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In essence, the &#946;<sub>2</sub> adrenergic receptor (&#946;<sub>2</sub>AR) plays an antiproliferative role by increasing the intracellular cyclic 3',5'-adenosine monophosphate (cAMP) concentration through G<sub>&#945;s</sub> coupling, but interestingly, &#946;<sub>2</sub>AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the &#946;<sub>2</sub>AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired &#946;<sub>2</sub>AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of G<sub>&#945;s</sub> and G<sub>&#945;i</sub>, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in G<sub>&#945;s</sub>-G<sub>&#945;i</sub> coupling to &#946;<sub>2</sub>AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This G<sub>&#945;i</sub> coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not &#946;-arrestin2 or protein kinase A-mediated phosphorylation of &#946;<sub>2</sub>AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6'-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in G<sub>&#945;s</sub>-G<sub>&#945;i</sub> coupling to &#946;<sub>2</sub>AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in &#946;<sub>2</sub>AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA. Video Abstract.
Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous and common upper airway disease divided into various inflammatory endotypes. Recent epidemiological findings showed a T helper 2 (Th2)-skewed dominance in CRSwNP patients. Histone modification alterations can regulate transcriptional and translational expression, resulting in abnormal pathogenic changes and the occurrence of diseases. Trimethylation of histone H3 lysine 4 (H3K4me3) is considered an activator of gene expression through modulation of accessibility for transcription, which is closely related to CRSwNP. H3K4me3 levels in the human nasal epithelium may change under Th2-biased inflammatory conditions, resulting in exaggerated local nasal Th2 responses via the regulation of na&#239;ve CD4<sup>+</sup> T-cell differentiation. Here, we revealed that the level of SET and MYND domain-containing protein 3 (SMYD3)-mediated H3K4me3 was increased in NPs from Th2 CRSwNP patients compared with those from healthy controls. We demonstrated that SMYD3-mediated H3K4me3 is increased in human nasal epithelial cells under Th2-biased inflammatory conditions via S-adenosyl-L-methionine (SAM) production and further found that the H3K4me3<sup>high</sup> status of insulin-like growth factor 2 (IGF2) produced in primary human nasal epithelial cells could promote na&#239;ve CD4<sup>+</sup> T-cell differentiation into Th2 cells. Moreover, we found that SAM production was dependent on the c-Myc/methionine adenosyltransferase 2A (MAT2A) axis in the nasal epithelium. Understanding histone modifications in the nasal epithelium has immense potential utility in the development of novel classes of therapeutics targeting Th2 polarization in Th2 CRSwNP. Video Abstract.
Alterations in progranulin (PGRN) expression are associated with multiple neurodegenerative diseases (NDs), including frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), and lysosomal storage disorders (LSDs). Recently, the loss of PGRN was shown to result in endo-lysosomal system dysfunction and an age-dependent increase in the expression of another protein associated with NDs, glycoprotein non-metastatic B (GPNMB).
Chromobox protein homolog 7 (CBX7), a member of the Polycomb repressor complex, is a potent epigenetic regulator and gene silencer. Our group has previously reported that CBX7 functions as a tumor suppressor in ovarian cancer cells and its loss accelerated formation of carcinomatosis and drove tumor progression in an ovarian cancer mouse model. The goal of this study is to identify specific signaling pathways in the ovarian tumor microenvironment that down-regulate CBX7. Given that adipocytes are an integral component of the peritoneal cavity and the ovarian tumor microenvironment, we hypothesize that the adipose microenvironment is an important regulator of CBX7 expression.
The World Health Organization (WHO) prioritizes pneumococcal disease as a vaccine-preventable disease and recommends the inclusion of pneumococcal conjugate vaccines (PCV) in national immunization programs worldwide. However, PCV is not included in the National Immunization Program in China and has low vaccination coverage due to its high cost. To address this, Weifang City implemented an innovative strategy for a 13-valent PCV (PCV13) on June 1, 2021. This strategy aimed to provide one dose of PCV13 free of charge for children aged 6 months to 2 years in registered households and to adopt a commercial insurance model with one dose of PCV13 free of charge in 2023 for children over 2 years old. The Health Commission of Weifang and other departments conducted a comprehensive investigation and considered various factors, such as vaccine effectiveness, safety, accessibility, vaccine price, and immunization schedules, for eligible children (under 5 years old). Stakeholder opinions were also solicited before implementing the policy. The Commission negotiated with various vaccine manufacturers to maximize its negotiating power and reduce vaccine prices. The implementation plan was introduced under the Healthy Weifang Strategy. Following the implementation of this strategy, the full course of vaccination coverage increased significantly from 0.67 to 6.59%. However, vaccination coverage is still lower than that in developed countries. Weifang's PCV13 vaccination innovative strategy is the first of its kind in Chinese mainland and is an active pilot of non-immunization program vaccination strategies. To further promote PCV13 vaccination, Weifang City should continue to implement this strategy and explore appropriate financing channels. Regions with higher levels of economic development can innovate the implementation of vaccine programs, broaden financing channels, improve accessibility to vaccination services, and advocate for more localities to incorporate PCV13 into locally expanded immunization programs or people-benefiting projects. A monitoring and evaluation system should also be established to evaluate implementation effects.
Protein complex assembly facilitates the combination of individual protein subunits into functional entities, and thus plays a crucial role in biology and biotechnology. Recently, we developed quasi-twodimensional, silicon-based compartmental biochips that are designed to study and administer the synthesis and assembly of protein complexes. At these biochips, individual protein subunits are synthesized from locally confined high-density DNA brushes and are captured on the chip surface by molecular traps. Here, we investigate single-gene versions of our quasi-twodimensional synthesis systems and introduce the trap-binding efficiency to characterize their performance. We show by mathematical and computational modeling how a finite trap density determines the dynamics of protein-trap binding and identify three distinct regimes of the trap-binding efficiency. We systematically study how protein-trap binding is governed by the system's three key parameters, which are the synthesis rate, the diffusion constant and the trap-binding affinity of the expressed protein. In addition, we describe how spatially differential patterns of traps modulate the protein-trap binding dynamics. In this way, we extend the theoretical knowledge base for synthesis, diffusion, and binding in compartmental systems, which helps to achieve better control of directed molecular self-assembly required for the fabrication of nanomachines for synthetic biology applications or nanotechnological purposes.
Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease and one of the major causes of death in the global pig industry. PRRS virus (PRRSV) strains have complex and diverse genetic characteristics and cross-protection between strains is low, which complicates vaccine selection; thus, the current vaccination strategy has been greatly compromised. Therefore, it is necessary to identify effective natural compounds for the clinical treatment of PRRS. A small molecule library composed of 720 natural compounds was screened in vitro, and we found that Sanggenon C (SC) was amongst the most effective natural compound inhibitors of PRRSV infection. Compared with ribavirin, SC more significantly inhibited PRRSV infection at both the gene and protein levels and reduced the viral titres and levels of protein expression and inflammatory cytokine secretion to more effectively protect cells from PRRSV infection and damage. Mechanistically, SC inhibits activation of the NF-κB signalling pathway by promoting TRAF2 expression, thereby reducing PRRSV replication. In conclusion, by screening natural compounds, we found that SC suppresses PRRSV infection by regulating the TRAF2/NF-κB signalling pathway. This study contributes to a deeper understanding of the therapeutic targets and pathogenesis of PRRSV infection. More importantly, our results demonstrate that SC has potential as a candidate for the treatment of PRRS.
ATP7B is a copper-transporting protein that contributes to the chemo-resistance of human cancer cells. It remains unclear what the molecular mechanisms behind ATP7B are in cancer, as well as its role in human pan-cancer studies.
To investigate the expression of polo-like kinase 1 protein (PLK1) and its phosphorylation level (p-PLK1) in extranodal NK/T cell lymphoma (NKTCL) and their correlation with clinical characteristics and prognosis.
Extracellular amyloid-β (Aβ) plaques and intracellular aggregates of tau protein in form of neurofibrillary tangles (NFT) are pathological hallmarks of Alzheimer's disease (AD). The exact mechanism how these two protein aggregates interact in AD is still a matter of debate. Neuritic plaques (NP), a subset of Aβ plaques containing dystrophic neurites (DN), are suggested to be unique to AD and might play a role in the interaction of Aβ and tau. Quantifying NP and non-NP in postmortem brain specimens from patients with increasing severity of AD neuropathological changes (ADNC), we demonstrate that the total number of Aβ plaques and NP increase, while the number of non-NP stagnates. Furthermore, investigating the correlation between NP and NFT, we identified unexpected brain region-specific differences when comparing cases with increasingly more severe ADNC. In neocortical regions NFT counts increase in parallel with NP counts during the progression of ADNC, while this correlation is not observed in hippocampus. These data support the notion that non-NP are transformed into NP during the progression of ADNC and indicate that NP might drive cortical NFT formation. Next, using spatial transcriptomics, we analyzed the gene expression profile of the microenvironment around non-NP and NP. We identified an upregulation of neuronal systems and Ca-dependent event pathways around NP compared to non-NP. We speculate that the upregulation of these transcripts may hint at a compensatory mechanism underlying NP formation. Our studies suggest that the transformation of non-NP to NP is a key event in ADNC progression and points to regenerative failure as a potential driving force of this process.
Gain-of-function (GOF) variants give rise to increased/novel protein functions whereas loss-of-function (LOF) variants lead to diminished protein function. Experimental approaches for identifying GOF and LOF are generally slow and costly, whilst available computational methods have not been optimized to discriminate between GOF and LOF variants. We have developed LoGoFunc, a machine learning method for predicting pathogenic GOF, pathogenic LOF, and neutral genetic variants, trained on a broad range of gene-, protein-, and variant-level features describing diverse biological characteristics. LoGoFunc outperforms other tools trained solely to predict pathogenicity for identifying pathogenic GOF and LOF variants and is available at https://itanlab.shinyapps.io/goflof/ .
Endoplasmic reticulum stress (ER stress) is suggested to promote cardiomyocyte apoptosis and ultimately lead to ischemic injury. Inhibition of ER stress-induced apoptosis may be a therapeutic strategy for MI injury. Astragaloside-IV (AST) from <i>Astragalus membranaceus</i> (Fisch) Bge, was reported to have cardioprotective properties. In this study, we investigated the protective effect of AST on cardiomyocytes against hypoxia injury by regulating ER stress and inhibiting apoptosis. H9c2 cardiomyocytes were divided into three groups, normal group, hypoxia group and AST group. Cell viability was determined by CCK-8 assay. Intracellular reactive oxygen species (ROS) production was detected by DCFH-DA (2,7- dichloro-dihydrofluorescein diacetate) florescent staining. The study showed that AST treatment could significantly increase the cell viability of H9c2 cells exposed to hypoxia. Furthermore, AST could restrain cell apoptosis and decrease the production of ROS. Compared with normal group, the protein levels of Bax, caspase-3, caspase-9, GRP78, p-eIF2&#945;, and CHOP were enhanced in the hypoxia group, whereas the protein level of Bcl-2 was dramatically reduced. Compared with hypoxia group, AST markedly inhibited the phosphorylation of eIF2&#945; and the expression of caspase-3, caspase-9 and CHOP, and promoted the protein expression of Bcl-2. Thus, AST can inhibit the ER stress-mediated apoptosis, partly through the eIF2&#945;/CHOP pathway suppression to inhibit ER stress.
