogenesis after peripheral nerve injury (PNI) [##REF##35431954##3##]. Therefore, adequate vascularization is not only important for providing essential nutrients for nerve regeneration and clearing degenerative and necrotic substances from injury site, but also essential for guiding SCs migration and specific reconnection from axons to target organs. The coordination between ECs and SCs within nerve injury sites is crucial in this process, however, it still remains unclear. In this case, it is significantly necessary to investigate the interaction of ECs and SCs during regeneration, particularly the modulation of repair-related SC phenotype, which is remarkable in developing new therapeutic strategies for promoting peripheral nerve repair.</p>", "<p id=\"Par51\">Exosomes are nano-sized liposomes that originate from invagination of endosomal membranes and are important components of the paracrine secretion of cells. They are formed from multivesicular bodies with a diameter of 50–150 nm [##UREF##0##10##, ##REF##24912806##11##]. Exosomes have been proven to participate in the transport of biochemicals such as proteins, cytokines, mRNAs, and miRNAs and, as a result, mediate a key mechanism of cell-to-cell communication [##REF##23522040##12##, ##REF##26874206##13##]. Exosomes derived from ECs have been demonstrated to protect neural cells against injury by promoting cell growth and migration, and inhibiting cell apoptosis [##REF##28849073##14##, ##REF##30662605##15##]. These findings indicated that EC-derived exosomes (EC-EXO) might be developed as a promising therapeutic target for neural damage. However, in peripheral nerve injury, the effect and regulatory mechanism of EC-EXO on repair-related cell phenotypes in SCs remains unclear.</p>", "<p id=\"Par52\">The aim of this study was to elucidate the effect and mechanisms of EC-EXO on regulating SCs phenotypes and to determine its role in functional recovery after peripheral nerve injury. We found that EC-EXO enhanced SCs proliferation, migration and the secretion of immune factors and neurotrophic factors. And we determined that miR199-5p was a critical role in the effect of EC-EXO on SCs. We also demonstrated that EC-EXO activated PI3K/AKT/PTEN signaling pathway to boost the repair phenotypes of SCs and the PI3K inhibitor was used to further verify it. In vivo experiments confirmed that EC-EXO possessed a favorable neuronal affinity and could be present in the nerves for a prolonged time. Subsequently, we identified the promoting effect of EC-EXO on functional recovery in rats with sciatic nerve injury by testing sciatic nerve function index (SFI) analysis, evoked compound muscle action potential (CMAP) and weight ratio of gastrocnemius muscle. Furthermore, immunofluorescent staining and transmission electron microscopy (TEM) were used to determine the effects of EC-EXO on nerve repair including axon regeneration, myelination and angiogenesis following sciatic nerve injury. Our study elucidated the active role of EC-EXO in regulating the repair phenotypes of SCs and suggested that EC-EXO could serve as a promising therapeutic target for peripheral nerve injury.</p>" ]
[ "<title>Materials and methods</title>", "<title>Cell culture</title>", "<p id=\"Par78\">Human umbilical vein ECs (HUVECs) were purchased from Procell Life Science&amp;Technology Co., Ltd (Wuhan, China). HUVECs were cultured in EC medium (ScienCell, USA) supplemented with 10% exosome-depleted foetal bovine serum (SBI, USA), 1% EC growth supplement (ScienCell) and 1% penicillin/streptomycin solution (ScienCell), and cells were cultured at 37 ℃ in humidified air containing 5% CO<sub>2</sub>. HUVECs were performed for the experiments in passages 4–6.</p>", "<p id=\"Par79\">Rat SCs (RSC96 cells) were purchased from Procell Life Science&amp;Technology Co., Ltd. SCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, USA). Moreover, the culture mediums were supplemented with 10% exosome-depleted FBS (SBI). RSC96 cells were performed for the experiments in passages 4–6.</p>", "<title>Isolation and characterization of EC-derived exosomes</title>", "<p id=\"Par80\">ECs (Passage 4–6) were cultured in 10% exosome-depleted FBS DMEM for 48 h to produce enough exosomes. Then the media was collected and centrifuged at 800 ×<italic>g</italic> for 10 min and 5000 ×<italic>g</italic> for 10 min to remove cells and cell debris. The supernatant was filtered by a 0.22-μM filter (Millipore). The supernatant was centrifuged at 100,000 <italic>g</italic> for 2 h at 4 ℃ to pellet exosomes (Micro Ultracentrifuge, Himac, Japan). Next, the exosomes were washed with PBS and centrifuged at 100,000 ×<italic>g</italic> for 2 h. Exosomes were eventually resuspended by 100 μL PBS. Exosomes were measured with the Bicinchoninic Acid (BCA) Protein Assay Kit (Solarbio, China), about 32.1 μg exosomes could be extracted from 1 mL EC culture medium. Then exosomes used for transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were stored at 4 ℃ and used within 48 h. Other exosomes were stored at – 80 ℃ until use.</p>", "<p id=\"Par81\">The exosomal markers TSG101, CD9, CD81 and GAPDH were analysed by western blot for ECs and exosomes derived from ECs. In addition, NTA measured exosome size distribution and zeta potential with the NICOMP Nano-ZLS Z3000 instrument (Beckman Coulter, USA). Exosomes were fixed with 2% glutaraldehyde stationary liquid. Exosome suspension was dropped onto the copper grid with carbon film for 5 min, and 2% phosphotungstic acid was dropped on the copper grid to stain for 5 min at room temperature. The exosomes are observed under TEM (HITACHI, Japan).</p>", "<title>Exosomes labeling and in vitro uptake</title>", "<p id=\"Par82\">Exosomes were labelled with 10 mg/mL 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Beyotime) at a volume ratio of 1:100 for 30 min in the dark at 37 ℃ according to the manufacturer’s procedures. Then the labelled exosomes suspension was dialyzed for 12 h in a 100 KD-aperture dialysis bag (Shanghai yuanye Bio-Technology) to remove the residual fluorescent dye. SCs were seeded in the 20 mm glass-bottom cell culture dish (Nest, China) and incubated with DiI-labelled exosomes for 0 h, 2 h, 6 h, 12 h and 24 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Servicrbio, China), and cells were observed under confocal laser scanning microscopy (LSM880, Zeiss, Germany).</p>", "<title>Cell proliferation and colony-forming assay</title>", "<p id=\"Par83\">SCs were seeded on 96-well plates at a density of 2 × 10<sup>4</sup> cells/mL overnight. EC-EXO of different concentrations (1, 10, 50 and 100 μg/mL) were added to each well and cultured for 24 h and 48 h. Then 10 μL of Cell Counting Kit-8 (Dojindo, Japan) was added into each well and incubated for 2 h at 37 ℃ in humidified air containing 5% CO<sub>2</sub>. The cell proliferation was determined using a full-wavelength microplate reader at 450 nm. We also used the CCK8 assay to study the growth curve of SCs in different groups. Furthermore, the 5-ethynyl-2ʹ-deoxyuridine (EdU) Cell Proliferation Assay Kit (Ribobio) was also used to measure the cell proliferation of SCs in different groups. EdU-labelled cells were counted manually in three fields of view randomly chosen from each well to calculate the percentages. To study the clonogenic ability of SCs in different groups, cells with different treatments were seeded to 6-well plates (1000 cells/well). SCs were cultured for 10 days and stained with 0.1% Crystal Violet Stain solution (Solarbio). The numbers of colonies were counted manually, and the inverted microscope detected the morphology of the colonies from different groups.</p>", "<title>Cell cycle and apoptosis analyses</title>", "<p id=\"Par84\">SCs were seeded into 6-well plates (1 × 10<sup>5</sup> cells/mL) and 50 μg/mL EC-EXO or PBS were added in the corresponding well for 24 h. The cells were collected and resuspended by PBS. Then the Cell Cycle Staining Kit (MultiSciences Biotech, China) was used for cell cycle analyses and the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme Biotech, China) was used for cell apoptosis analyses according to the manufacturer’s protocol. The cell cycle and apoptosis were detected by flow cytometry analysis. Moreover, data acquisition and analysis were performed using NovoExpress software.</p>", "<title>Migration assay</title>", "<p id=\"Par85\">The migration of SCs was evaluated using a transwell with 8 μm pores (Corning, USA). To study the effect of ECs on SC migration and the role of exosomes play in this process, we carried out a coculture of ECs and SCs in the transwell system. First, SCs were seeded at 1 × 10<sup>4</sup> cells in 100 μL in DMEM supplemented with 1% FBS onto the upper chamber [##REF##34596354##66##]. Then, ECs, ECs with GW4869 (Umibio), the inhibitor of exosome secretion, PBS and PBS with GW4869 were added to the lower chambers. Besides, we also filled the lower chamber of the transwell with a medium including EC-EXO of different concentrations (1, 10, 50, 100 μg/mL) to further estimate the influence of EC-EXO on SC migration more directly. First, cells were incubated for 24 h at 37 ℃ and non-migrated cells were removed using cotton swabs. Next, migrated cells were fixed with 4% paraformaldehyde (Solarbio) for 30 min. Next, PBS washed the fixed cells. Then cells were stained with 0.1% Crystal Violet Stain solution for 30 min. Following 12 h of drying, stained cells were observed with an inverted microscope, and the number of migrated cells was counted using ImageJ software.</p>", "<title>Sequencing of miRNAs and data analysis</title>", "<p id=\"Par86\">Total RNA was extracted from SCs treated with EC-EXO or PBS for 24 h by RNAiso Plus (Takara, Japan) and analyzed for RNA integrity and total amount with 2100 bioanalyzer (Agilent, CA, USA). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI Group, China). Following acquiring the raw data, the differentially expressed miRNAs were calculated using the <italic>t</italic> test. The data with ≥ twofold upregulation and a P value &lt; 0.05 were regarded as significantly different.</p>", "<title>MiRNA real-time quantitative PCR</title>", "<p id=\"Par87\">Total RNAs in SCs treated with EC-EXO for 24 h were isolated using RNAiso Plus (Takara, Japan) and recerse transcribed using a miRNA first-strand cDNA synthesis kit (Takara, Japan) according to the manufacturer's guidelines. RT–qPCR was performed in a 20 μL reaction system involving forward/reverse primers, cDNA, and NovoStart SYBR qPCR SuperMix Plus (Novoprotein Scientific, China) according to manufacturer’s instructions. We set three replicates in each group and used the 2<sup>−ΔΔCT</sup> method. The primers were purchased from Ribobio Biotech.</p>", "<title>Transfection of miRNA mimic, mimic negative control, miRNA inhibitor and inhibitor negative control</title>", "<p id=\"Par88\">For transfection of the mimic negative control (Mi-NC), miR199-5p mimic, inhibitor negative control (In-NC) and miR199-5p inhibitor (Ribobio, China) at a final concentration of 50 nM using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions. The sequence of miR199-5p is: ACUGGACUUGGAGUCAGAAG.