This review aimed to validate the therapeutic potential of Bushen Tiansui decoction (BSTSD), a traditional Chinese formulation, in treating delayed union of fractures. Comprehensive database searches identified randomized controlled trials up to September 13, 2022, assessing BSTSD's efficacy in delayed fracture healing. Outcomes were bone metabolism indexes and Harris hip scores. Quality and risk assessments were conducted using the Cochrane Collaboration's tools. Data were analyzed using RevMan software, with sensitivity analysis through Stata. BSTSD significantly improved bone GLA protein (SMD=1.76, P<0.00001) and alkaline phosphatase (SMD=1.31, P<0.00001). Additionally, Harris hip scores for pain, function, deformity, and motion showed marked improvement. BSTSD treatment also demonstrated enhanced clinical efficiency (RR=1.27, P<0.00001) with fewer complications. Sensitivity analyses indicated consistent results. BSTSD shows promise in treating delayed fracture unions, yet conclusions necessitate further high-quality research for validation.
NUT carcinoma (NC) is one of the most common types of undifferentiated carcinomas affecting young adults with a dismal prognosis. NUT carcinomas often involve chromosomal translocations, leading to the production of BRD4-NUT fusion protein that generates large domains of hyperactive chromatin and activates oncogenic gene expression. Bromodomain and extraterminal domain (BET) bromodomain inhibitors offer a direct means to block BRD4-mediated gene activation but have shown limited clinical efficacy in patients. In this issue of Cancer Research, Huang and colleagues report an unexpected discovery of a synthetic lethal NC dependency on Polycomb repressive complex 2 (PRC2)-mediated gene repression, including EZH2, the catalytic subunit of PRC2. EZH2 is highly expressed in NC patient tumors and a specific inhibitor of its methyltransferase activity, tazemetostat, exhibits potent antitumor cell activity. While the repressed and activated chromatin domains in NC cells are distinct, the resultant gene expression changes exhibit convergent features, including dysregulation of CDKN2A and the E2F-RB1 axis. As a result, combined treatment of NC tumors with tazemetostat and the BET inhibitor mivebresib produces marked antitumor therapeutic synergy in vitro and in vivo, associated with enhanced suppression of RB1 function through convergent remodeling of NC gene expression. This study advances epigenetic cooperativity as a distinct mode of gene expression dysregulation in NC and nominates a compelling combination epigenetic strategy for investigation in clinical trials for patients. See related article by Huang et al., p. 3956.
An international collaborative study was run within the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the Commission of the European Union to establish replacement batches for European Pharmacopoeia (Ph. Eur.) Heparin Low-Molecular-Mass (LMM) for calibration Chemical Reference Substance batch 3 (CRS3) used for the characterisation of LMM heparins by high performance size-exclusion chromatography. Two candidate batches (A, cCRS4 and B, cCRS5) were filled using the same material as the existing official calibrants, adopted with either an assigned number-average molecular mass (Mna) or a broad standard table (BST). Fifteen laboratories evaluated the suitability of these candidate batches for use as calibrants with the pharmacopoeial dual refractive index/ultraviolet (RI/UV) detector calibration method, as well as with a modified mobile phase and the BST calibration method. Seven preparations of LMM heparin were tested. The results confirmed that the proposed batches are suitable for use with the same characteristic Mna as CRS3 and with the BST established for the World Health Organization (WHO) 2<sup>nd</sup> International Standard (IS). The BST calibration method gave comparable results to the RI/UV method, while showing better reproducibility, being easier to perform and requiring no calibrant with UV absorbance. The modified mobile phase had no impact on the calculated values while improving separation between the calibrant and salt peaks. The two candidate batches were adopted as Ph. Eur. Heparin LMM for calibration CRS batches 4 and 5, respectively, with the assigned Mna value of 3800 and a BST. In anticipation of the depletion of the calibrant required for use with the RI/UV method, and taking into account the unlikely procurement of a new lot of suitable starting material, it was recommended to include the BST method in Ph. Eur. monograph <i>0828, Heparins, low-molecular-mass</i>. In order to improve peak separation, it was also recommended to include the use of ammonium acetate solution as mobile phase in the monograph, both for the Ph. Eur. RI/UV and the proposed BST calibration methods. Further to this study, Ph. Eur. monograph <i>0828</i> was revised to replace the RI/UV method by the BST method. This contributed to the harmonisation of methods across regions, thereby facilitating a concerted global action for the development and establishment of the next batches of calibrants for the quality control of LMM heparins.
By controlling the passage of small molecules across lipid bilayers, membrane transporters influence not only the uptake and efflux of nutrients, but also the metabolic state of the cell. With more than 450 members, the Solute Carriers (SLCs) are the largest transporter super-family, clustering into families with different substrate specificities and regulatory properties. Cells of different types are, therefore, able to tailor their transporter expression signatures depending on their metabolic requirements, and the physiological importance of these proteins is illustrated by their mis-regulation in a number of disease states. In cancer, transporter expression is heterogeneous, and the SLC family has been shown to facilitate the accumulation of biomass, influence redox homeostasis, and also mediate metabolic crosstalk with other cell types within the tumour microenvironment. This Review explores the roles of membrane transporters in physiological and malignant settings, and how these roles can affect drug response, through either indirect modulation of sensitivity or the direct transport of small-molecule therapeutic compounds into cells.
Nonalcoholic steatohepatitis (NASH) is emerging as the primary driver of liver disease&#8209;induced fibrosis. The imperative need for noninvasive biomarkers to ascertain disease progression stage is evident. The present study elucidated the biological roles of hub genes that could potentially serve as diagnostic markers for NASH. Using an in&#160;vivo approach, C57BL/6J mice were subjected to a high&#8209;fat and fructose diet (HFFD) for 6, 10, 14, 18 or 22&#160;weeks. Serological biochemical indices were assessed and liver specimens were obtained to identify potential markers linked to the NASH process, employing a comprehensive strategy that combined transcriptomic and histopathological analyses. The HFFD regimen induced hyperlipidemia, obesity and insulin resistance, progressively culminating in NASH with fibrosis over time. The transcriptomic analyses indicated temporal patterns of pivotal gene sets intricately connected to NASH progression, which encompassed processes such as glucose homeostasis, inflammatory responses, reactive oxygen species&#8209;mediated damage, lipid metabolism disruptions and the formation of fibrotic tissue. Among these genes, Serpine1 and Mmp9 demonstrated promising diagnostic potential for NASH, with their intrahepatic mRNA expression levels serving as robust indicators. Moreover, the levels of PAI&#8209;1 (encoded by the Serpine1 gene) and MMP&#8209;9 in the serum of mice demonstrated a parallel increase with the duration of HFFD intervention. <i>In&#160;vitro</i> experiments utilizing HepG2 cells further validated these findings, demonstrating a significant elevation in the protein expression levels of both PAI&#8209;1 and MMP&#8209;9 upon exposure to free fatty acids, in agreement with the results of the animal study. Consequently, PAI&#8209;1 and MMP&#8209;9 are promising noninvasive biomarkers for assessing the progression of NASH.
The occurrence of herpes zoster (HZ) correlates with declining memory T cells that had responded to earlier infection with varicella-zoster virus (VZV). There are especially lower T cell responses to the single immunodominant VZV protein glycoprotein E (gE) in people over 50 years of age, although antibody responses to VZV persist. Therefore, a live attenuated zoster vaccine (ZVL) aimed at restoring T cell responses was developed. Surprisingly, a recombinant zoster vaccine (RZV) consisting of gE combined with the AS01B adjuvant system proved superior in efficacy and durability. In this issue of the JCI, Laing, Ford, and colleagues showed that both vaccines stimulated preimmunization naive CD4+ T cells, not just memory CD4+ T cells, to gE, and recruited these naive responses into the overall memory response. However, compared with ZVL, RZV stimulated this response to a much greater degree. These results will help guide development of more effective and durable vaccines for older individuals.
Sarcoidosis is a disease of unknown etiology in which granulomas form throughout the body and is typically treated with glucocorticoids, but there are no approved steroid-sparing alternatives. Here, we investigated the mechanism of granuloma formation using single-cell RNA-Seq in sarcoidosis patients. We observed that the percentages of triggering receptor expressed on myeloid cells 2-positive (TREM2-positive) macrophages expressing angiotensin-converting enzyme (ACE) and lysozyme, diagnostic makers of sarcoidosis, were increased in cutaneous sarcoidosis granulomas. Macrophages in the sarcoidosis lesion were hypermetabolic, especially in the pentose phosphate pathway (PPP). Expression of the PPP enzymes, such as fructose-1,6-bisphosphatase 1 (FBP1), was elevated in both systemic granuloma lesions and serum of sarcoidosis patients. Granuloma formation was attenuated by the PPP inhibitors in in vitro giant cell and in vivo murine granuloma models. These results suggest that the PPP may be a promising target for developing therapeutics for sarcoidosis.
Non‑coding RNAs with a length of 22‑24 nt are known as microRNAs (miRNAs or miRs), which are critical regulators of protein translation. Over the past 10 years, the roles of miRNAs have been extensively investigated in several human cancer types. There is evidence to indicate that miRNAs regulate gene expression by concentrating on a number of substances that have an impact on the physiology and development of cancer cells. Thus, miRNAs as regarded as effective targets for further studies on the design of novel therapeutic strategies. Hepatocellular carcinoma, breast, prostate, and ovarian cancer are only a few of the cancers that miR‑124 suppresses. Furthermore, it has been shown that miR‑124 is linked to the development and aggressive spread of malignancies. The aim of the present review was to clarify and highlight the role of miR‑124 in the development and progression of cancer, emphasizing recent research illustrating how miR‑124 has been used as a therapeutic agent against cancer, as well as the diagnostic potential, regulatory mechanisms and clinical application of miR‑124.
The high catalytic activity and specificity of enzymes can be used to pretreat biomass. Herein, the resourceful, reproducible, cheap, and crude protein-rich cottonseed meal (CM) is selected as a precursor and the protease in the K2CO3-KHCO3 buffer solution is used as the enzyme degradation substance to pretreat CM. The crude protein content is significantly reduced by the protease degradation, and, meanwhile, it results in a looser and porous structure of CM. What is more, it significantly reduces the amount of activator. In the subsequent carbonization process, the K2CO3-KHCO3 in the buffer solution is also used as an activating agent (the mass ratio of CM to activator is 2:1), and after carbonization, the O, S, and N doped porous carbon is obtained. The optimized PCM-800-4 exhibits high heteroatom contents and a hierarchical porous structure. The specific capacitance of the prepared porous carbon reaches up to 233 F g-1 in 6M KOH even when 10 mg of active material is loaded. In addition, a K2CO3-KHCO3/EG based gel electrolyte is prepared and the fabricated flexible capacitor exhibits an energy density of 15.6 Wh kg-1 and a wide temperature range (-25 to 100 °C). This study presents a simple enzymatic degradation and reduced activator dosage strategy to prepare a cottonseed meal derived carbon material and looks forward to preparing porous carbon using other biomass.
A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing <i>Caenorhabditis elegans</i> somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.
Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, in vitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.
Human antigen R (HuR) is a universally expressed RNA-binding protein that plays an essential role in governing the fate of mRNA transcripts. Accumulating evidence indicated that HuR is involved in the development and functions of several cell types. However, its role in cerebral ischemia/reperfusion injury (CIRI) remains unclear. In this study, we found that HuR was significantly upregulated after CIRI. Moreover, we found that silencing HuR could inhibit the inflammatory response of microglia and reduce the damage to neurons caused by oxygen-glucose deprivation/reperfusion treatment. In vivo, we found that microglial HuR deficiency significantly ameliorated CIRI and reduced NLRP3-mediated inflammasome activation. Mechanistically, we found that HuR could regulate NLRP3 mRNA stability by binding to the AU-rich element (ARE) region within the 3' untranslated region (UTR) of NLRP3 mRNA. In addition, we found that the upregulation of HuR was dependent on the upregulation of NADPH oxidase-mediated ROS accumulation. Collectively, our studies revealed that HuR could regulate NLRP3 expression and that HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke. Targeting HuR may be a novel therapeutic strategy for cerebral ischemic stroke treatment.
Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients.
Nutritional biomarkers like serum prealbumin, transferrin, retinol-binding protein (RBP), C-reactive protein (CRP), leptin, and insulin-like growth factor 1 (IGF1) have the inherent ability to diagnose undernutrition objectively before it is clinically manifested. The primary objective of the study was to evaluate the diagnostic efficacy of the specific nutritional biomarkers in predicting post-operative complications.
Systems biology utilizes computational approaches to examine an array of biological processes, such as cell signaling, metabolomics and pharmacology. This includes mathematical modeling of CAR T cells, a modality of cancer therapy by which genetically engineered immune cells recognize and combat a cancerous target. While successful against hematologic malignancies, CAR T cells have shown limited success against other cancer types. Thus, more research is needed to understand their mechanisms of action and leverage their full potential. In our work, we set out to apply information theory on a mathematical model of NFκB signaling initiated by the CAR following antigen encounter. First, we estimated channel capacity for CAR-4-1BB-mediated NFκB signal transduction. Next, we evaluated the pathway's ability to distinguish contrasting "low" and "high" antigen concentration levels, depending on the amount of variability in protein concentrations. Finally, we assessed the fidelity by which NFκB activation reflects the encountered antigen concentration, depending on the prevalence of antigen-positive targets in tumor population. We found that in most scenarios, fold change in the nuclear concentration of NFκB carries a higher channel capacity for the pathway than NFκB's absolute response. Additionally, we found that most errors in transducing the antigen signal through the pathway skew towards underestimating the concentration of encountered antigen. Finally, we found that disabling IKKβ deactivation could increase signaling fidelity against targets with antigen-negative cells. Our information-theoretic analysis of signal transduction can provide novel perspectives on biological signaling, as well as enable a more informed path to cell engineering.Kindly check and confirm whether the corresponding affiliation is correctly identified.this is correct.
Concerted atomic motions are requisite for sarcomere protein function and may become disrupted in HCM pathologies. Computational approaches such as molecular dynamics simulation can resolve such dynamics with unrivalled spatial and temporal resolution. This chapter describes methods to model structural and dynamical changes in biomolecules with HCM-associated perturbations.
Patient-specific modeling of atrial electrical activity enables the execution of simulations that can provide mechanistic insights and provide novel solutions to vexing clinical problems. The geometry and fibrotic remodeling of the heart can be reconstructed from clinical-grade medical scans and used to inform personalized models with detail incorporated at the cell- and tissue-scale to represent changes in image-identified diseased regions. Here, we provide a rubric for the reconstruction of realistic atrial models from pre-segmented 3D renderings of the left atrium with fibrotic tissue regions delineated, which are the output from clinical-grade systems for quantifying fibrosis. We then provide a roadmap for using those models to carry out patient-specific characterization of the fibrotic substrate in terms of its potential to harbor reentrant drivers via cardiac electrophysiology simulations.
Proteases serve essential roles in numerous biological processes and signaling cascades by cleaving their substrates in a restricted manner or via degradation. It is important to determine which proteins are protease substrates and where their cleavage sites are located to characterize the impact of proteolysis on the molecular mechanisms of their substrates. N-terminomics is a branch of proteomics that enriches the N-terminal sequence of proteins. A proteome-wide collection of these sequences has been broadly applied to comprehend proteolytic cascades and for genome annotation. Terminal Amine Isotopic Labeling of Substrates (TAILS) is a combined N-terminomics and proteomics technique that has been applied for protein N-terminal characterization and quantification of natural and neo-N-termini of proteins using liquid chromatography and tandem mass spectrometry (LC-MS/MS). TAILS uses negative selection to enrich both original mature protein N-termini and neo-N-termini produced from proteolysis in a proteome labeled with isotopic tags. This approach has been applied to the investigation of protease function and substrate identification in cell culture systems, animal disease models, and, most recently, clinical samples such as blood and tumor tissues from cancer patients.
In 2001, the release of the first draft of the human genome marked the beginning of the Big Data era for biological sciences. Since then, the complexity of datasets generated by laboratories worldwide has increased exponentially. Public repositories such as the Protein Data Bank, which has exceeded the 200000 entries in 2023, have been instrumental not only to collect, organize, and distill this enormous research output but also to promote further research enterprises. The achievements of artificial intelligence programs such as AlphaFold would not have been possible without the collective efforts of countless researchers who made their work publicly available. Here, I provide a practical, but far from exhaustive, list of resources useful to investigate protease function.
Introducing an N-linked glycosylation motif into recombinant proteins at specific sites is a useful tool in probing protein-protein interactions and epitope mapping. Due to their large size, a new N-glycan can block protein-protein interactions if it is introduced by site-directed mutagenesis on the same face as a ligand or antibody binding site. Recombinant mutant proteins containing these engineered glycans can then be studied using binding or functional assays to establish if the new glycan causes steric hindrance, prevents an important protein-protein interaction, or blocks (auto)antibody binding. In this book chapter, we provide guides and protocols for inserting engineered glycans, including how to use AlphaFold models to choose amino acid residues on the surface of protein domains that are suitable for mutagenesis into N-linked glycosylation motifs as well as protocols for site-directed mutagenesis and recombinant protein expression of the N-glycan variants.
ADAMTS8 (A Disintegrin-like and Metalloproteinase with Thrombospondin motifs 8) is a secreted zinc-dependent metalloproteinase whose expression is downregulated in a variety of solid tumors. Xenografts expressing high levels of ADAMTS8 have a poor capacity to invade and migrate in nude mice. While this data highlights a beneficial, anti-cancerogenic role of ADAMTS8, the mechanism behind this activity is still not fully elucidated. So far, the only reported substrate for ADAMTS8 is osteopontin (OPN), an extracellular matrix protein widely implicated in multiple steps of cancer progression, albeit, similar to other ADAMTS family members, it is very likely that ADAMTS8 cleaves a variety of substrates. The availability of purified ADAMTS8 may enlighten the biological role of this metalloproteinase.Here we describe methods for expression and purification of recombinant ADAMTS8 in HEK293T cells as well as a convenient assay to test ADAMTS8 proteolytic activity using OPN as a substrate.
Osteopontin (OPN) is a matricellular protein containing binding sites for a variety of ligands including an RGD sequence for binding to &#945;<sub>v</sub>&#946;<sub>3</sub> integrins. OPN is a conserved substrate for thrombin, the effector protease of the coagulation cascade. Thrombin cleaves OPN at a single site revealing new functionalities such as a previously cryptic &#945;<sub>4</sub>&#946;<sub>1</sub> and &#945;<sub>9</sub>&#946;<sub>1</sub> integrin-binding site. That integrin-binding site is abolished upon treatment with a basic carboxypeptidase. The thrombin cleavage of OPN has been demonstrated to play a role in regulating tumor growth.This report describes methods for production of full-length OPN as well as the enzymatically cleaved OPN fragments resulting from thrombin and carboxypeptidase treatments. Quantification procedures for the various OPN proteins are described as well as functional assays on mouse melanoma and myeloid cell lines.
Silencing expression with short interfering RNA (siRNA) is a rapid and cost-effective way to analyze the involvement of target genes in a range of biological processes. Here we describe isolation of primary human monocytes from peripheral blood and their in vitro differentiation to macrophages, followed by electroporation with siRNA to silence expression of a disintegrin and metalloproteinase 17 (ADAM17). This enables evaluation of ADAM17's role in cleaving transmembrane proteins, such as its prototypic substrate tumor necrosis factor (TNF), by enzyme-linked immunosorbent assay (ELISA), flow cytometry, or immunoblotting.
The membrane-bound matrix metalloproteinase 14 (MMP14, also known as MT1-MMP) plays important roles in the remodeling of the extracellular matrix during various cellular processes such as cancer metastasis, angiogenesis, and wound healing through its proteolytic activity. There are no known MMP14-specific inhibitors to date, and hence identification of MMP14-specific inhibitors will be beneficial for finding potential therapeutics for various diseases, including cancer and inflammation. High-throughput screening (HTS) assays have become a common way to search for new small compounds, peptides, and natural products. Enzymatic assays are highly amenable to HTS because most enzyme activities are quantifiable with the effect of many small molecules of interest on a specific target enzyme. Here, we describe a fluorescence-based enzymatic assay that can be applied as a large-scale HTS and a follow-up enzyme kinetics assay to find MMP14-specific inhibitors.
Targeting dysregulated protease expression and/or abnormal substrate proteolysis, highly selective inhibition of pathogenic proteases by monoclonal antibodies (mAbs) presents an attractive therapeutic approach for the treatment of diseases including cancer. Herein, we report a functional selection method for protease inhibitory mAbs by periplasmic co-expression of three recombinant proteins-a protease of interest, an antibody Fab library, and a modified β-lactamase TEM-1. We validate this approach by isolation of highly selective and potent mAbs inhibiting human matrix metalloproteinase 9 (MMP9).