</p>", "<title>RNA sequencing</title>", "<p id=\"Par89\">RNA was extracted from SCs treated with or without EC-EXO for 24 h by RNAiso Plus (Takara, Japan) and analyzed for RNA integrity and total amount with 2100 bioanalyzer (Agilent, CA, USA). RNA sequencing library was prepared and sequenced on Illumina HiSeq 6000 (Illumina, CA, USA). The sequencing service was provided by Novogene (Beijing, China). The DESeq2 R package (1.20.0) was used to analyze two groups’ differential expression genes (DEGs). Moreover, the DEGs were screened out on the ground of the threshold of P-value ≤ 0.05 and |log2FoldChange|≥ 1. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene set enrichment analysis (GSEA) of DEGs was implemented by the clusterProfiler R package (3.8.1).</p>", "<title>Real-time quantitative polymerase chain reaction (RT–qPCR)</title>", "<p id=\"Par90\">SCs in each group were used for RNA extration with RNAiso Plus. Total RNA was transcribed into complementary DNAs (cDNAs) by the NovoScriptPlus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge; Novoprotein Scientific, China). RT–qPCR was performed in a 20 μL reaction system involving forward/reverse primers, cDNA, and NovoStart SYBR qPCR SuperMix Plus according to manufacturer's instructions. We set three replicates in each group and used the 2<sup>−ΔΔCT</sup> method. All primers used in this study were listed in (Additional file ##SUPPL##0##1##: Table S1).</p>", "<title>Western blot analysis</title>", "<p id=\"Par91\">Exosomes lysate and cell lysate were prepared by RIPA Lysis Buffer (Yazyme, China) added with Protease Inhibitor Cocktail (Yazyme) and Phosphatase Inhibitor Cocktail (Yazyme). Total proteins were separated in SDS-polyacryla-mide gel (Yazyme) and transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime). The membranes were blocked using Protein Free Rapid Blocking Buffer (Yazyme) for 30 min at room temperature. The blocked membranes were incubated overnight at 4 ℃ with antibodies specific for the TSG101 (Abcam, 1:1000), CD9 (Abcam, 1:1000), CD81 (Abcam, 1:1000), GAPDH (Abcam, 1:1000), BAX (Abcam, 1:1000), Bcl2 (Abcam, 1:1000), PCNA (CST, 1:1000), c-Jun (Abcam, 1:1000), STAT3 (CST, 1:1000), p-STAT3 (CST, 1:1000), PI3K (Abcam, 1:1000), p-PI3K(Affinity, China, 1:1000), PTEN (Abcam, 1:1000), AKT (Abcam, 1:10,000), p-AKT (Abcam, 1:5000), NGF (Abcam, 1:1000), VEGFA (Abcam, 1:1000), CNTF (Abcam, 1:1000), BDNF (Abcam, 1:5000) and GDNF (Abcam, 1:1000) and β-actin (Solarbio, 1:1000). Then the membranes were washed and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (Solarbio). The blots were detected using Amersham Imager 600. GAPDH or β-actin was used as the loading control, and the interested protein's relative intensity was normalized to that of the control group. LY294002, a PI3K inhibitor (PI3Ki, GLPBIO) was also used as a well-known PI3K signaling pathway inhibitor.</p>", "<title>Animal model and EC-EXO delivery</title>", "<p id=\"Par92\">Adult male Sprague–Dawley rats (300–400 g) were obtained from Beijing Vital River Laboratory Animal Technology Co.,Ltd (China, Beijing). The living conditions and experimental procedures conformed to the National Institutes of Health (NIH) Guide Concerning the Cre and Use of Laboratory Animals. In addition, the whole animal experiment was approved by the Animal Experimentation Ethics Committee of Zhengzhou University. Five rats per cage were kept in the specific-pathogen-free (SPF) room with constant temperature (23–24 ℃), humidity (55 ± 5%), and light (12 h light–dark cycle). All rats had free access to food and water.</p>", "<p id=\"Par93\">The rats were randomly divided into three experimental groups (n = 5 for each group): a sham group, a PNI group, and an exosome treatment (EXO) group. Following an effective inhalation of ether, the rat was intraperitoneally injected with 2.0 mL per kg body weight of 2% pentobarbital sodium. Then the right nerve of the rat was exposed using the gluteal muscle dissection method. First, the PNI model was established at a location 5 mm away from the sciatic notch using Dumont No. Five forceps three times (10 s each time, 10 s intervals). Subsequently, the PNI group received multi-site injections of 20 μL of PBS without EXO under the epineurium of the sciatic nerve by using a micro syringe (Hamilton, USA). After each injection, the needle was indwelled for 30 s to prevent leaking out. Then a 2-mm-long translucent band was formed at the injury site, which was marked with a 10–0 nylon epineural suture for later identification. Next, the EXO group received multi-site injections of 20 μL of 50 μg/mL EC-EXO under the epineurium of the sciatic nerve by using a micro syringe [##REF##35351151##16##]. Finally, the rats in the sham group underwent the same procedure without suffering any sciatic nerve damage.</p>", "<title>Bioluminescence imaging</title>", "<p id=\"Par94\">Exosomes were stained with the liposomal dye DiR (US Everbright, China) according to the manufacturer's instructions to visualize their distribution in vivo. Then the labelled exosomes suspension was dialyzed in a 100 KD-aperture dialysis bag for 12 h to remove the residual fluorescent dye. As for the control group, the DiR dye was diluted in PBS, then the solution was also dialyzed in a 100 KD-aperture dialysis bag for 12 h to verify the interference of vestigital DiR to this experiment. Injection of DiR-labelled exosomes and control solution under the epineurium of the sciatic nerve was performed in the rats by using a micro syringe (dosage per rat: 1 μg of DiR-labelled exosomes, in 20 μL of PBS), and 20 μL of DiR solution was used as the control (n = 3 for each group). An IVIS imaging system (PerkinElmer, USA) was used to perform living and sciatic nerve tissue imaging 1 day, 3 day, 7 day, 14 day and 28 day after the injection.</p>", "<title>Exosomes labeling and in vivo uptake</title>", "<p id=\"Par95\">To further visualize the distribution of exosomes in sciatic nerve, exosomes were prestained with the DiI according to the manufacturer's instructions. Then the labelled exosomes suspension was dialyzed in a 100 KD-aperture dialysis bag to remove the residual fluorescent dye. For the control group, the DiI dye was diluted in PBS, then the solution was also dialyzed in a 100 KD-aperture dialysis bag for 12 h as above. As stated above,we used a micro syringe to inject DiI-labelled exosomes locally under the epineurium of the sciatic nerve (dosage per rat: 1 μg of DiI-labelled exosomes, in 20 μL of PBS), and 20 μL of DiI solution was used as the control. After 1 day, 3 day, 7 day, 14 day and 28 day, the rats were sacrificed and the sciatic nerves were embedded in Tissue-Tek O.C.T. (Leica, Wetzlar, Germany) to make frozen blocks for fluorescent staining. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy.</p>", "<title>TUNEL staining</title>", "<p id=\"Par96\">The apoptosis rate in each group of rat sciatic nerve was detected using a TUNEL staining kit (Vazyme) according to the manufacturer’s instructions. Nuclei were stained with DAPI. TUNEL-labelled cells were stained with green fluorescence and counted manually in three fields of view randomly chosen from each well to calculate the percentages.</p>", "<title>Walking track analysis</title>", "<p id=\"Par97\">To evaluate the motion function following nerve injury, walking track analysis was used on the injury model rats postoperatively at 3 days before operation, 7, 14, and 28 days following operation. In this trial, the plantar surfaces of both hind paws were painted with Eosin Y solution (Solarbio), and the rat was allowed to walk along a narrow corridor with white paper on the base towards a dark compartment at the end. Paw length (PL), the toe-spread distance between toes 1 and 5 (TS), and toe-spread distance (IT) between toes 2 and 4 were recorded from the normal (N) and experimental (E) hind limbs. Sciatic Functional Index (SFI) was calculated as the following formula [##REF##2909054##67##].The rat footprint measurements could evaluate the functional muscle status of the hind limbs according to the walking track analysis [##REF##15072638##68##]. In general, a SFI value of 0 indicates normal neurological function, while a SFI value of <bold>-</bold>100 indicates complete loss of motor function.</p>", "<title>Electrophysiological assessment</title>", "<p id=\"Par98\">The electrophysiological assessment was conducted using previously developed methods [##REF##33300246##69##]. MD3000-C multichannel physiological signal acquisition and processing system (Anhui Zhenghua Biological Instrument, China) was used to evaluate functional recovery 28 days after the operation [##REF##33025785##70##]. First, the rats were anesthetized, and the sciatic nerve tissues were exposed. Bipolar electrodes were placed at the proximal end of the crushed site to send single electrical stimulation. In the meantime, a recording electrode was inserted into the homolateral gastrocnemius muscle. The recorded nerve’s compound muscle action potentials (CMAPs) were obtained to perform a comparative analysis of the three groups.</p>", "<title>Histological and morphological analysis of regenerated nerve</title>", "<p id=\"Par99\">At 28 days after the operation, the sciatic nerves were removed and fixed overnight in 4% paraformaldehyde (PFA), then dehydrated in gradient grade ethanol, and embedded in paraffin. Longitudinal and transverse sections (5 μm) were de-waxed and hydrated after paraffin embedding. The nerve sections were stained with Hematoxylin–eosin (HE) and Masson staining according to manufacturer's instructions. At last, slides were fixed with neutral resin and capped. Images of stained sections were acquired with a light microscope (Olympus, Japan).</p>", "<title>Electron microscopy and TB staining assessments</title>", "<p id=\"Par100\">The sciatic nerves 3–5 mm distal to the injury site were harvested and put in 2.5% glutaraldehyde overnight. The tissues were immersed in 1% osmic acid for 2 h and dehydrated with acetone. Then the samples were encapsulated in epoxy resin and oven-dried. The tissues were sectioned into 0.5 μm semi-thin cross-sections and 70.0 nm ultrathin cross-sections. The semi-thin sections were stained with toluidine blue (TB) and observed using a light microscope. The ultrathin cross-sections were examined using a projection electron microscope (Hitachi). ImageJ software measured myelinated axon number, G-ratio (inner axonal diameter to fiber diameter ratio), and myelin sheath thickness. We randomly selected 3 representative TEM pictures and counted the mean thickness and G-ratio of all the myelin sheath in each picture.