The yeast surface display platform provides a powerful approach for screening protein diversity libraries to identify binders with an enhanced affinity toward a binding partner. Here, we describe an adaptation of the approach to identify binders with enhanced specificity toward one among multiple closely related binding partners. Specifically, we describe methods for engineering selective matrix metalloproteinase (MMP) inhibitors via yeast surface display of a tissue inhibitor of metalloproteinase (TIMP) diversity library coupled with a counter-selective screening strategy. This protocol may also be employed for developing selective protein binders or inhibitors toward other targets.
Protease inhibitors of the alpha-macroglobulin family (&#945;M) have a unique mechanism that allows them to trap proteases that is dependent not on the protease's class, but rather on its cleavage specificity. Proteases trigger a conformational change in the &#945;M protein by cleaving within a "bait region," resulting in the sequestering of the protease inside the &#945;M molecule. This nonspecific inhibitory mechanism appears to have arisen early in the &#945;M family, and the broad protease-trapping capacity that it allows may play a role in pathogen defense.Human &#945;<sub>2</sub>-macroglobulin (A2M) is a tetrameric &#945;M whose bait region is permissive to cleavage by most proteases, making it a broad-spectrum protease inhibitor. Recent work has demonstrated that the inhibitory capacity of A2M derives directly from its bait region sequence: modifying the bait region sequence to introduce or remove protease cleavage sites will modify A2M's inhibition of the relevant proteases accordingly. Thus, changing the amino acid sequence of the bait region presents an effective avenue for protein engineering of new protease inhibitors if the substrate specificity of the target protease is known. The design of new A2M-based protease inhibitors with tailored inhibitory capacities has potential applications in basic research and the clinic. In this chapter, we describe the general approach and considerations for the bait region engineering of A2M.
The tripartite interaction motif (TRIM) family of proteins is known for their antiviral activity through different mechanisms, such as interfering with viral components, regulating immune responses, and participating in autophagy-mediated defense pathways. In this study, we investigated the role of tripartite interaction motif 26 (TRIM26), which is encoded by a major histocompatibility complex (MHC) gene, in regulating Epstein-Barr virus (EBV) infection of nasopharyngeal epithelial cells. We found that TRIM26 expression was induced upon EBV infection and that it indirectly targeted EphA2, a crucial epithelial receptor for EBV entry. Our results showed that TRIM26 interacted with heat shock protein 90-beta (HSP-90β) and promoted its polyubiquitination, which led to its degradation via the proteasome pathway. This, in turn, affected EphA2 integrity and suppressed EBV infection. These findings suggest that TRIM26 could be a valuable target for developing therapeutic interventions against EBV infection and its associated pathogenesis.
Urea cycle disorders (UCDs) are a group of rare inborn diseases caused by a deficiency in one of the six enzymes or one of the two transporters involved in the urea cycle. The most common biochemical feature is elevated blood ammonia levels, which can be toxic at high levels, especially to the brain and may manifest as encephalopathy if left untreated. Glycerol phenylbutyrate (GPB) is currently approved for use in the USA and Europe for patients of all ages with UCD who cannot be managed with protein restriction and/or amino acid supplementation alone. This article presents the author's experience in different exemplary settings and depicts the most efficient management of UCDs with GPB.
Enhancing protein thermal stability is important for biomedical and industrial applications as well as in the research laboratory. Here, we describe a simple machine-learning method which identifies amino acid substitutions that contribute to thermal stability based on comparison of the amino acid sequences of homologous proteins derived from bacteria that grow at different temperatures. A key feature of the method is that it compares the sequences based not simply on the amino acid identity, but rather on the structural and physicochemical properties of the side chain. The method accurately identified stabilizing substitutions in three well-studied systems and was validated prospectively by experimentally testing predicted stabilizing substitutions in a polyamine oxidase. In each case, the method outperformed the widely used bioinformatic consensus approach. The method can also provide insight into fundamental aspects of protein structure, for example, by identifying how many sequence positions in a given protein are relevant to temperature adaptation.
In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10<sup>vWA</sup> complex at 2.17 &#197; resolution and of the SAP05-SPL5<sup>ZnF</sup> complex at 2.20 &#197; resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two &#946;-sheets, loops, and &#945;-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.
Prokaryotic viruses, also known as bacteriophages, play crucial roles in regulating microbial communities and have the potential for phage therapy applications. Accurate prediction of phage-host interactions is essential for understanding the dynamics of these viruses and their impacts on bacterial populations. Numerous computational methods have been developed to tackle this challenging task. However, most existing prediction models can be constrained due to the substantial number of unknown interactions in comparison to the constrained diversity of available training data. To solve the problem, we introduce a model for prokaryotic virus host prediction with graph contrastive augmentation (PHPGCA). Specifically, we construct a comprehensive heterogeneous graph by integrating virus-virus protein similarity and virus-host DNA sequence similarity information. As the backbone encoder for learning node representations in the virus-prokaryote graph, we employ LGCN, a state-of-the-art graph embedding technique. Additionally, we apply graph contrastive learning to augment the node representations without the need for additional labels. We further conducted two case studies aimed at predicting the host range of multi-species phages, helping to understand the phage ecology and evolution.
The matrix metalloproteinase MMP9 influences cellular morphology and function, and plays important roles in organogenesis and disease. It exerts both protective and deleterious effects in renal pathology, depending upon its specific substrates. To explore new functions for MMP9 in kidney cysts formation and disease progression, we generated a mouse model by breeding juvenile cystic kidney (jck) mice with MMP9 deficient mice. Specifically, we provide evidence that MMP9 is overexpressed in cystic tissue where its enzymatic activity is increased 7-fold. MMP9 deficiency in cystic kidney worsen cystic kidney diseases by decreasing renal function, favoring cyst expansion and fibrosis. In addition, we find that periostin is a new critical substrate for MMP9 and in its absence periostin accumulates in cystic lining cells. As periostin promotes renal cyst growth and interstitial fibrosis in polycystic kidney diseases, we propose that the control of periostin by MMP9 and its associated intracellular signaling pathways including integrins, integrin-linked kinase and focal adhesion kinase confers to MMP9 a protective effect on the severity of the disease.
Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters in plasma from high density lipoprotein (HDL) to very low density lipoprotein and low density lipoprotein. Loss-of-function variants in the CETP gene cause elevated levels of HDL cholesterol. In this study, we have determined the functional consequences of 24 missense variants in the CETP gene. The 24 missense variants studied were the ones reported in the Human Gene Mutation Database and in the literature to affect HDL cholesterol levels, as well as two novel variants identified at the Unit for Cardiac and Cardiovascular Genetics, Oslo University Hospital in subjects with hyperalphalipoproteinemia.
Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.
Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, we carried out a bioinformatics-based investigation of cytb561 family members from nine species representing eight insect orders. We performed a phylogenetic analysis to classify insect cytb561 ortholog groups. We then conducted sequence analyses and analyzed protein models to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. Our study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. We found that CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Our investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved "KXXXXKXH" non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects.
Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors. Here, we describe an approach using platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) to enable exogenous protein expression in human and rat platelets. Within the library of mRNA-LNP tested, exogenous protein expression did not require nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. We expect this technology will expand the therapeutic potential of platelets.
Mitochondrial antiviral signaling protein (MAVS)-mediated cytosolic RNA sensing plays a central role in tumor immunogenicity. However, the effects of host MAVS signaling on antitumor immunity remain unclear. Here, we demonstrate that the host MAVS pathway supports tumor growth and impairs antitumor immunity, whereas MAVS deficiency in dendritic cells (DCs) promotes tumor-reactive CD8<sup>+</sup> T cell responses. Specifically, CD8<sup>+</sup> T cell priming capacity was enhanced by MAVS ablation in a type I interferon-independent, but IL-12-dependent, manner. Mechanistically, loss of the RIG-I/MAVS cascade activated the noncanonical NF-&#954;B pathway and in turn induced IL-12 production by DCs. MAVS-restrained IL-12 promoted cross-talk between CD8<sup>+</sup> T cells and DCs, which was licensed by IFN-&#947;. Moreover, ablation of host MAVS sensitized tumors to immunotherapy and attenuated radiation resistance, thereby facilitating the maintenance of effector CD8<sup>+</sup> T cells. These findings demonstrate that the host MAVS pathway acts as an immune regulator of DC-driven antitumor immunity and support the development of immunotherapies that antagonize MAVS signaling in DCs.
Differentiation between benign and malignant thyroid nodules has been a challenge in clinical practice. Exploring a novel biomarker to determine the malignancy of thyroid nodules has important implications. We semi-quantitatively determined the DNA methylation levels of four CpG sites located at the gene body of HYAL1 in formalin-fixed paraffin-embedded (FFPE) tissue samples from 190 early-stage papillary thyroid cancer (PTC) cases and 190 age- and gender-matched subjects with benign thyroid nodule (BTN). HYAL1 expression was evaluated by immunohistochemical (IHC) staining in another cohort of 55 PTC and 55 matched BTN cases. Covariates-adjusted odds ratios (ORs) for 10% increased methylation were calculated by binary logistic regression. A 165 bp amplicon covering four CpG sites at the second exon of HYAL1 gene was designed. After adjusted for all covariates, higher methylation level of HYAL1_CpG_3,4 in the FFPE tissue was associated with PTC (OR per 10% increased methylation=1.53, p=0.025), even with stage І PTC (OR per 10% increased methylation=1.58, p=0.021). Hypermethylation of HYAL1_CpG_3,4 had a significant association with early-stage PTC in the females (OR per 10% increased methylation=1.60, p=0.028) rather than in the males. Besides, we found the higher expression of HYAL1 protein in PTC than that in BTN patients (IHC score: 2.3 vs. 0.5, p=1.00E-06). Our study suggested altered methylation and expression of HYAL1 could be a novel and potential biomarker in distinguishing malignant and benign thyroid nodules.
Pancreatic ductal adenocarcinoma (PDAC) is a challenging target for immunotherapy because it has an immunosuppressive tumor microenvironment. Neoadjuvant chemoradiotherapy can increase tumor-infiltrating lymphocyte (TIL) density, which may predict overall survival (OS). We hypothesized that adding programmed cell death protein 1 (PD-1) blockade to chemoradiotherapy would be well tolerated and increase TILs among patients with localized PDAC.