</p>", "<title>Histological assessment of muscle</title>", "<p id=\"Par101\">At 28 days after the operation, bilateral gastrocnemius muscles were harvested from rats and weighed promptly to acquire the muscle relative wet weight ratio by calculating the ratio of ipsilateral muscle weight. Then the experimental gastrocnemius muscle belly was fixed, embedded in paraffin, and stained with HE and Masson staining. Finally, the representative images of stained sections were observed with a light microscope. We randomly selected 3 representative HE staining pictures and counted the mean muscle fiber diameter in each picture.</p>", "<title>Immunofluorescence staining and immunofluorescence evaluation</title>", "<p id=\"Par102\">At 28 days following the operation, the sciatic nerve tissues containing the area of crush injury site (5 mm away from the sciatic notch) were harvested and fixed with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue were prepared. Moreover, the sections were stained with NF200 (CST, 1:200), S100β (Abcam, 1:200), TuJ1 (Abcam, 1:200), MBP (Abcam, 1:200), CD31 (Abcam, 1:200), CD34 (Abcam, 1:200), VEGFR (Abcam, 1:200), GAPDH (Abcam, 1:200), Akt (Abcam, 1:500), p-AKT (Abcam, 1:500), PI3K (Abcam, 1:500), p-PI3K (ThermoFisher, 1:500) and PTEN (Abcam, 1:500). Secondary antibodies were as follows:Alexa Fluor568–conjugated Goat Anti-Rabbit IgG (Abcam), CoraLite594-conjugated Goat Anti-Mouse IgG (Proteintech, China), CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech), CY3-labeled goat anti-rabbit (Servicebio) and AlexaFluor594-labeled goat anti-rabbit IgG (Abcam). Besides, we also used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence intensity. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy. The percentages of the markers positive areas were calculated by dividing integrated option density by selected region area, then multiplied by 100%. All parameters were measured using ImageJ.</p>", "<title>Statistical analysis</title>", "<p id=\"Par103\">Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). The results were presented as mean ± SD. One-way ANOVA was used for comparisons within multiple groups, and a two-tailed unpaired Student's test was used for comparisons between two groups. P values &lt; 0.05 were considered statistically significant.</p>" ]
[ "<title>Results</title>", "<title>Characterization of EC-EXO</title>", "<p id=\"Par53\">EC-EXO were characterized according to morphology, specific markers, size distribution and surface charge. First, we isolated exosomes derived from ECs and used TEM to characterize the morphology of a single exosome. The EC-EXO showed a round shape with a bilayer structure (Fig. ##FIG##0##1##A). Then we used NTA to measure the size distribution of exosomes, and the mean diameter was 128.8 nm (Fig. ##FIG##0##1##B). Moreover, the mean zeta potential of EC-EXO was − 5.50 mV (Fig. ##FIG##0##1##C). Then we used western blot to examine for the existence of several “exosome markers”: three transmembrane/lipid-bound proteins (CD9 and CD81) and a cytosolic protein (TSG101). Moreover, GAPDH was used as the loading control (Fig. ##FIG##0##1##D).</p>", "<title>EC-EXO boosted repair-related phenotypes of SCs in vitro</title>", "<p id=\"Par54\">To verify the effect of EC-EXO on repair-related phenotypes of SCs, we labeled the EC-EXO with DiI and added them to SCs to observe the interactions between EC-EXO and SCs. Then, we proved that EC-EXO could boost a battery of changes in phenotypes in SCs through in vitro experiments. These phenotypic changes in SCs are considered to be critical for nerve repair in the process of nerve regeneration, which include: (1) enhanced proliferation and the anti-apoptotic ability for the survival of injured neurons, (2) enhanced migration capability for the formation of bands of Büngner, (3) upregulation of growth factors and neurotrophic factors including NGF, VEGFA, CNTF, BDNF and GDNF for angiogenesis and nerve regeneration in the nerve injury site, and (4) upregulated expression of immune-related cytokines for the clearance of axonal and myelin debris [##REF##35351151##16##–##REF##21878125##20##].</p>", "<title>EC-EXO regulated cell proliferation, cycle, and apoptosis in SCs</title>", "<p id=\"Par55\">First, we tested the ability of SCs to internalize EC-EXO in vitro. EC-EXO labelled with DiI were incubated with SCs for 0 h, 2 h, 6 h, 12 h and 24 h, then spots with red fluorescence presented in the cytoplasm of SCs which suggested that SCs have an efficient ability to internalize EC-EXO (Fig. ##FIG##1##2##A). Next, to investigate EC-EXO' effect on the proliferation of SCs, we treated SCs with EC-EXO of different concentrations (1, 10, 50 and 100 μg/mL) for 24 and 48 h. The results of the CCK8 assay showed that the absorbance of SCs treated with EC-EXO was higher than those treated with PBS (Additional file ##SUPPL##0##1##: Fig. S1A). The EdU staining results were performed to evaluate further EC-EXO' intensive function to SCs proliferation (Fig. ##FIG##1##2##B, C). Next, to investigate EC-EXO' role in the colony formation of SCs, the EC-EXO with different concentrations were incubated with SCs. After 10 days, the SCs treated with EC-EXO aggregated into more colonies than the control group treated with PBS. (Fig. ##FIG##1##2##D, E). Based on the effects of different concentrations of EC-EXO on SCs, we found that EC-EXO can enhance SC proliferation, which is concentration-dependent. According to these results, we finally chose 50 μg/mL EC-EXO as the experimental group in the following experiments. To further study the proliferation promotion effect of EC-EXO on SCs at different times, we used the CCK8 assay to analyze the cell growth curve of SCs treated with EC-EXO. The results showed that the pro-proliferative effect of EC-EXO on SCs began to differ significantly on the second day (Additional file ##SUPPL##0##1##: Fig. S1B). Moreover, compared with the control group, EC-EXO increased cell counts in the S phase (from 34.60% to 46.54%) and decreased cell counts in the G0/G1 phase (from 44.59% to 36.05%) in SCs (Fig. ##FIG##1##2##F, G). Furthermore, flow cytometry analysis revealed that EC-EXO significantly reduced the apoptosis rate of SCs (from 4.01% to 2.07%) (Additional file ##SUPPL##0##1##: Fig. S1C, D). We also used western blot to detect the protein expression levels of apoptosis-related biomarkers, including BAX and Bcl2. As shown in (Additional file ##SUPPL##0##1##: Fig. S1E, F), the expression of pro-apoptotic protein BAX in the EXO-treated group was significantly lower than those in the control group, and the anti-apoptotic protein Bcl2 in the EXO-treated group was significantly higher than those of control group. These results indicated that EC-EXO significantly improved the anti-apoptotic ability of SCs.</p>", "<title>EC-EXO upregulated the expression of growth factors and neurotrophic factors in SCs</title>", "<p id=\"Par56\">The expression levels of growth factors and neurotrophic factors associated with angiogenesis and axon regeneration including NGF, VEGFA, CNTF, BDNF and GDNF were increased in the EXO-treated SCs compared with the control SCs (Fig. ##FIG##1##2##H). The western blot results indicated that EC-EXO could upregulate the expression of these repair-supporting molecules in SCs, which might be beneficial in creating a microenvironment conductive to nerve regeneration.</p>", "<title>EC-EXO upregulated the expression of immune-related cytokines in SCs</title>", "<p id=\"Par57\">The upregulation of immune-related cytokines including LIF, Gal-3 and MCP-1 could be beneficial in activating the innate immune response, thereby recruiting macrophages for the clearance of redundant myelin and the formation of blood vessels [##REF##26279190##9##, ##REF##18803324##19##, ##REF##17311007##21##, ##REF##24107955##22##]. Therefore, we used RT–qPCR to determine the expression levels of the cytokines (LIF, Gal-3 and MCP-1) in SCs. The results showed that the expression levels of these cytokines in the EXO group were significantly higher than those in the control group (Additional file ##SUPPL##0##1##: Fig. S1G).</p>", "<title>EC-EXO enhanced the migration capacity of SCs</title>", "<p id=\"Par58\">The recruitment and migration of SCs to nerve injury sites is essential for forming bands of Büngner and axon regeneration. To investigate the influence of ECs on SC migration and exosomes’ role in this process, we carried out a coculture of ECs and SCs in the transwell system (Fig. ##FIG##1##2##J). The results showed that the coculture of SCs with ECs significantly increased the migratory ability of SCs. To evaluate the effect of exosomes derived from ECs in this process, we added GW4869 (an inhibitor to exosome secretion) in the coculture system. The results showed that the involvement of GW4869 significantly faded the increased cell migration in the coculture (Fig. ##FIG##1##2##K, L). These results indicated that ECs facilitate the migration of SCs partly through exosomes. To further investigate EC-EXO' effect on SC migration, we filled the lower chamber of the transwell with a medium including EC-EXO of different concentrations (1, 10, 50 and 100 μg/mL) (Fig. ##FIG##1##2##M). The results showed that EC-EXO concentration-dependently facilitated SCs migration to the lower chamber (Fig. ##FIG##1##2##N, O).</p>", "<title>EC-EXO could promote the proliferation and migration of SCs better than SC-EXO</title>", "<p id=\"Par59\">It has been demonstrated that SC-exosomes (SC-EXO) represent an important mechanism to support axonal maintenance and regeneration following nerve damage via communication with neighboring axons during regenerative processes [##REF##24038411##23##]. SC-EXO were characterized according to morphology, specific markers, size distribution and surface charge using TEM, NTA and western blot (Additional file ##SUPPL##0##1##: Fig. S2A–C). The EdU assay demonstrated that SC-EXO could also enhance the proliferation of SCs, while this effect was slightly weaker than that of EC-EXO (Additional file ##SUPPL##0##1##: Fig. S2D, E). And the transwell assay showed EC-EXO were more capable of promoting migration in SCs than SC-EXO (Additional file ##SUPPL##0##1##: Fig. S2F, G). Next, the colony formation assay revealed that the SCs treated with EC-EXO could aggregate more colonies than the group with SC-EXO (Additional file ##SUPPL##0##1##: Fig. S2H, I).</p>", "<title>MiR199-5p was upregulated in SCs treated with EC-EXO and promoted SC repair-related phenotypes</title>", "<p id=\"Par60\">To identify EC-EXO-related miRNAs, we analyzed the expression profile consisting of SCs treated with EC-EXO and the SCs treated with PBS as control group by miRNA sequencing. The result of the differential expression of SCs in each group was intersected by Venn analysis (Fig. ##FIG##2##3##A). GO enrichment analysis showed the GO terms associated with nerve regeneration, which included axon, neuron projection, signaling receptor binding, axon guidance, angiogenesis and PI3K signaling (Fig. ##FIG##2##3##B). The KEGG enrichment analysis indicated the significant signaling pathways related with the regulation of repair-supportive cell phenotypes, including PI3K-AKT signaling pathway and JAK-STAT signaling pathway (Fig. ##FIG##2##3##C). Besides, it was found that miR199-5p was significantly upregulated in EC-EXO group (Fig. ##FIG##2##3##D, E). Then we used RT–qPCR to validate that the miR199-5p level was increased in SCs treated with EC-EXO compared to that in control group (Fig. ##FIG##2##3##F).</p>", "<p id=\"Par61\">We investigated the effect of miR199-5p on SCs by transfecting miR199-5p mimics and miR199-5p inhibitor. The transfection efficiencies were determined by RT–qPCR (Fig. ##FIG##2##3##G). The cell growth curve of SCs and results of EdU assay in different groups indicated that overexpression of miR199-5p significantly improved the proliferation ability of SCs, and the inhibition of miR199-5p inhibited the proliferation ability of SCs (Fig. ##FIG##2##3##H–J). Additionally, the ability of colony formation and migration was also increased in miR199-5p-overexpressed SCs, and this ability was inhibited in miR199-5p-inhibited SCs (Fig. ##FIG##2##3##K–N).</p>", "<title>EC-EXO regulated the molecular mechanism and signaling pathways associated with SC repair-related phenotypes in vitro</title>", "<title>EC-EXO regulated molecular mechanism associated with repair-related phenotypes of SC</title>", "<p id=\"Par62\">We carried out RNA-seq analysis on SCs treated with EC-EXO for 24 h and the cells treated with PBS to expose the molecular mechanism. The differentially expressed genes from the two groups were illustrated using heat maps (Fig. ##FIG##3##4##A). Comparing the control group and the EC-EXO-treated group, GO enrichment analysis showed the GO terms associated with nerve regeneration, which included neuron apoptotic process, neuron projection cytoplasm, and S100 protein binding (Fig. ##FIG##3##4##B). As shown in (Fig. ##FIG##3##4##C), there were two important signal pathways related with the regulation of repair-supportive cell phenotypes, including the JAK-STAT signaling pathway and PI3K-AKT signaling pathway. GSEA indicated that EC-EXO treatment might contribute to the activation of signaling pathways associated with axon regeneration, which including neuron projection cytoplasm signaling pathway (NSE = 1.31, FDR = 0.16), axon cytoplasm signaling pathway (NSE = 1.40, FDR = 0.08) and growth factor activity (NSE = 1.30, FDR = 0.12) (Fig. ##FIG##3##4##D). We selected five significant genes associated with nerve survival and regeneration from the RNA-seq data, GDF15, CCN1 and JunD have been shown to positively mediate nerve repair, while TXNIP and KLF10 have been shown to inhibit axon regrowth (Fig. ##FIG##3##4##E) [##REF##26077927##24##–##REF##20155803##28##]. We verified the sequencing results by RT–qPCR assay, and the results were consistent with sequencing data (Fig. ##FIG##3##4##F). Primer sequences of all related genes were listed in (Additional file ##SUPPL##0##1##: Table S1).</p>", "<title>EC-EXO activated the transcription factors associated with repair phenotypes in SCs</title>", "<p id=\"Par63\">Activating the transcriptional repair program in SCs is crucial for enforcing the SC injury response and axon regeneration [##REF##26864683##7##, ##REF##25033179##29##, ##REF##28320842##30##]. The transcription factors c-Jun and STAT3 regulate of transcriptional mechanisms associated with the repair program. Therefore, the excitation of these transcription factors following injury is critical for generating repair-related SCs. We used Western blot to detect the expression of STAT3, tyrosine 705 STAT3 phosphorylation (p-STAT3-Tyr705) and c-Jun between the control group and EXO-treated group. The results showed that EC-EXO significantly upregulated the expression of p-STAT3-Tyr705 and c-Jun in SCs, confirming that EC-EXO could provide an effective strategy to induce repair phenotypes in SCs (Fig. ##FIG##3##4##G, H).</p>", "<title>EC-EXO activated PI3K/AKT/PTEN signaling pathway to boost the repair-related phenotypes of SCs in vitro</title>", "<p id=\"Par64\">Multiple signals are involved in regulating SC repair phenotypes following nerve injury. Recent studies showed that SCs’ PI3K/AKT signaling pathways play an essential role in their differentiation, myelination, and later PNS pathology [##REF##34157170##31##, ##REF##27658374##32##]. It has been demonstrated that PI3K is necessary for a full mitogenic response and cell proliferation, survival, and protein synthesis [##REF##19453943##33##]. Activated PI3K induces the phosphorylation of 4,5-biphosphate (PIP2) to generate a second messenger, phosphatidylinositol 3, 4, 5, triphosphate (RIP3), and the lipid phosphatase PTEN acts as a negative mediator in the pathway and reverses this reaction [##REF##16847462##34##]. Western blot showed that EC-EXO significantly elevated the phosphorylation of PI3K and AKT and downregulated the expression of PTEN (Fig. ##FIG##3##4##I, J). These results confirmed that PI3K/AKT/PTEN signaling pathway contributed to EC-EXO-regulated repair-related cell phenotypes in SCs.</p>", "<title>PI3K inhibitor could depress the proliferative and migratory phenotype of SCs induced by EC-EXO</title>", "<p id=\"Par65\">To further investigate the role of PI3K/AKT/PTEN signaling pathway in the regulation of SC repair phenotypes, we used LY294002, a PI3K inhibitor (PI3Ki), to inhibit the PI3K/AKT/PTEN signaling pathway and observed the changes of SC phenotypes [##REF##28320092##35##]. The western blot results indicated that the protein levels of PI3K, p-PI3K, AKT and p-AKT in EXO + PI3Ki group were significantly down-regulated. Meanwhile, the PTEN expression level in the group of EXO + PI3Ki was higher than that of EXO group (Additional file ##SUPPL##0##1##: Fig. S3A, B). Then we carried out series of experiments and demonstrated that PI3Ki could significantly depress the promoting effects of EC-EXO on SC proliferation (Additional file ##SUPPL##0##1##: Fig. S3C, D), migration (Additional file ##SUPPL##0##1##: Fig. S3E, F) and colony formation (Additional file ##SUPPL##0##1##: Fig. S3G, H).</p>", "<title>EC-EXO possessed a favorable neuronal affinity and could be present in the nerves for at least 28 days</title>", "<p id=\"Par66\">To trace the distribution and the length of exosomes stayed in the sciatic nerve, the DiR-labelled EC-EXO were injected into the sciatic nerve of SD rats, and then in vivo imaging was performed 1 day, 3 day, 7 day, 14 day and 28 day post-injection. The imaging demonstrated that EC-EXO had a fair distribution and a long duration in the sciatic nerve (Fig. ##FIG##4##5##A–D). In addition, the SD rat model was injected with DiI-labelled exosomes. Following 1 day, 3 day, 7 day, 14 day and 28 day, the sciatic nerve was removed and made into frozen sections, then the nerve was stained to visualize the nucleus with DAPI and was detected by confocal laser scanning microscopy. The red fluorescence presented in the EXO-treated group suggested that the EC-EXO possessed favorable neuronal affinity in the sciatic nerve (Fig. ##FIG##4##5##E).</p>", "<title>EC-EXO promoted the anti-apoptotic ability of injured nerve tissue</title>", "<p id=\"Par67\">We constructed a sciatic nerve crush model in rats as shown in (Additional file ##SUPPL##0##1##: Fig. S4A). Following the successful construction of the sciatic nerve crush model in SD rats, the anti-apoptotic function of EC-EXO in injured sciatic nerve was detected and analyzed by TUNEL staining (Additional file ##SUPPL##0##1##: Fig. S4B, C). The results showed that EC-EXO could significantly decrease the apoptotic levels of nerve tissue. In addition, we used western blot to detect the protein expression levels of apoptosis-related biomarkers, including BAX and Bcl2. EC-EXO significantly reduced the expression of the pro-apoptotic protein BAX while increasing the expression of the anti-apoptotic protein Bcl2 (Additional file ##SUPPL##0##1##: Fig. S4D, E). These results indicated that EC-EXO significantly improved the anti-apoptotic ability of injured nerves.</p>", "<title>EC-EXO promoted functional recovery and prevented gastrocnemius muscle atrophy following PNI</title>", "<p id=\"Par68\">To assess EC-EXO' effect on the functional recovery of PNI rat models, we evaluated SFI 3 days before the operation, 7, 14 and 28 days following the operation using walking track analysis. As shown in (Fig. ##FIG##5##6##A), at 28 days after operation, severe foot contractures were developed in the group of PNI. On the contrary, footprints in sham and EXO groups were clear and flared, indicating the remarkable functional recovery of the rats. Furthermore, the data showed no significant difference in SFI within seven days following the operation, while the SFI values of the EXO group were higher than those in the PNI group at 14 days after the operation. This difference was further expanded 28 days after operation (Fig. ##FIG##5##6##B). Furthermore, we used electrophysiological analysis to assess the functional recovery of regenerative nerves. The compound muscle action potential (CMAP) was evoked by the stimulating electrode in the sciatic nerve and recorded by the receiving electrode in the gastrocnemius muscle (GM) (Fig. ##FIG##5##6##C). Amplitudes in the EXO group were higher than those in PNI group, which further indicated that the treatment of EC-EXO enhanced the functionality of the regenerated sciatic nerve (Fig. ##FIG##5##6##D).</p>", "<p id=\"Par69\">We evaluated the gastrocnemius muscle atrophy by the relative weight of the left gastrocnemius muscle atrophy and the muscle fiber mean diameter in each group. The gastrocnemius muscle in the EXO group was larger than that of the PNI group at 28 days following the operation, showing that the treatment of EC-EXO prevented the atrophy of gastrocnemius muscle (Fig. ##FIG##5##6##E). In addition, the weight ratio of the gastrocnemius muscle in the EXO group was higher than that in the PNI group (Fig. ##FIG##5##6##F). Moreover, the results of HE and Masson trichrome staining of gastrocnemius muscle showed that the muscle fiber mean diameter in the EXO group was larger than that in the PNI group (Fig. ##FIG##5##6##G, H). These results demonstrated that EC-EXO could prevent gastrocnemius atrophy following sciatic nerve injury.</p>", "<title>EC-EXO promoted nerve repair following sciatic nerve injury</title>", "<p id=\"Par70\">HE and Masson staining were performed on longitudinal and transverse sections of sciatic nerves to observe the morphology of sciatic nerves and the histological changes in the injured nerve fibers. As shown in (Fig. ##FIG##6##7##A), the EXO group had a more tightly and orderly arranged structure, less edema and vacuolization, and more newly formed vessels than the PNI group. The TB staining results of sciatic nerve transverse sections in (Fig. ##FIG##6##7##B) showed that the EXO group had more myelinated axons than the PNI group (Fig. ##FIG##6##7##D). To further evaluate EC-EXO' effect on myelin regeneration following the operation, we performed transmission electron microscopy to observe the histological changes in regenerated myelin and count diameters of the regenerated axons in each group (Fig. ##FIG##6##7##C). The histological changes, including myelin sheath axons, myelinated axons, and newly-formed vessels could be observed at low magnification, and diameters of the axons and G-ratio (inner axonal diameter to fiber diameter ratio) were accurately measured at high magnification (Fig. ##FIG##6##7##E). Axons in the EXO group had thicker myelin sheaths and lower G-ratio than the PNI group, indicating a significant regeneration of the injured sciatic nerves with the EC-EXO treatment (Fig. ##FIG##6##7##F).