Fibroblast activation protein &#945; (FAP) is highly expressed on cancer-associated fibroblasts of epithelial-derived cancers. Breast, colon, and pancreatic tumors often show strong desmoplastic reactions, which result in a dominant presence of stromal cells. FAP has gained interest as a target for molecular imaging and targeted therapies. Single-domain antibodies (sdAbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and targeted therapy. <b>Methods:</b> We describe the generation, selection, and characterization of a sdAb against FAP. In mice, we assessed its imaging and therapeutic potential after radiolabeling with tracer-dose <sup>131</sup>I and <sup>68</sup>Ga for SPECT and PET imaging, respectively, and with <sup>131</sup>I and <sup>225</sup>Ac for targeted radionuclide therapy. <b>Results:</b> The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [<sup>68</sup>Ga]Ga-DOTA-4AH29, [<sup>225</sup>Ac]Ac-DOTA-4AH29, and [<sup>131</sup>I]I-guanidinomethyl iodobenzoate (GMIB)-4AH29, displayed radiochemical purities exceeding 95% and effectively bound to recombinant human FAP protein and FAP-positive GM05389 human fibroblasts. These radiolabeled compounds exhibited rapid and specific accumulation in human FAP-positive U87-MG glioblastoma tumors, with low but specific uptake in lymph nodes, uterus, bone, and skin (&#8764;2-3 percentage injected activity per gram of tissue [%IA/g]). Kidney clearance of unbound [<sup>131</sup>I]I-GMIB-4AH29 was fast (&lt;1 %IA/g after 24&#8201;h), whereas [<sup>225</sup>Ac]Ac-DOTA-4AH29 exhibited slower clearance (8.07&#8201;&#177;&#8201;1.39 %IA/g after 24&#8201;h and 2.47&#8201;&#177;&#8201;0.18 %IA/g after 96&#8201;h). Mice treated with [<sup>225</sup>Ac]Ac-DOTA-4AH29 and [<sup>131</sup>I]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. <b>Conclusion:</b> [<sup>68</sup>Ga]Ga-DOTA-4AH29 and [<sup>131</sup>I]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [<sup>225</sup>Ac]Ac-DOTA-4AH29 and [<sup>131</sup>I]I-GMIB-4AH29 exhibit strong and sustained tumor targeting, resulting in dose-dependent therapeutic effects in FAP-positive tumor-bearing mice, albeit with kidney toxicity observed later for [<sup>225</sup>Ac]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.
Computing tools and machine learning models play an increasingly important role in biology and are now an essential part of discoveries in protein science. The growing energy needs of modern algorithms have raised concerns in the computational science community in light of the climate emergency. In this work, we summarize the different ways in which protein science can negatively impact the environment and we present the carbon footprint of some popular protein algorithms: molecular simulations, inference of protein-protein interactions, and protein structure prediction. We show that large deep learning models such as AlphaFold and ESMFold can have carbon footprints reaching over 100 tonnes of CO<sub>2</sub>e in some cases. The magnitude of these impacts highlights the importance of monitoring and mitigating them, and we list actions scientists can take to achieve more sustainable protein computational science.
Pancreatic cancer is a globally recognized highly aggressive malignancy, posing a significant threat to human health and characterized by pronounced heterogeneity. In recent years, researchers have uncovered that the development and progression of cancer are often attributed to the accumulation of somatic mutations within cells. However, cancer somatic mutation data exhibit characteristics such as high dimensionality and sparsity, which pose new challenges in utilizing these data effectively. In this study, we propagated the discrete somatic mutation data of pancreatic cancer through a network propagation model based on protein-protein interaction networks. This resulted in smoothed somatic mutation profile data that incorporate protein network information. Based on this smoothed mutation profile data, we obtained the activity levels of different metabolic pathways in pancreatic cancer patients. Subsequently, using the activity levels of various metabolic pathways in cancer patients, we employed a deep clustering algorithm to establish biologically and clinically relevant metabolic subtypes of pancreatic cancer. Our study holds scientific significance in classifying pancreatic cancer based on somatic mutation data and may provide a crucial theoretical basis for the diagnosis and immunotherapy of pancreatic cancer patients.
Designing 3D molecules with high binding affinity for specific protein targets is crucial in drug design. One challenge is that the atomic interaction between molecules and proteins in 3D space has to be taken into account. However, the existing target-aware methods solely model the joint distribution between the molecules and proteins, disregarding the binding affinities between them, which leads to limited performance. In this paper, we propose an explainable diffusion model to generate molecules that can be bound to a given protein target with high affinity. Our method explicitly incorporates the chemical knowledge of protein-ligand binding affinity into the diffusion model, and uses the knowledge to guide the denoising process towards the direction of high binding affinity. Specifically, an SE(3)-invariant expert network is developed to fit the Vina scoring functions and jointly trained with the denoising network, while the domain knowledge is distilled and conveyed from Vina functions to the expert network. An effective guidance is proposed on both continuous atom coordinates and discrete atom types by taking advantages of the gradient of the expert network. Experiments on the benchmark CrossDocked2020 demonstrate the superiority of our method. Additionally, an atom-level explanation of the generated molecules is provided, and the connections with the domain knowledge are established.
Aurora-A kinase interacting protein 1 (AURKAIP1) has been proved to take an intermediary role in cancer by functioning as a negative regulator of Aurora-A kinase. However, it remains unclear whether and how AURKAIP1 itself would directly engage in regulating malignancies. The expression levels of AURKAIP1 were detected in triple negative breast cancer (TNBC) by immunohistochemistry and western blots. The CCK8, colony formation assays and nude mouse model were conducted to determine cell proliferation whereas transwell and wound healing assays were performed to observe cell migration. The interaction of AURKAIP1 and DEAD-box helicase 5 (DDX5) were verified through co-immunoprecipitation and successively western blots. From the results, we found that AURKAIP1 was explicitly upregulated in TNBC, which was positively associated with tumor size, lymph node metastases, pathological stage and unfavorable prognosis. AURKAIP1 silencing markedly inhibited TNBC cell proliferation and migration in vitro and in vivo. AURKAIP1 directly interacted with and stabilized DDX5 protein by preventing ubiquitination and degradation, and DDX5 overexpression successfully reversed proliferation inhibition induced by knockdown of AURKAIP1. Consequently, AURKAIP1 silencing suppressed the activity of Wnt/β-catenin signaling in a DDX5-dependent manner. Our study may primarily disclose the molecular mechanism by which AURKAIP1/DDX5/β-catenin axis modulated TNBC progression, indicating that AURKAIP1 might serve as a therapeutic target as well as a TNBC-specific biomarker for prognosis.
Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.
Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters-electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Methacrylate phosphopeptides derived from 14-3-3-binding proteins irreversibly label 14-3-3σ via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibit pan-isoform binding of 14-3-3 proteins both in lysates and in extracellular media. Finally, we apply this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly binds to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.
Hyposalivation is a common complaint among the elderly, but no established treatment prevents age-induced hyposalivation. Platelet derivatives such as platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and plasma rich in growth factor (PRGF), are used widely in different areas of regenerative medicine to enhance the wound healing processes. This study examined whether the local injection of the supernatant of activated PRP (saPRP) into the salivary gland (SG) could help prevent aging-induced SG dysfunction and explored the mechanisms responsible for the protective effects on the SG hypofunction. The platelets were separated from the blood of male SD rats (220 ± 20 g). saPRP was manufactured by removing the fibrin clot after activating platelet with calcium ionophore 10 μM (A23187). The total protein and TGF-β1 levels were significantly higher in saPRP than in PRP. Human salivary gland epithelial cell(hSGEC) was treated with saPRP or PRP after senescence through irradiation. The significant proliferation of hSGEC was observed in saPRP treated group compared to irradiation only group and irradiation + PRP group. Cellular senescence, apoptosis, and inflammation significantly reduced in saPRP group. The SG function and structural tissue remodeling by the saPRP were investigated with naturally aged mice. The mice were divided into three groups: 3 months old (3 M), 22 months old (22 M), and 22 months old treated with saPRP (22 M + saPRP). Salivary flow rate and lag time were significantly improved in 22 M + saPRP group compared to 22 M group. The histologic examinations showed the significant proliferation of acinar cell in 22 M + saPRP group. The decrease of senescence, apoptosis, and inflammation observed by western blot in 22 M + saPRP group. The saPRP induced the proliferation of hSGECs, leading to a significant decrease in cellular senescence via decrease inflammation and apoptosis, in vitro. Moreover, the acini cells of the salivary gland were regenerated, and the salivary function increased in aged mice. These results showed that saPRP could be a treatment agent against aging-induced SG dysfunction.
Macromolecular complexes are essential functional units in nearly all cellular processes, and their atomic-level understanding is critical for elucidating and modulating molecular mechanisms. The Protein Data Bank (PDB) serves as the global repository for experimentally determined structures of macromolecules. Structural data in the PDB offer valuable insights into the dynamics, conformation, and functional states of biological assemblies. However, the current annotation practices lack standardised naming conventions for assemblies in the PDB, complicating the identification of instances representing the same assembly. In this study, we introduce a method leveraging resources external to PDB, such as the Complex Portal, UniProt and Gene Ontology, to describe assemblies and contextualise them within their biological settings accurately. Employing the proposed approach, we assigned standard names to over 90% of unique assemblies in the PDB and provided persistent identifiers for each assembly. This standardisation of assembly data enhances the PDB, facilitating a deeper understanding of macromolecular complexes. Furthermore, the data standardisation improves the PDB's FAIR attributes, fostering more effective basic and translational research and scientific education.
Hyperaccumulators are a group of plant species that accumulate high concentrations of one or more metal(loid)s in their above-ground tissues without showing any signs of toxicity. Several hyperaccumulating species belong to the Brassicaceae family, among them the Cd and Zn hyperaccumulator Noccaea praecox. In this paper, we present de novo transcriptome assembled from two naturally occurring N. praecox populations growing in (i) metal-enriched soil and (ii) soil non-contaminated with metals (control site). Total RNA was extracted from the leaves of both populations. We obtained 801,935,101 reads, which were successfully assembled and annotated. The resulting assembly contains 135,323 transcripts, with 103,396 transcripts (76.4%) annotated with at least one function and encoding 53,142 putative proteins. Due to its close relationship with the hyperaccumulating model species N. cearulescens, it will be possible to derive protein functions from sequence comparisons with this species. Comparisons will highlight common and differing pathways of metal acquisition, storage, and detoxification which will allow us to expand our knowledge of these processes.
Renin-angiotensin system inhibitors (RASi), particularly angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs), are commonly used in the treatment of hypertension and are recommended for kidney protection. Uncertainty remains about the effectiveness of RASi being used as first-line antihypertensive therapy on eGFR maintenance compared to its alternatives, especially for those with no or early-stage chronic kidney disease (CKD). We conducted a retrospective cohort study of 19,499 individuals (mean age 64.1, 43.5% males) from primary care in Singapore with 4.5 median follow-up years. The study cohort included newly diagnosed individuals with hypertension (whose eGFR was mainly in CKD stages G1-G2) and initiated on ACEIs, ARBs, beta-blockers (BBs), calcium channel blockers (CCBs) or diuretics (Ds) as first-line antihypertensive monotherapy. We compared the estimated glomerular filtration rate (eGFR) curve before/after the drug initiation over time of patients under different drug classes and analyzed the time to declining to a more advanced stage CKD. Inverse probability of treatment weighting (IPTW) was used to adjust for baseline confounding factors. Two key findings were observed. First, after initiating antihypertensive drugs, the eGFR almost maintained the same as the baseline in the first follow-up year, compared with dropping 3&#160;mL/min/1.73&#160;m<sup>2</sup> per year before drug initiation. Second, ARBs were observed to be slightly inferior to ACEIs (HR&#8201;=&#8201;1.14, 95% CI&#8201;=&#8201;(1.04, 1.23)) and other antihypertensive agents (HR&#8201;=&#8201;1.10, 95% CI&#8201;=&#8201;(1.01, 1.20)) in delaying eGFR decline to a more advanced CKD stage in the study population. Our results showed that initiating antihypertensive agents can significantly maintain eGFR for those newly diagnosed patients with hypertension. However, RASi may not be superior to other antihypertensive agents in maintaining eGFR levels for non-CKD or early stages CKD patients.
P-glycoprotein (P-gp), a membrane transport protein overexpressed in certain drug-resistant cancer cells, has been the target of numerous drug discovery projects aimed at overcoming drug resistance in cancer. Most characterized P-gp inhibitors bind at the large hydrophobic drug binding domain (DBD), but none have yet attained regulatory approval. In this study, we explored the potential of designing inhibitors that target the nucleotide binding domains (NBDs), by computationally screening a large library of 2.6 billion synthesizable molecules, using a combination of machine learning-guided molecular docking and molecular dynamics (MD). 14 of the computationally best-scoring molecules were subsequently tested for their ability to inhibit P-gp mediated calcein-AM efflux. In total, five diverse compounds exhibited inhibitory effects in the calcein-AM assay without displaying toxicity. The activity of these compounds was confirmed by their ability to decrease the verapamil-stimulated ATPase activity of P-gp in a subsequent assay. The discovery of these five novel P-gp inhibitors demonstrates the potential of in-silico screening in drug discovery and provides a new stepping point towards future potent P-gp inhibitors.
Reducing the loss of oligodendrocytes (OLs) is a major goal for neuroprotection after spinal cord injury (SCI). Therefore, the OL translatome was determined in Ribotag:Plp1-CreERT2 mice at 2, 10, and 42 days after moderate contusive T9 SCI. At 2 and 42 days, mitochondrial respiration- or actin cytoskeleton/cell junction/cell adhesion mRNAs were upregulated or downregulated, respectively. The latter effect suggests myelin sheath loss/morphological simplification which is consistent with downregulation of cholesterol biosynthesis transcripts on days 10 and 42. Various regulators of pro-survival-, cell death-, and/or oxidative stress response pathways showed peak expression acutely, on day 2. Many acutely upregulated OL genes are part of the repressive SUZ12/PRC2 operon suggesting that epigenetic de-silencing contributes to SCI effects on OL gene expression. Acute OL upregulation of the iron oxidoreductase Steap3 was confirmed at the protein level and replicated in cultured OLs treated with the mitochondrial uncoupler FCCP. Hence, STEAP3 upregulation may mark mitochondrial dysfunction. Taken together, in SCI-challenged OLs, acute and subchronic enhancement of mitochondrial respiration may be driven by axonal loss and subsequent myelin sheath degeneration. Acutely, the OL switch to oxidative phosphorylation may lead to oxidative stress that is further amplified by upregulation of such enzymes as STEAP3.
Acrolein, a respiratory irritant, induces systemic neuroendocrine stress. However, peripheral metabolic effects have not been examined. Male and female WKY rats were exposed to air (0 ppm) or acrolein (3.16 ppm) for 4 h, followed by immediate serum and liver tissue collection. Serum metabolomics in both sexes and liver transcriptomics in males were evaluated to characterize the systemic metabolic response. Of 887 identified metabolites, > 400 differed between sexes at baseline. An acrolein biomarker, 3-hydroxypropyl mercapturic acid, increased 18-fold in males and 33-fold in females, indicating greater metabolic detoxification in females than males. Acrolein exposure changed 174 metabolites in males but only 50 in females. Metabolic process assessment identified higher circulating free-fatty acids, glycerols, and other lipids in male but not female rats exposed to acrolein. In males, acrolein also increased branched-chain amino acids, which was linked with metabolites of nitrogen imbalance within the gut microbiome. The contribution of neuroendocrine stress was evident by increased corticosterone in males but not females. Male liver transcriptomics revealed acrolein-induced over-representation of lipid and protein metabolic processes, and pathway alterations including Sirtuin, insulin-receptor, acute-phase, and glucocorticoid signaling. In sum, acute acrolein inhalation resulted in sex-specific serum metabolomic and liver transcriptomic derangement, which may have connections to chronic metabolic-related diseases.
Circular RNAs (circRNAs) are linked to cancer, but it's still not clear what role they play in prostatic cancer. Through high-throughput sequencing, the goal of this study was to compare how circRNAs are expressed at different stages of prostate cancer. 12 patients attending the Department of Urology at the Second Affiliated Hospital of Nanchang University between June 2020 and October 2021 were used for RNA sequencing, and 14 patients were used for real-time fluorescent quantitative PCR (qRT-PCR). The expression profiles of prostate cancer circRNAs were constructed by sequencing with the help of next-generation high-throughput sequencing technology, and the differentially expressed circRNAs were analyzed by targeting microRNA (miRNA) loci and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the genes from which circRNAs originated. Finally, the expression of target circRNAs in two prostate tissues was verified by qRT-PCR. Following high-throughput sequencing, 13,047 circRNAs were identified, and 605 circRNAs with significant differential expression were identified, of which 361 circRNAs were up-regulated, and 244 circRNAs were down-regulated. Analysis of circRNA-originated genes using GO and the KEGG enrichment analysis showed that circRNA host genes can regulate and influence multiple signaling pathways in prostate cancer with important biological functions. And the circRNA-miRNA network was constructed. The highest number of differentially expressed circRNA-binding miRNAs were: hsa_circ_000 7582 (52), hsa_circ_000 6198 (37), hsa_circ_000 6759 (28), hsa_circ_000 5675 (25), and hsa_circ_000 2172 (22). Moreover, we further screened out the circRNA (hsa_circ_0005692) that was significantly differentially expressed and common to all groups and verified by qRT-PCR that the expression of the target circRNA (hsa_circ_0005692) was significantly downregulated in prostate cancer compared with benign prostatic hyperplasia (BPH) tissues.
As modern agricultural practices increase their use of chemical pesticides, it is inevitable that we will find a number of these xenobiotics within drinking water supplies and disseminated throughout the food chain. A major problem that arises from this pollution is that the effects of most of these pesticides on cellular mechanisms in general, and how they interact with each other and affect human cells are still poorly understood. In this study we make use of cultured human cancer cells to measure by qRT-PCR how pesticides affect gene expression of stress pathways. Immunoblotting studies were performed to monitor protein expression levels and activation of signaling pathways. We make use of immunofluorescence and microscopy to visualize and quantify DNA damage events in those cells. In the current study, we evaluate the potential of a subset of widely used pesticides to activate the dioxin receptor pathway and affect its crosstalk with estrogen receptor signaling. We quantify the impact of these chemicals on the p53-dependent cellular stress response. We find that, not only can the different pesticides activate the dioxin receptor pathway, most of them have better than additive effects on this pathway when combined at low doses. We also show that different pesticides have the ability to trigger crosstalk events that may generate genotoxic estrogen metabolites. Finally, we show that some, but not all of the tested pesticides can induce a p53-dependent stress response. Taken together our results provide evidence that several xenobiotics found within the environment have the potential to interact together to elicit significant effects on cell systems. Our data warrants caution when the toxicity of substances that are assessed simply for individual chemicals, since important biological effects could be observed only in the presence of other compounds, and that even at very low concentrations.
Accurately capturing domain-domain interactions is key to understanding protein function and designing structure-based drugs. Although AlphaFold2 has made a breakthrough on single domain, it should be noted that the structure modeling for multi-domain protein and complex remains a challenge. In this study, we developed a multi-domain and complex structure assembly protocol, named DeepAssembly, based on domain segmentation and single domain modeling algorithms. Firstly, DeepAssembly uses a population-based evolutionary algorithm to assemble multi-domain proteins by inter-domain interactions inferred from a developed deep learning network. Secondly, protein complexes are assembled by means of domains rather than chains using DeepAssembly. Experimental results show that on 219 multi-domain proteins, the average inter-domain distance precision by DeepAssembly is 22.7% higher than that of AlphaFold2. Moreover, DeepAssembly improves accuracy by 13.1% for 164 multi-domain structures with low confidence deposited in AlphaFold database. We apply DeepAssembly for the prediction of 247 heterodimers. We find that DeepAssembly successfully predicts the interface (DockQ ≥ 0.23) for 32.4% of the dimers, suggesting a lighter way to assemble complex structures by treating domains as assembly units and using inter-domain interactions learned from monomer structures.
Circulating tumor HPV DNA (ctHPV16) assessed in liquid biopsy may be used as a marker of cancer in patients with HPV-associated oropharyngeal cancer (HPV + OPC). Factors influencing the initial ctHPV16 quantity are not well recognized. In this study we aimed to establish what factors are related to the level of ctHPV16 at the time of diagnosis. 51 patients (37 men and 14 women, median age of 57 years old) with HPV + OPC prior to definitive treatment were included. ctHPV16 was measured by qPCR. Tumor and nodal staging were assessed according to AJCC8. Blood derived factors included squamous cell carcinoma antigen (SCC-Ag), serum soluble fragment of cytokeratin 19 (CYFRA 21-1), C-reactive protein (CRP), albumin level (Alb), neutrophils (Neut), thrombocytes (Plt) and lymphocyte (Lym) count, Neut/Lym ratio were assessed. The volumes of the primary tumor (TV) and involved lymph nodes (NV) were calculated using MRI, CT or PET-CT scans. Data were analysed using parametric and nonparametric methods. Variables for multivariable linear regression analysis were chosen based on the results from univariable analysis (correlation, univariable regression and difference). There were 9 (18%), 10 (19%) and 32 (63%) patients who had TV and NV assessed in MRI, CT or PET respectively. Primary tumor neither as T-stage nor TV was related to ctHPV16 level. Significant differences in the ctHPV16 between patients with high vs low pain (P = 0.038), NV (P = 0.023), TV + NV (P = 0.018), CYFRA 21-1 (P = 0.002), CRP (P = 0.019), and N1 vs N3 (P = 0.044) were observed. ctHPV16 was significantly associated with CYFRA 21-1 (P = 0.017), N stage (P = 0.005), NV (P = 0.009), TV + NV (P = 0.002), CRP (P = 0.019), and pain (P = 0.038). In univariable linear regression analysis the same variables predicted ctHPV16 level. In multivariable analyses, CYFRA 21-1 and CRP (both as categorical variables) were predictors of ctHPV16 level even above NV. ctHPV16 at presentation is driven by tumor volume measured mostly by N. CYFRA 21-1 and CRP are additional factors related to ctHPV16 prior to the treatment.