</p>", "<title>EC-EXO enhanced axon regeneration, myelination and angiogenesis of the injured sciatic nerve</title>", "<p id=\"Par71\">To evaluate the regeneration efficiency of neurofilaments and SCs, we performed the neurofilament-200 (NF200) and S100β double staining of sciatic nerve longitudinal sections and cross-sections (Fig. ##FIG##7##8##A and G). Besides, we also conducted the class III β-tubulin (TuJ-1) and myelin basic protein (MBP) double staining to assess further the neural expression of axons and myelinated fibers (Fig. ##FIG##7##8##B and H). As a result, the data showed that NF200, S100β, TuJ-1, and MBP had a higher expression in the EXO group than in the PNI group, showing a more regenerative capacity in axon and myelination of an injured nerve (Fig. ##FIG##7##8##C–F, I–L). To study the effect of EC-EXO on the angiogenesis of the injured nerve, we performed immunofluorescence staining for angiogenesis markers CD31, VEGFR and CD34 of sciatic nerve cross-sections (Fig. ##FIG##7##8##M–O). The results indicated that EC-EXO promoted the expression of CD31, VEGFR, and CD34 which showed a powerful angiogenesis ability of the injured nerve treated with EC-EXO (Fig. ##FIG##7##8##P).</p>", "<title>EC-EXO activated PI3K/AKT/PTEN signaling pathway to promote nerve regeneration</title>", "<p id=\"Par72\">Previous studies depicted that EC-EXO could boost repair-related cell phenotypes of SCs through PI3K/AKT/PTEN signaling pathway. To further investigate the signaling pathways mediated by EC-EXO in vivo associated with the regeneration of injured nerve, we used western blot to detect the activated state of PI3K/AKT/PTEN signaling pathway in sciatic nerve tissue. According to the analysis of these proteins' expression and phosphorylation enrichment levels, EC-EXO significantly upregulated the expression of p-PI3K and p-AKT and down-regulated the expression level of PTEN (Fig. ##FIG##8##9##A, B). In addition, we also used immunofluorescence to detect the activation status of the PI3K/AKT/PTEN signaling pathway in vivo. The results showed that the percentages of positive p-PI3K and p-AKT in the EXO group were significantly higher than in the PNI group (Fig. ##FIG##8##9##C-F). Moreover, as the negative regulator of PI3K/AKT signaling pathway, the percentage of positive PTEN was lower in EXO group (Fig. ##FIG##8##9##G, H). The immunofluorescence results were consistent with those in western blot.</p>" ]
[ "<title>Discussion</title>", "<p id=\"Par73\">Regeneration of injured peripheral nerves is a complex process involving coordinated action of neuronal axons, SCs, ECs, macrophages and fibroblasts [##REF##20887890##36##]. SCs have been proven to play an essential role in axonal regeneration after peripheral nerve injury. Repair-related phenotypes of SCs can accelerate debris clearance, proliferate and migrate to form Büngner bands, guide axon regrowth, secrete growth factors and neurotrophic factors, and remyelinate regenerated axons [##REF##35282045##37##]. These repair SCs with special repair supporting phenotypes are specialized for repair and differ from other cells in the SC lineage such as myelin and Remak states [##REF##26864683##7##, ##REF##30632639##17##]. Arthur-Farraj et al. found the absence of c-Jun results in the formation of a dysfunctional repair cell, striking failure of functional recovery, and neuronal death, which indicated that c-Jun plays a key role in the activation of a repair program in SCs and the creation of a cell specialized to support regeneration [##REF##22920255##38##]. Liu et al. indicated that RSC96 cells can sense external, magnetically driven mechanical forces and transduce them to intracellular biochemical signals that promote nerve regeneration by inducing and maintaining the repair phenotypes of SCs [##REF##35351151##16##]. Indeed, it would be interesting to explore whether EC-EXO play a critical role on transition of SCs between different subtype of SCs except repair SCs, such as immature SCs, myelin SCs and nonmyelin (Remak). And we consider this a promising strategy to further explore the critical role of EC-EXO in regulation of SC biology. Numerous studies have elucidated the active effect and related mechanisms of axon-glia interaction in peripheral nerve regeneration [##REF##32628805##39##]. Notably, it is becoming increasingly apparent that ECs also play a critical role in this process, however, the underlying mechanism still remains unclear [##REF##29085283##40##]. Hobson et al. reported that VEGF could enhance vascularization and indirectly promote nerve regeneration [##REF##11197533##41##]. Ramos et al. suggested that the migration of ECs could be enhanced by SCs after nerve injury, and ECs secreted VEGF to participate the regulation of SCs and to promote nerve regeneration [##REF##26491999##42##]. Cattin et al. first reported that macrophage-derived VEGFA induced a polarized vasculature within the injured site and blood vessels directed the migrating cords of SCs, and disrupting the organization of the newly formed blood vessels in vivo could compromise SCs directionality resulting in defective nerve repair [##REF##26279190##9##]. Thus, previous studies mainly focused on explaining the ECs-to-SCs interaction largely due to the effect of VEGF, few studies explored the regulatory effect of ECs on SCs from the aspect of exosomes, which played an essential role in the cell-to-cell interaction. As the essential messengers and information mediators, exosomes from different resource cells carry different contents including proteins, lipids and various RNA species and interact with target cells to modulate their cell phenotypes [##REF##22510790##43##–##REF##27053351##46##]. Numerous researches have shown that exosomes are crucial in promoting nerve regeneration. For example, exosomes from SCs, mesenchymal stem cells, and macrophages have been demonstrated to be effective in accelerating peripheral nerve regeneration [##REF##25135977##47##, ##REF##25824139##48##]. Exosome therapy is more satisfactory for clinical treatment than direct stem cell transplantation because of its advantages, including low immunogenicity, improved safety, ease of storage and management, and mass production [##REF##26725829##49##, ##REF##27650895##50##]. In this study, we found that EC-EXO boosted repair-related phenotypes of SCs including: enhanced proliferation, migration and the anti-apoptotic ability, the upregulation of growth factors, neurotrophic factors and immune-related cytokines.</p>", "<p id=\"Par74\">Moreover, in the rat model of sciatic nerve injury, EC-EXO possessed favorable neuronal affinity and could inhibit apoptosis, enhance axon regeneration, myelination and angiogenesis, and promote functional recovery. Zhou et al. intraventricularly injected microvascular ECs (bEnd.3) derived exosomes into the acute middle cerebral artery occlusion model and found that after exosomes treatment, neurobehavioral outcomes were improved, neural progenitor cell proliferation and migration were activated, and cell apoptosis was attenuated, suggesting microvascular ECs derived exosomes played an essential role for brain protection in the ischemia/reperfusion injury [##REF##32502570##51##]. Moreover, Zhang et al. isolated exosomes from cerebral endothelial cells of nonischemic and ischemic rats (nCEC-exos and isCEC-exos) and found that isCEC-exos enhanced axonal growth and exhibited more robust elevation of select miRNAs than nCEC-exos, indicating facilitative effect of nCEC-exos and isCEC-exos on axonal growth by altering miRNAs [##REF##33138691##52##]. Interestingly, we found that EC-EXO could promote the proliferation and migration of SCs better than SC-EXO. Liu et al. isolated exosomes from human adipose derived mesenchymal stem cells (hADMSCs) with and without differentiation (dExo vs uEXO), and demonstrated that dExo protected rat SCs from oxidative stress and enhanced HUVEC migration and angiogenesis [##REF##35310346##53##]. Xiao et al. reported that HUVECs derived exomes could directly protect SH-SY5Y nerve cells against cerebral ischemia injury [##REF##28849073##14##]. Consequently, these results demonstrated that EC-EXO had a significant effect on boosting repair-related phenotypes of SCs and promoting nerve regeneration and functional recovery. However, the regulatory mechanism of EC-EXO on repair-related cell phenotypes in SCs remains unclear. Members of miR199 family have been shown to positively promote nerve repair, angiogenesis, and muscle regeneration. Liu et al. isolated exosomes from human adipose derived mesenchymal stem cells (hADMSCs) with and without differentiation (dExo vs uEXO), and demonstrated that the miR199b-5p upregulated in dExo than in uExo was highly related to neuroprotection and angiogenesis [##REF##35310346##53##]. Chen et al. reported that miR199b regulated the phenotypic switch during vascular cell differentiation derived from induced pluripotent stem (iPS) cells through critical signaling angiogenic responses [##REF##25535084##54##]. Fukuoka et al. verified systemic administration of miR199 mimics to the mice of Duchenne muscular dystrophy (DMD) significantly enhanced muscle regeneration and ameliorated muscular dystrophy [##REF##33782502##55##]. To further explore the mechanism of EC-EXO promoting SCs repair-related phenotypes, we determined miR199-5p was a critical role in the communication mechanism between the EC-EXO and SCs. We demonstrated that miR199-5p was upregulated in SCs treated with EC-EXO and enhanced the proliferation and migration of SCs in vitro. We consider that further investigation surrounding the effect of miR199-5p in the in vivo nerve tissues would be interesting, which will be a remarkable direction in our future work. Moreover, we demonstrated that EC-EXO activated PI3K/AKT/PTEN signaling pathway to boost the repair phenotypes of SCs. In our study, the PI3K inhibitor was used to further verify the crucial role of PI3K signaling in promoting the repair phenotype of SCs by EC-EXO. Numerous studies had shown that SCs miRNA expression levels are drastically changed following injury and miRNA could regulate SCs proliferation and axon myelination. Li et al. found that SCs miR340 boosted debris clearance following nerve crush injury in a rat sciatic model, and that the dysregulation of the miR450 expression in the injury site perturbed cell debris removal and axonal regrowth [##REF##27344331##56##]. Yu et al. also reported that up-regulation of miR-221 and miR-222 cluster was correlated with injury-induced SCs phenotypic modulation [##REF##22393241##57##]. Gao et al. demonstrated that EC-EXO promoted functional motor recovery and reconstruction of synaptic function in ischemic brain injury, and miR126-3p from EC-EXO could serve as a treatment for nerve damage [##REF##32574032##58##]. Venkat et al. also suggested that miR126 might mediate EC-EXO-induced neurorestorative effects by inducing capillary tube formation and axonal outgrowth [##REF##31394992##59##]. Moreover, Xie et al. reported that ADSC-EXO promoted proliferation and migration and inhibited apoptosis of PC12 through the activation of the PI3K/AKT pathway [##REF##34691190##60##]. Taken together, we can conclude that EC-EXO promoting SCs repair-related phenotypes via PI3K/AKT/PTEN signaling pathway.