Iron overload negatively affects bone mass and strength. However, the impact of iron excess on osteocytes-important bone cells for mechanotransduction and remodeling-is poorly understood. Herein, we examined the effects of iron exposure on osteocytes during their maturation process. We discovered that iron overload caused apoptosis of osteocytes in early and late stages of differentiation. Notably, the expression of key proteins for iron entry was downregulated during differentiation, suggesting that mature osteocytes were less susceptible to iron toxicity due to limited iron uptake. Furthermore, iron overload also enriched a subpopulation of mature osteocytes, as indicated by increased expression of Dmp1, a gene encoding protein for bone mineralization. These iron-exposed osteocytes expressed high levels of Sost, Tnfsf11 and Fgf23 transcripts. Consistently, we demonstrated that exogenous FGF23 stimulated the formation and survival of osteoclasts, suggesting its regulatory role in bone resorption. In addition, iron overload downregulated the expression of Cx43, a gene encoding gap junction protein in the dendritic processes, and impaired YAP1 nuclear translocation in response to fluid flow in differentiated osteocytes. It can be concluded that iron overload induces cellular adaptation in differentiating osteocytes, resulting in insensitivity to mechanical stimulation and potential disruption of the balance in bone remodeling.
Enzyme therapy can be an appropriate treatment option for celiac disease (CeD). Here, we developed Bromelain-Loaded Nanocomposites (BLNCs) to improve the stability and retention of bromelain enzyme activity. After the characterization of BLNCs, the cytotoxicity of BLNCs was determined on the Caco-2 cell line. The effect of BLNCs on gliadin degradation and the production of pro-inflammatory cytokines and anti-inflammatory molecules in peripheral blood mononuclear cells (PBMCs) obtained from celiac patients were assessed. Furthermore, the expression of CXCR3 and CCR5 genes was measured in CaCo-2 cells treated with gliadin, gliadin-digested with BLNCs, and bromelain. Our study demonstrated that the Bromelain entrapment efficiency in these nanoparticles was acceptable, and BLNCs have no toxic effect on cells. SDS-PAGE confirmed the digestion effect of bromelain released from nanocomposites. When Caco-2 cells were treated with gliadin digested by free bromelain and BLNCs, the expression of CXCR3 and CCR5 genes was significantly decreased. PBMCs of celiac patients treated with Bromelain and BLNCs decreased inflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-γ) production compared to untreated PBMCs. This treatment also increased IL-10 and CTLA-4 in PBMCs of CeD patients. According to the promising results of this study, we can hope for the therapeutic potential of BLNCs for CeD.
The H9N2 subtype of the avian influenza virus (AIV) is one of the main subtypes of low pathogenic AIV, and it seriously affects the poultry breeding industry. Currently, vaccination is still one of China's main strategies for controlling H9N2 avian influenza. In this study, we selected MW548848.1 on the current popular main branch h9.4.2.5 as the reference strain, and we optimized the amino acid sequence of HA1 to make it suitable for expression in Bacillus subtilis. The B. subtilis expression vector showed good safety and stress resistance; therefore, this study constructed a recombinant B. subtilis expressing H9N2 HA1 protein and evaluated its immunogenicity in mice. The following results were obtained: the sIgA level of HA1 protein in small intestine fluid and the IgG level of PHT43-HA1/B. subtilis in serum were significantly improved (P < 0.01); PHT43-HA1/B. subtilis can cause a special immune response in mice; and cytokine detection interferon-gamma (IFN-γ) (P < 0.05) and Interleukin 2 (IL-2) (P < 0.01) expressions significantly increased. Additionally, the study found that PHT43-HA1/B. subtilis can alleviate the attack of H9N2 AIV in the spleen, lungs, and small intestine of mice. This study was the first to use an oral recombinant B. subtilis-HA1 vaccine candidate, and it provides theoretical data and technical reference for the creation of a new live vector vaccine against H9N2 AIV.
Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP).
Breast cancer is the most common malignant tumour in women. The early silk-splitting inhibitor protein 1 Emi1 is responsible for mediating ubiquitin protein degradation. The present study investigated the effects of the decreased expression of the Emil gene on the proliferation and invasion of breast cancer cells. The interference efficiency of small interfering ribonucleic acid (siRNA) was quantitatively verified using fluorescence real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, and the effect of Emi1 gene silencing on cell vitality and invasion was determined using MTT and Transwell assays, respectively. The expression of the proliferation genes programmed cell death receptor 4 (PDCD-4), fatty acid synthase ligand (FasL), PTEN and RhoB, along with the invasive genes Maspin, TIMP3 and RECK, was measured using fluorescence RT-qPCR. In breast cancer cells, siRNA successfully reduced the expression of the Emi1 gene, and the expression level of the cell proliferation genes PDCD-4, FasL, PTEN and RhoB, along with invasive genes Maspin, TIMP3 and RECK, decreased significantly (P < 0.05). Furthermore, Emi1 gene silencing reduced the proliferation and invasion abilities of MDA-MB-231 and SUM149PT cells by reducing the expression of proliferative and invasive genes.
Automated coaches (eCoach) can help people lead a healthy lifestyle (e.g., reduction of sedentary bouts) with continuous health status monitoring and personalized recommendation generation with artificial intelligence (AI). Semantic ontology can play a crucial role in knowledge representation, data integration, and information retrieval.
Common bean (Phaseolus vulgaris) is one of the legume crops most consumed worldwide and bean rust is one of the most severe foliar biotrophic fungal diseases impacting its production. In this work, we searched for new sources of rust resistance (Uromyces appendiculatus) in a representative collection of the Portuguese germplasm, known to have accessions with an admixed genetic background between Mesoamerican and Andean gene pools. We identified six accessions with incomplete hypersensitive resistance and 20 partially resistant accessions of Andean, Mesoamerican, and admixed origin. We detected 11 disease severity-associated single-nucleotide polymorphisms (SNPs) using a genome-wide association approach. Six of the associations were related to partial (incomplete non-hypersensitive) resistance and five to incomplete hypersensitive resistance, and the proportion of variance explained by each association varied from 4.7 to 25.2%. Bean rust severity values ranged from 0.2 to 49.1% and all the infection types were identified, reflecting the diversity of resistance mechanisms deployed by the Portuguese germplasm.The associations with U. appendiculatus partial resistance were located in chromosome Pv08, and with incomplete hypersensitive resistance in chromosomes Pv06, Pv07, and Pv08, suggesting an oligogenic inheritance of both types of resistance. A resolution to the gene level was achieved for eight of the associations. The candidate genes proposed included several resistance-associated enzymes, namely β-amylase 7, acyl-CoA thioesterase, protein kinase, and aspartyl protease. Both SNPs and candidate genes here identified constitute promising genomics targets to develop functional molecular tools to support bean rust resistance precision breeding.
The enzyme phenylalanine ammonia lyase (PAL) controls the transition from primary to secondary metabolism by converting L-phenylalanine (L-Phe) to cinnamic acid. However, the function of PAL in pear plants (Pyrus bretschneideri) has not yet been fully elucidated.
The uc.291 transcript controls keratinocytes differentiation by physical interaction with ACTL6A and subsequent induction of transcription of the genes belonging to the epidermal differentiation complex (EDC). Uc.291 is also implicated in the dedifferentiation phenotype seen in poorly differentiated cutaneous squamous cell carcinomas. Here, we would like to investigate the contribution of uc.291 to the unbalanced differentiation state of keratinocytes observed in hyperproliferative skin disorders, e. g., psoriasis. Psoriasis is a multifactorial inflammatory disease, caused by alteration of keratinocytes homeostasis. The imbalanced differentiation state, triggered by the infiltration of immune cells, represents one of the events responsible for this pathology. In the present work, we explore the role of uc.291 and its interactor ACTL6A in psoriasis skin, using quantitative real-time PCR (RT-qPCR), immunohistochemistry and bioinformatic analysis of publicly available datasets. Our data suggest that the expression of the uc.291 and of EDC genes loricrin and filaggrin (LOR, FLG) is reduced in lesional skin compared to nonlesional skin of psoriatic patients; conversely, the mRNA and protein level of ACTL6A are up-regulated. Furthermore, we provide evidence that the expression of uc.291, FLG and LOR is reduced, while ACTL6A mRNA is up-regulated, in an in vitro psoriasis-like model obtained by treating differentiated keratinocytes with interleukin 22 (IL-22). Furthermore, analysis of a publicly available dataset of human epidermal keratinocytes treated with IL-22 (GSE7216) confirmed our in vitro results. Taken together, our data reveal a novel role of uc.291 and its functional axis with ACTL6A in psoriasis disorder and a proof of concept that biological inhibition of this molecular axis could have a potential pharmacological effect against psoriasis and, in general, in skin diseases with a suppressed differentiation programme.
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display.
Friedreich ataxia is the most common inherited ataxia in Europe and is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene that lead to a decrease in frataxin protein levels. Rarely, affected individuals carry either a large intragenic deletion or whole-gene deletion of FXN on one allele and a full-penetrance expanded GAA repeat on the other allele.
Classical swine fever virus (CSFV) is an ancient pathogen that continues to pose a threat to animal agriculture worldwide. The virus belongs to the genus Pestivirus and the family Flaviviridae. It causes a multisystemic disease that affects only pigs and is responsible for significant economic losses. CSFV infection is probably a multistep process that involves the proteins in the virus envelope and more than one receptor in the membrane of permissive cells. To date, the cellular receptors essential for CSFV entry and their detailed functions during this process remains unknown. All the viral envelope proteins Erns, E1 and E2 are involved in the entry process to some extent and the experimental approaches conducted until now have helped to unveil their contributions. This review aims to provide an overview of current knowledge on cellular molecules described to be involved in CSFV entry, including complement regulatory protein 46 (CD46), heparan sulphate (HS), Laminin receptor, Integrin ß3, Annexin II, MERKT and ADAM17. This knowledge would not only help to understand the molecular mechanisms involved in pestivirus infection, but also provide a rational basis for the development of nonvaccinal alternatives for CSFV control.
Acanthamoeba spp. are opportunistic pathogens that cause inflammation, mostly in the brain, lungs and cornea. Recent reports indicate kidney dysfunction in hosts with systemic acanthamoebiasis. The aim of the study was to analyze the gene expression and protein concentration of NADPH oxidase 2 and 4 (NOX2 and NOX4, respectively) and nuclear erythroid 2-related factor (Nrf2) in the kidneys of hosts with systemic acanthamoebiasis. We also aimed to determine the protein and gene expressions of Bcl2, Bax, caspases 3 and 9.