</p>", "<p id=\"Par75\">In summary, our study showed that EC-EXO could boost and maintain the SCs repair-related phenotypes resulting in promoting axonal regeneration, remyelination and angiogenesis. Furthermore, the mechanism may be relevant to the up-regulated expression of miR199-5p and activation of PI3K/AKT/PTEN signaling pathway leading to a satisfactory functional recovery after peripheral nerve injury. Our study elucidated the active role of EC-EXO in regulating the repair phenotypes of SCs and suggested that EC-EXO could serve as a promising therapeutic target for peripheral nerve injury.</p>", "<p id=\"Par76\">In our manuscript, EC-EXO induced the repair-supportive phenotypes in SCs, and repair SCs have a positive feedback effect on EC activation. The previous study showed that the cell coordination in nerve injury sites between perineurial cells and SCs, between macrophages and endothelial cells, and between macrophages and SCs have all been reported [##REF##26279190##9##, ##REF##20869108##61##, ##REF##30726731##62##]. Therefore, we speculate besides repair SCs, there may be some other feedback loop toward endogenous EC activation and exosomes may play a critical role in this process. The influence of ECs and EC-EXO on other cells in nerve repair microenvironment, such as macrophages, fibroblasts and axons, as well as the possible feedback regulatory pathways, will be a critical direction of our future research. Despite promising results of EC-EXO in peripheral nerve regeneration, the translation of EC-EXO is challenged by massive production, purification, modification and storage. With the research techniques for separation and concentration of exosomes are gradually developed, there are currently lots of different techniques available employing principles of density, buoyant density, size, membrane and immunoaffinity, and among them, differential ultracentrifugation has been the most commonly used technique [##REF##27802845##63##]. To achieve higher recovery and specificity, a combination of techniques is recommended such as precipitation or filtration can be combined with size exclusion chromatography to eliminate non-EV components [##REF##33936570##64##]. Additionally, although there is currently no standardized procedure about the amount of exosomes for treatment purpose, it is recommended to perform dose–response studies with additional control such as non-EV or EV-depleted fractions [##REF##30637094##65##]. In accordance with the features of specificity, intrinsic homing abilities and non-immunogenicity, EC-EXO can be used as therapeutic vehicles to deliver molecules directly to neurons to improve nerve regeneration following injuries. Although more investigation is required to move forward the clinical application of EC-EXO, recent developments have shown us the powerful potential of EC-EXO in nerve regeneration that could support clinical use. The understanding of the relevance of EC-EXO in SCs biology and peripheral nerve regeneration is still in its infancy. Continued research in this field will definitely allow a better understanding of mechanisms in pursuit of new therapeutics.</p>" ]
[ "<title>Conclusion</title>", "<p id=\"Par77\">In this study, we isolated exosomes derived from ECs, and determined the beneficial effect of EC-EXO on the SCs repair-related phenotypes via up-regulating expression of miR199-5p and activating PI3K/AKT/PTEN signaling pathway. EC-EXO could promote functional recovery after PNI by enhancing axonal regeneration, remyelination and angiogenesis. Our research demonstrates that EC-EXO can be an effective therapeutic tool for regulating repair-supportive cell phenotypes associated with nerve regeneration. We hope this study will provide a valuable therapeutic strategy for the regeneration and repair of peripheral nerve injury.</p>" ]
[ "<title>Background</title>", "<p id=\"Par1\">Schwann cells (SCs) respond to nerve injury by transforming into the repair-related cell phenotype, which can provide the essential signals and spatial cues to promote axonal regeneration and induce target reinnervation. Endothelial cells (ECs) contribute to intraneural angiogenesis contributing to creating a permissive microenvironment. The coordination between ECs and SCs within injury sites is crucial in the regeneration process, however, it still unclear. As the intercellular vital information mediators in the nervous system, exosomes have been proposed to take a significant role in regulating regeneration. Thus, the main purpose of this study is to determine the facilitative effect of ECs-derived exosomes on SCs and to seek the underlying mechanism.</p>", "<title>Results</title>", "<p id=\"Par2\">In the present study, we collected exosomes from media of ECs. We demonstrated that exosomes derived from ECs possessed the favorable neuronal affinity both in vitro and in vivo. Further research indicated that EC-exosomes (EC-EXO) could boost and maintain repair-related phenotypes of SCs, thereby enhancing axonal regeneration, myelination of regenerated axons and neurologically functional recovery of the injured nerve. MiRNA sequencing in EXO-treated SCs and control SCs indicated that EC-EXO significantly up-regulated expression of miR199-5p. Furthermore, this study demonstrated that EC-EXO drove the conversion of SC phenotypes in a PI3K/AKT/PTEN-dependent manner.</p>", "<title>Conclusion</title>", "<p id=\"Par3\">In conclusion, our research indicates that the internalization of EC-EXO in SCs can promote nerve regeneration by boosting and maintaining the repair-related phenotypes of SCs. And the mechanism may be relevant to the up-regulated expression of miR199-5p and activation of PI3K/AKT/PTEN signaling pathway.</p>", "<title>Graphical Abstract</title>", "<p id=\"Par4\">\n\n</p>", "<title>Supplementary Information</title>", "<p>The online version contains supplementary material available at 10.1186/s12951-023-01767-9.</p>", "<title>Keywords</title>" ]
[ "<title>Supplementary Information</title>", "<p>\n</p>" ]
[ "<title>Acknowledgements</title>", "<p>We appreciate the technical help from the Academy of Medical Science, Zhengzhou University during performing experiments.</p>", "<title>Author contributions</title>", "<p>JH: conceived the experimental designs, performed most experiments and wrote the paper. GZ: participated in animal experiments and data analysis. SL: participated in animal experiments. JL: participated in cellular experiments. WW: participated in data analysis. JX: participated in initial subject design. YW and MF: participated in most revision process of manuscript. NZ: provided experimental ideas, supervised the project, critically reviewed and wrote this paper. All authors approved the manuscript. All authors read and approved the final manuscript.</p>", "<title>Funding</title>", "<p>This work was supported by the National Natural Science Foundation of China (82071388), Postdoctoral Science Foundation of China (2019M660175), Excellent Youth Science Foundation of Henan Province (212300410077), Young and middle-aged Health Science and Technology Innovation Talent of Henan Province (YXKC2020048), Key Research Project of Henan Educational Committee (21A320033 &amp; 23A320063), and Jilin Provincial Natural Science Foundation (No. 20200201531JC).</p>", "<title>Data availability</title>", "<p>The data that support the findings of this study are available from the corresponding author upon reasonable request.</p>", "<title>Declarations</title>", "<title>Ethics approval and consent to participate</title>", "<p id=\"Par104\">The animal study was reviewed and approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University.</p>", "<title>Consent for publication</title>", "<p id=\"Par105\">All authors agree to be published.</p>", "<title>Competing interests</title>", "<p id=\"Par106\">The authors declare no potential competing interest.</p>" ]
[ "<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Characterization of EC-EXO. <bold>A</bold> Representative transmission electron microscopy (TEM) images of EC-EXO. Scale bar, 200 nm (left) and 40 nm (right). <bold>B</bold> Particle size distribution of EC-EXO measured by nanoparticle tracking analysis (NTA), inset showing representative exosome images captured from the NTA video frames. <bold>C</bold> Zeta potential measurements of EC-EXO. <bold>D</bold> Protein immunoblots of exosomes, including the typical markers (TSG101, CD9 and CD81) and GAPDH</p></caption></fig>", "<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>EC-EXO induced repair-related phenotypes of SCs. <bold>A</bold> SCs were incubated with DiI-labelled EC-EXO for 0 h, 2 h, 6 h, 12 h and 24 h, and representative fluorescence images show the delivery of DiI-labelled EC-EXO (red) into SCs. Scale bar, 20 μm. <bold>B</bold> Representative EdU staining images of control group and EXO-treated groups with different concentration (1, 10, 50 and 100 μg/mL) for 24 h. Scale bar, 100 μm. <bold>C</bold> Statistical evaluation of percentage of EdU-positive SCs. The data are expressed as mean ± SD (n = 3). <bold>D</bold> The colony formation of SCs treated with different concentrations of EC-EXO for 10 days. Scale bar, 500 μm. <bold>E</bold> Statistical results of the colony formation in each group. The data are expressed as mean ± SD (n = 3). <bold>F</bold> The cell cycle of SCs was detected by flow cytometry assay. <bold>G</bold> Statistical results of the cell cycle. The data are expressed as mean ± SD (n = 3). <bold>H</bold> The protein levels of factors associated with nerve regeneration (NGF, VEGFA, CNTF, BDNF and GDNF) were detected by western blot of SCs in each group. <bold>I</bold> Quantification of NGF, VEGFA, CNTF, BDNF and GDNF protein levels in each group. The data are expressed as mean ± SD (n = 3). <bold>J</bold> Abridged general view of coculture system ECs and SCs in the transwell migration assay. <bold>K</bold> Representative images of vertical migration of SCs with different treatments for 24 h. Scale bar, 100 μm. <bold>L</bold> The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). <bold>M</bold> Schematic diagrams of co-incubation of EC-EXO and SCs in the transwell migration assay. <bold>N</bold> Representative images of vertical migration of SCs with different concentrations of EC-EXO for 24 h. Scale bar, 100 μm. <bold>O</bold> The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001</p></caption></fig>", "<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>MiR199-5p was a key EC-EXO cargo in promoting SC repair-related phenotypes. The miRNAs in SCs treated with EC-EXO were profiled by the miRNA sequencing analysis. <bold>A</bold> Different miRNAs were detected in EXO-treated and control groups on a co-expression Venn diagram. <bold>B</bold> Gene Ontology (GO) term analyses of significantly changed miRNAs in control group and EXO-treated group regarding their involvement in biological processes (BP), cellular components (CC) and molecular functions (MF). <bold>C</bold> Significantly enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways related to the differentially expressed miRNAs obtained from the comparison of control group and EXO-treated group. <bold>D</bold> The heat map displayed the expression ratios of different microRNAs between control group and EXO-treated group. <bold>E</bold> Volcano plots showed the up-regulated microRNAs (red), down-regulated microRNAs (green) and non-changing microRNAs (gray) between EXO-treated SCs and control SCs. <bold>F</bold> Real-time qRT-PCR validated novel-miR199-5p expression in EXO-treated SCs. Data are expressed as mean ± SD (n = 3). <bold>G</bold> Quantitative PCR analysis of novel-miR199-5p expression levels in SCs transfected with mimic, mimic negative control (Mi-NC), inhibitor and inhibitor negative control (In-NC). Data are expressed as mean ± SD (n = 3). <bold>H</bold> Cell growth curve was detected by CCK-8 assay in different groups. The data are expressed as mean ± SD (n = 3). <bold>I</bold> Representative EdU staining images in different groups. Scale bar, 100 μm. <bold>J</bold> Statistical evaluation of percentage of EdU-positive SCs. The data are expressed as mean ± SD (n = 3). <bold>K</bold> Representative images of the colony formation in different groups. Scale bar, 500 μm. <bold>L</bold> Statistical results of the colony formation in each group. The data are expressed as mean ± SD (n = 3). <bold>M</bold> Representative images of vertical migration of SCs in different groups for 24 h. Scale bar, 100 μm. <bold>N</bold> The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001</p></caption></fig>", "<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>EC-EXO regulated the molecular mechanism and signaling pathways associated with SC repair-related phenotypes in vitro. <bold>A</bold> The heat map displayed the expression ratios of different genes between control group and EXO-treated group. <bold>B</bold> GO term analyses of significantly changed genes in control group and EXO-treated group regarding their involvement in BP, CC and MF. <bold>C</bold> Significantly enriched KEGG pathways related to the differentially expressed genes (DEGs) obtained from the comparison of control group and EXO-treated group. <bold>D</bold> Gene set enrichment analysis (GSEA) of nerve regeneration-associated pathways in different groups. NES, normalized enrichment score. FDR, false discovery rate. <bold>E</bold> The bar graph depicted the significant genes associated with SC repair-related phenotypes from RNA-seq data, including GDF15, CCN1, JunD, TXNIP and KLF10. Data are expressed as mean ± SD (n = 3). <bold>F</bold> PCR validation of differentially expressed mRNAs in control group and EXO-treated group. <bold>G</bold> The relative protein expression of SC repair phenotype-related transcription factors (c-Jun, STAT3 and p-STAT3) in different groups was detected through western blot. <bold>H</bold> Quantification of c-Jun, STAT3 and p-STAT3 protein levels in each group. Data are expressed as mean ± SD (n = 3). <bold>I</bold>, <bold>J</bold> Western blot and statistical analysis of PI3K, p-PI3K, AKT, p-AKT and PTEN. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001</p></caption></fig>", "<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>EC-EXO possessed favorable neuronal affinity and could be present in the nerves for a prolonged time. To study the neuronal affinity of EC-EXO, 50 μg/mL EC-EXO (20 μL) were injected locally under the epineurium of sciatic nerve. <bold>A</bold> Representative pictures of living imaging in each group at 1 day, 3 days, 7 days, 14 days and 28 days following surgery. <bold>B</bold> Radiant efficiency in different groups at different days. The data are expressed as mean ± SD (n = 3). <bold>C</bold> The rats were sacrificed on Day 28. Sciatic nerves were harvested and examined by bioluminescent imaging. <bold>D</bold> Luminescence of sciatic nerves at Day 28 were quantified. The data are expressed as mean ± SD (n = 3). <bold>E</bold> SD rat model reached the injection with DiI-labelled EC-EXO under the epineurium of sciatic nerve. Then at 1 day, 3 days, 7 days, 14 days and 28 days following surgery, the nerves were harvested and stained to visualize nucleus with DAPI. Representative images to visualize the distribution of EC-EXO in sciatic nerve at 1 day, 3 days, 7 days, 14 days and 28 days following surgery. Scale bar, 20 μm. *p &lt; 0.05, **p &lt; 0.01</p></caption></fig>", "<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Functional analysis of the regenerated sciatic nerves and histological assessment of the gastrocnemius muscle. <bold>A</bold> Representative images of footprints in sham, PNI and EXO groups at 28 days following operation. <bold>B</bold> Sciatic nerve function index (SFI) analysis in each group at 3 days before operation, 7 days and 14 days following operation. The data are expressed as mean ± SD (n = 5). <bold>C</bold> Evoked compound muscle actionpotential (CMAP) in each group at 28 days after operation. <bold>D</bold> Amplitude of CMAP in each group. The data are expressed as mean ± SD (n = 5). <bold>E</bold> Representative images of harvested gastrocnemius muscles in sham, PNI and EXO groups. <bold>F</bold> Statistical analysis of weight ratio of gastrocnemius muscle in different groups. The data are expressed as mean ± SD (n = 5). <bold>G</bold> Hematoxylin eosin (HE) and Masson trichrome staining of gastrocnemius muscle in each group. Scale bar, 20 μm. <bold>H</bold> Statistical analysis of muscle fiber mean diameter. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001</p></caption></fig>", "<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>Histological changes in injured sciatic nerve with the treatment of EC-EXO. <bold>A</bold> Representative images of HE and Masson staining sciatic nerve longitudinal and cross sections in sham, PNI and EXO groups 28 days following the treatment of EC-EXO (n = 3). Scale bar, 20 μm. <bold>B</bold> Representative images of toluidine blue (TB) staining sciatic nerve in each group (n = 3). Scale bar, 20 μm. <bold>C</bold> Representative TEM images of sciatic nerve in each group (n = 3). Scale bar, 10, 2 and 1 μm. <bold>D</bold> Statistical analysis of the number of axons with myelin. The data are expressed as mean ± SD (n = 3). <bold>E</bold> Statistical analysis of the thickness of myelin sheath. The data are expressed as mean ± SD (n = 3). <bold>F</bold> Statistical analysis of G-ratio; The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001</p></caption></fig>", "<fig id=\"Fig8\"><label>Fig. 8</label><caption><p>Immunofluorescence analysis of sciatic nerves. <bold>A</bold> NF200/S100β and <bold>B</bold> TuJ1/MBP double immunofluorescence staining of sciatic nerve longitudinal sections at 28 days following the operation. Nuclei were stained with DAPI. Scale bar, 20 μm. <bold>C-F</bold> The statistical results of percentages of positive. NF200, S100β, TuJ1 and MBP in sham, PNI and EXO groups. The data are expressed as mean ± SD (n = 3). <bold>G</bold>, <bold>H</bold> Representative immunofluorescence images of sciatic nerve cross-sections stained with NF200/S100β and TuJ1/MBP. Nuclei were stained with DAPI. Scale bar, 20 μm. <bold>I</bold>–<bold>L</bold> Statistical analysis of different groups. The data are expressed as mean ± SD (n = 3). <bold>M</bold> Representative images of immunofluorescence staining for CD31, CD34 and VEGFR of sciatic nerve cross-sections in each group. Nuclei were stained with DAPI. Scale bar, 100 μm. <bold>N</bold> Statistical analysis of vessel area. The data are expressed as mean ± SD (n = 3). *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001.</p></caption></fig>", "<fig id=\"Fig9\"><label>Fig. 9</label><caption><p>EC-EXO promoted nerve regeneration by activating PI3K/AKT/PTEN signaling pathways. <bold>A</bold> The expression levels of biomarkers of PI3K/AKT/PTEN pathway (PI3K, p-PI3K, AKT, p-AKT and PTEN) in sciatic nerves in different experimental groups were detected by western blot 28 days following the operation. <bold>B</bold> Statistical analysis of western blot results. The data are expressed as mean ± SD (n = 3). Representative images of PI3K/p-PI3K/GAPDH (<bold>C</bold>), AKT/p-AKT/GAPDH (<bold>E</bold>) and PTEN/GAPDH (<bold>G</bold>) multiple fluorescence staining of sciatic nerve at 28 days following the operation. Nuclei were stained with DAPI. Scale bar, 20 μm. The statistical results of percentages of positive PI3K/p-PI3K/GAPDH staining (<bold>D</bold>), percentages of positive AKT/p-AKT/GAPDH (<bold>F</bold>) and percentages of positive PTEN/GAPDH staining (<bold>H</bold>). The data are presented as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001</p></caption></fig>" ]
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[ "<fn-group><fn><p>The original online version of this article has been revised: the fig. 2d is corrected.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>" ]
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[ "<media xlink:href=\"12951_2023_1767_MOESM1_ESM.docx\"><caption><p><bold>Additional file 1: Figure S1.</bold> The effect of EC-EXO on cell phenotypes of SCs. <bold>A</bold> The proliferation of SCs treated with EC-EXO of different concentration (1, 10, 50 and 100 μg/mL) for 24 and 48 h was detected by CCK8 assay. The data are expressed as mean ± SD (n = 3). <bold>B</bold> Cell growth curve was detected by CCK-8 assay in 50 μg/mL EXO-treated and control groups. The data are expressed as mean ± SD (n = 3). <bold>C</bold> SC apoptotic levels determined by flow cytometry assay. The data are expressed as mean ± SD (n = 3). <bold>D</bold> Statistical results of cell apoptotic. The data are expressed as mean ± SD (n = 3). <bold>E</bold> The protein levels of proliferation-related protein (PCNA) and apoptosis-related protein (BAX and Bcl2) were analyzed by western blot following incubation of SCs to EC-EXO for 24 h. <bold>F</bold> Quantification of PCNA, BAX and Bcl2 levels in EXO-treated and control groups. The data are expressed as mean ± SD (n = 3). <bold>G</bold> The mRNA levels of immune factors (LIF, Gal-3 and MCP-1) were detected by RT–qPCR of SCs in each group. The mRNA levels are expressed as fold change of the control. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. <bold>Figure S2.</bold> Comparison of the effects of EC-EXO and SC-EXO on the proliferation and migration phenotypes of SCs. <bold>A</bold> Representative transmission electron microscopy (TEM) images of SC-EXO. Scale bar, 100 nm. <bold>B</bold> Protein immunoblots of exosomes, including the typical markers (TSG101, CD9 and CD81) and GAPDH. <bold>C</bold> Particle size distribution of SC-EXO measured by nanoparticle tracking analysis (NTA), inset showing representative exosome images captured from the NTA video frames. <bold>D</bold> Representative EdU staining images in different groups. Scale bar, 50 μm. <bold>E</bold> Statistical evaluation of percentage of EdU-positive SCs. The data are expressed as mean ± SD (n = 3). <bold>F</bold> Representative images of vertical migration of SCs in different groups for 24 h. Scale bar, 100 μm. <bold>G</bold> The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). <bold>H</bold> Representative images of the colony formation in indicated groups. Scale bar, 500 μm. <bold>I</bold> Statistical results of the colony formation in each group. The data are expressed as mean ± SD (n = 3). *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. <bold>Figure S3.</bold> PI3K inhibitor (PI3Ki) could depress the proliferative and migratory phenotype of SCs induced by EC-EXO. <bold>A</bold> Western blot of PI3K, p-PI3K, AKT, p-AKT and PTEN in control, EXO and EXO + PI3Ki groups. <bold>B</bold> Quantification of PI3K, p-PI3K, AKT, p-AKT and PTEN protein levels in each group. Data are expressed as mean ± SD (n = 3). <bold>C</bold> Representative EdU staining images in different groups. Scale bar, 50 μm. <bold>D</bold> Statistical evaluation of percentage of EdU-positive SCs. The data are expressed as mean ± SD (n = 3). <bold>E</bold> Representative images of vertical migration of SCs in different groups for 24 h. Scale bar, 100 μm. <bold>F</bold> The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). <bold>G</bold> Representative images of the colony formation in indicated groups. Scale bar, 500 μm. <bold>H</bold> Statistical results of the colony formation in each group. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. <bold>Figure S4.</bold> EC-EXO improved the anti-apoptotic ability of injured nerve tissue. To study the effect of EC-EXO in vivo, 50 μg/mL EC-EXO (20 μL) were injected locally under the epineurium of injured sciatic nerve. <bold>A</bold> The left picture is a schematic illustration of a sciatic nerve crush model on rats. The right picture illustrated the injured sciatic nerve during the surgery. <bold>B, C</bold> TUNEL-positive (green) cells were detected in sciatic nerves by TUNEL staining and the statistical results. The data are expressed as mean ± SD (n = 3). Scale bar, 20 μm. <bold>D</bold> The protein levels of BAX and Bcl2 of sciatic nerves in each group were analyzed by western blot. <bold>E</bold> Quantification of BAX and Bcl2 levels in each group. The data are expressed as mean ± SD (n = 3). *p &lt; 0.05, **p &lt; 0.01.</p></caption></media>" ]
[{"label": ["10."], "surname": ["Witwer", "Buz\u00e1s", "Bemis", "Bora", "L\u00e4sser", "L\u00f6tvall", "Nolte-'t Hoen", "Piper", "Sivaraman", "Skog"], "given-names": ["KW", "EI", "LT", "A", "C", "J", "EN", "MG", "S", "J"], "article-title": ["Standardization of sample collection, isolation and analysis methods in extracellular vesicle research"], "source": ["J Extracell Vesicles"], "year": ["2013"], "volume": ["2"], "fpage": ["20360"], "pub-id": ["10.3402/jev.v2i0.20360"]}]
{ "acronym": [ "SCs", "ECs", "EC-EXO", "SC-EXO", "HUVECs", "PNI", "TEM", "NTA", "TSG101", "DiI", "VEGF", "NGF", "CNTF", "BDNF", "GDNF", "PI3K", "PIP2", "RIP3", "PTEN", "SFI", "CMAP", "NF200", "TuJ-1", "MBP", "mTOR", "EdU", "Mi-NC", "In-NC", "DEGs", "GO", "BP", "CC", "MF", "KEGG", "GSEA", "NES", "FDR", "RT–qPCR", "cDNAs", "WB", "PVDF", "GM", "HE", "TB" ], "definition": [ "Schwann cells", "Endothelial cells", "EC-exosomes", "SC-exosomes", "Human umbilical vein ECs", "Peripheral nerve injury", "Transmission electron microscopy", "Nanoparticle tracking analysis", "Tumor susceptibility gene 101", "1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate", "Vascular endothelial growth factor", "Nerve growth factor", "Ciliary neurotrophic factor", "Brain-derived neuotrophyic factor", "Glial cell derived neurotrophic factor", "PhosphoInositide-3 Kinase", "Phosphorylation of 4,5-biphosphate", "Phosphatidylinositol 3, 4, 5, triphosphate", "Phosphatase and tensin homolog", "Sciatic nerve function index", "Compound muscle action potential", "Neurofilament-200", "Class III β-tubulin", "Myelin basic protein", "Mammalian target of the rapamycin", "5-Ethynyl-2ʹ-deoxyuridine", "Mimic negative control", "Inhibitor negative control", "Differential expression genes", "Gene Ontology", "Biological processes", "Cellular components", "Molecular functions", "Kyoto Encyclopedia of Genes and Genomes", "Gene set enrichment analysis", "Normalized enrichment score", "False discovery rate", "Real-time quantitative polymerase chain reaction", "Complementary DNAs", "Western blot", "Polyvinylidene fluoride", "Gastrocnemius muscle", "Hematoxylin–eosin", "Toluidine blue" ] }
70
CC BY
no
2024-01-13 23:36:46
J Nanobiotechnology. 2023 Jan 9; 21:10
oa_package/fe/05/PMC9827708.tar.gz
PMC9915899
36778401
[]
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[]
[]
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[ "<title>Abstract</title>", "<p>\n<italic toggle=\"yes\">BRAF</italic>\n<sup>V600E</sup>\nmutation is a driver mutation in the serrated pathway to colorectal cancers. BRAF\n<sup>V600E</sup>\ndrives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a \"just-right\" level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in\n<italic toggle=\"yes\">BRAF</italic>\n<sup>V600E</sup>\n-mutant adenomas/polyps in mice and patients. In\n<italic toggle=\"yes\">Vill-Cre</italic>\n;\n<italic toggle=\"yes\">BRAF</italic>\n<sup>V600E/+</sup>\n;\n<italic toggle=\"yes\">Fak</italic>\n<sup>fl/fl</sup>\nmice,\n<italic toggle=\"yes\">Fak</italic>\ndeletion maximized BRAF\n<sup>V600E</sup>\n's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAF\n<sup>V600E</sup>\n-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a \"just-right\" ERK signaling optimal for\n<italic toggle=\"yes\">BRAF</italic>\n<sup>V600E</sup>\n-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.\n</p>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2024-01-13 23:35:04
Res Sq. 2024 Jan 5;:rs.3.rs-2531119
oa_package/e5/f2/PMC9915899.tar.gz
PMC9949255
36824869
[]
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[]
[]
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[ "<title>Abstract</title>", "<p>\nBacteria dysbiosis has been associated with an increased risk of HIV-1 transmission and acquisition. The prevalent idea is that bacteria dysbiosis compromises mucosal integrity and promotes inflammatory conditions to cause recruitment and activation of immune cells that harbor or are targeted by HIV-1. However, it is also possible that HIV-1 directly binds bacteria or bacterial products to impact virus infectivity and transmissibility. This study evaluated HIV-1 interactions with bacteria through glycan-binding lectins. The\n<italic toggle=\"yes\">Streptococcal</italic>\nSiglec-like lectin SLBR-N, which is part of the fimbriae shrouding the bacteria surface and recognizes α2,3 sialyated\n<italic toggle=\"yes\">O</italic>\n-linked glycans, was noted for its ability to enhance HIV-1 infectivity in the context of cell-free infection and cell-to-cell transfer. Enhancing effects were recapitulated with\n<italic toggle=\"yes\">O</italic>\n-glycan-binding plant lectins, signifying the importance of\n<italic toggle=\"yes\">O</italic>\n-glycans. Conversely,\n<italic toggle=\"yes\">N</italic>\n-glycan-binding bacterial lectins FimH and Msl had no effect. SLBR-N was demonstrated to capture and transfer infectious HIV-1 virions, bind to\n<italic toggle=\"yes\">O</italic>\n-glycans on HIV-1 Env, and increase HIV-1 resistance to broadly neutralizing antibodies targeting different regions of Env. Hence, this study highlights the potential contribution of\n<italic toggle=\"yes\">O</italic>\n-glycans in promoting HIV-1 infection through the exploitation of\n<italic toggle=\"yes\">O</italic>\n-glycan-binding lectins from commensal bacteria at the mucosa.\n</p>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2024-01-13 23:35:04
Res Sq. 2024 Jan 3;:rs.3.rs-2596269
oa_package/5c/2e/PMC9949255.tar.gz
PMC10055683
36993232
[]
[]
[]
[]
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[ "<title>Abstract</title>", "<p>\n<bold>Background:</bold>\nDiarrhea remains a leading cause of childhood illness throughout the world that is increasing due to climate change and is caused by various species of ecologically sensitive pathogens. The emerging Planetary Health movement emphasizes the interdependence of human health with natural systems, and much of its focus has been on infectious diseases and their interactions with environmental and human processes. Meanwhile, the era of big data has engendered a public appetite for interactive web-based dashboards for infectious diseases. However, enteric infectious diseases have been largely overlooked by these developments.\n<bold>Methods:</bold>\nThe Planetary Child Health and Enterics Observatory (Plan-EO) is a new initiative that builds on existing partnerships between epidemiologists, climatologists, bioinformaticians, and hydrologists as well as investigators in numerous low- and middle-income countries. Its objective is to provide the research and stakeholder community with an evidence base for the geographical targeting of enteropathogen-specific child health interventions such as novel vaccines. The initiative will produce, curate, and disseminate spatial data products relating to the distribution of enteric pathogens and their environmental and sociodemographic determinants.\n<bold>Discussion:</bold>\nAs climate change accelerates there is an urgent need for etiology-specific estimates of diarrheal disease burden at high spatiotemporal resolution. Plan-EO aims to address key challenges and knowledge gaps by making rigorously obtained, generalizable disease burden estimates freely available and accessible to the research and stakeholder communities. Pre-processed environmental and EO-derived spatial data products will be housed, continually updated, and made publicly available to the research and stakeholder communities both within the webpage itself and for download. These inputs can then be used to identify and target priority populations living in transmission hotspots and for decision-making, scenario-planning, and disease burden projection.\n<bold>Study registration:</bold>\nPROSPERO protocol #CRD42023384709\n</p>" ]
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{ "acronym": [], "definition": [] }
0
CC BY
no
2024-01-13 23:35:05
Res Sq. 2024 Jan 10;:rs.3.rs-2640564
oa_package/16/e4/PMC10055683.tar.gz