Parkinson's disease (PD), one of the most devastating neurodegenerative brain disorders, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of &#945;-synuclein aggregates. Currently, pharmacological interventions for PD remain inadequate. The cell necroptosis executor protein MLKL (Mixed-lineage kinase domain-like) is involved in various diseases, including inflammatory bowel disease and neurodegenerative diseases; however, its precise role in PD remains unclear. Here, we investigated the neuroprotective role of MLKL inhibition or ablation against primary neuronal cells and human iPSC-derived midbrain organoids induced by toxic &#945;-Synuclein preformed fibrils (PFFs). Using a mouse model (Tg-Mlkl<sup>-/-</sup>) generated by crossbreeding the SNCA A53T synuclein transgenic mice with MLKL knockout (KO)mice, we assessed the impact of MLKL deficiency on the progression of Parkinsonian traits. Our findings demonstrate that Tg-Mlkl<sup>-/-</sup> mice exhibited a significant improvement in motor symptoms and reduced phosphorylated &#945;-synuclein expression compared to the classic A53T transgenic mice. Furthermore, MLKL deficiency alleviated tyrosine hydroxylase (TH)-positive neuron loss and attenuated neuroinflammation by inhibiting the activation of microglia and astrocytes. Single-cell RNA-seq (scRNA-seq) analysis of the SN of Tg-Mlkl<sup>-/-</sup> mice revealed a unique cell type-specific transcriptome profile, including downregulated prostaglandin D synthase (PTGDS) expression, indicating reduced microglial cells and dampened neuron death. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing motor deficits in PD.
As one of the leading causes for dementia in the population, it is imperative that we discern exactly why Alzheimer's disease (AD) has a strong molecular association with beta-amyloid and tau. Although a clear understanding about etiology and pathogenesis of AD remains unsolved, scientists worldwide have dedicated significant efforts to discovering the molecular interactions linked to the pathological characteristics and potential treatments. Knowledge representations, such as domain ontologies encompassing our current understanding about AD, could greatly assist and contribute to disease research. This paper describes the construction and application of the integrated Alzheimer's Disease Ontology (ADO), combining selected concepts from the former version of the ADO and the Alzheimer's Disease Mapping Ontology (ADMO). In addition to the existing entities available from these knowledge models, essential knowledge about AD from public sources, such as newly discovered risk factor genes and novel treatments, was also integrated. The ADO can also be leveraged in text mining scenarios given that it is conceptually enriched with domain-specific knowledge as well as their relations. The integrated ADO consists of 39 855 total axioms. The ontology covers many aspects of the AD domain, including risk factor genes, clinical features, treatments and experimental models. The ontology complies with the Open Biological and Biomedical Ontology principles and was accepted by the foundry. In this paper, we illustrate the role of the presented ontology in extracting textual information from the SCAIView database and key measures in an ADO-based corpus. Database URL:  https://academic.oup.com/database.
<i>Stachys brachyclada</i> de No&#233; ex Coss. (Lamiaceae) is a quite rare medicinal plant endemic to the Mediterranean basin. In this study, seven secondary metabolites from a methanol extract of its leaves have been isolated and identified by a combination of chromatographic and spectroscopic methods (1D and 2D NMR experiments and ESIMS analysis). They include one ethyl 4-hydroxybenzoate (<b>1</b>), three acylated flavone glycosides (<b>2</b>-<b>4</b>), one diapigenin derivative (<b>5</b>) and two flavone aglycones (<b>6</b>-<b>7</b>). Stachysetin (<b>5</b>) was found the major compound of the extract (74.0&#8201;mg/g of dry matter). Moreover, the produced extract showed the ability in inhibiting the &#945;-glucosidase enzyme (IC<sub>50</sub> = 13.7&#8201;&#181;g/mL), in quenching the radical 1,1-diphenyl-2-picrylhydrazyl (EC<sub>50</sub> = 74.6&#8201;&#181;g/mL), and in reducing the intracellular oxidative stress level in Human Dermal Fibroblast (64% inhibition at 50&#8201;&#181;g/mL).
There is a need for a simple yet comprehensive tool to produce and edit pedagogical anatomy video courses, given the widespread usage of multimedia and 3D content in anatomy instruction. Anatomy teachers have minimal control over the present anatomical content generation pipeline. In this research, we provide an authoring tool for instructors that takes text written in the Anatomy Storyboard Language (ASL), a novel domain-specific language (DSL) and produces an animated video. ASL is a formal language that allows users to describe video shots as individual sentences while referencing anatomic structures from a large-scale ontology linked to 3D models. We describe an authoring tool that translates anatomy lessons written in ASL to finite state machines, which are then used to automatically generate 3D animation with the Unity 3D game engine. The proposed text-to-movie authoring tool was evaluated by four anatomy professors to create short lessons on the knee. Preliminary results demonstrate the ease of use and effectiveness of the tool for quickly drafting narrated video lessons in realistic medical anatomy teaching scenarios.
The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.
Label="BACKGROUND" NlmCategory="BACKGROUND">Olipudase alfa is a recombinant human acid sphingomyelinase enzyme replacement therapy for non-central-nervous-system manifestations of acid sphingomyelinase deficiency (ASMD). The ASCEND randomized placebo-controlled trial in adults with ASMD demonstrated reductions in sphingomyelin storage, organomegaly, interstitial lung disease and impaired diffusion capacity of the lung&#160;(DL<sub>CO</sub>), during the first year of olipudase alfa treatment. In an ongoing open-label extension of the ASCEND trial, individuals in the placebo group crossed over to olipudase alfa, and those in the olipudase alfa group continued treatment.
The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L (also known as UBA7). Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validate the structure and confirm the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enables the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study helps to define the molecular basis of these interactions and the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.
The aim of this study was to elucidate how different nursery production methods influence the composition of and relationship between soil and root community levels of Abies alba. In the Międzylesie Forest District, we quantified the responses of samples of both community-level fine roots and surrounding soil to environmental changes evoked by various seedling production methods. Fungi levels were identified based on their ITS 1 region and 5.8 S rDNA component. Analysis was conducted using Illumina SBS technology, and the obtained sequences were compared with reference samples deposited in the UNITE. Chemical analysis of the soil was also performed. Different nursery production methods resulted in a strong decoupling in the responses of fungal community levels between soil and roots. Changes in growth conditions imposed by production methods were significant in determining species composition. We found differences in fungal communities among functional groups of samples. In the soil, the dominant species of mycorrhizal fungi were Tylospora asterophora, Amanita rubescens, and Russula ionochlora. Mycorrhizal fungi in roots included Tuber anniae, Thelephoraceae sp., and Acephala applanata. Specific soil substrate conditions significantly influenced fungal community composition, leading to an increase in abundance of mycorrhizal fungi, specifically T. anniae.
Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient's cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.
Lentils are a significant source of plant protein and are cultivated across Asia, Europe, and North Africa. Plants are subjected to various environmental stresses, which can hinder growth, yield, and productivity. 5-aminolevulinic acid (ALA) is a compound that acts as a precursor in the biosynthesis of tetrapyrroles and can increase plant tolerance to different abiotic stressors. However, the effects of exogenously applied ALA on lentil growth, yield, and physiological parameters under rain-fed and supplemental irrigation conditions are not well-known. In this study, a split plot experiment was conducted to investigate the impact of ALA foliar application and supplemental irrigation on lentil (Lens culinaris Medik.). The experiment was designed based on a randomized complete block with three replications. The main plot included four levels of supplemental irrigation [(supplementary irrigation in the flowering and early seed-filling stages, supplementary irrigation in the flowering stage, supplementary irrigation in the early seed-filling along with rain-fed conditions (no irrigation)]. The subplot considered foliar application of ALA at varying levels [(0 (control), 50 and 100&#160;ppm)]. The results showed that water regimes and foliar spray with ALA significantly (P&#160;&#706;&#160;0.01) affected plant height, number of pods per plant, pod weight, number of seeds per pod and weight of 1000 seeds, biological yield, seed yield, and harvest index. The highest total chlorophyll content was observed in plants that were subjected to supplementary irrigation in flowering and early seed filling stages and foliar sprayed with 100&#160;ppm ALA. The study also found that exogenous ALA improved drought tolerance in lentil plants under rain-fed conditions mainly by regulating antioxidant enzymes, which ultimately protected the cellular membranes against overproduction of H<sub>2</sub>O<sub>2</sub>. Furthermore, ALA application increased total carbohydrate contents at all supplemental irrigation levels, but the rate was higher in complementary irrigation conditions during flowering and early seed-filling stages. Malondialdehyde (MDA), H<sub>2</sub>O<sub>2</sub>, and proline contents were increased in field-grown plants under rain-fed conditions without exogenous ALA application. In conclusion, this study sheds light on the effects of ALA foliar spray and supplemental irrigation on lentil growth, yield, and physiological parameters. The findings suggest that exogenous ALA can improve plant tolerance to various abiotic stressors and enhance plant growth, yield, and physiological parameters.
Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.
The large use of fish meal/fish oil in carnivorous fish feeds is the main concern regarding environmental sustainability of aquaculture. Here, we evaluated the effects of an innovative diet, designed to be (1) environmentally sustainable by lowering the marine protein content while being (2) cost effective by using sustainable alternative raw materials with acceptable cost and produced on an industrial scale, on growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax), a key species of the Mediterranean marine aquaculture, reared in sea cages. Results show that the specific growth rate of fish fed the low marine protein diet was significantly lower than those fed conventional diet (0.67% vs 0.69%). Fatty acid profile of fillets from fish fed a low marine protein diet presented significant lower n-6 and higher n-3 content when compared to conventional ones. Then, a significant increase in the abundance of Vibrio and reduction of Photobacterium were found in the gut of fish fed with the low marine protein diet but effects on sea bass health needs further investigation. Finally, no major health and welfare alterations for fish fed the low marine protein diet were observed, combined with a potential slight benefit related to humoral immunity. Overall, these results suggest that despite the low marine protein diet moderately affects growth performance, it nevertheless may enhance environmental and economic sustainability of the sea bass aquaculture.
Fabry disease (FD) is a multi-organ disorder caused by a deficiency of alpha-galactosidase (α-GLA) or reduced activity of the enzyme due to mutations in the GLA gene on the X chromosome, making it an X-linked hereditary disease. A 37-year-old man previously diagnosed with sudden deafness and cardiac hypertrophy was referred to our department after an abnormal urine finding during a public health checkup. A renal biopsy revealed characteristic findings, and he was diagnosed with FD with a novel GLA abnormality (c.714dupT (p.I239Yfs*11)). We are currently administering enzyme replacement therapy (ERT) with agalsidase α. This case shows that a novel genetic abnormality in FD can be overlooked for 37 years, even in the presence of typical symptoms. The significance of a renal biopsy in diagnosing FD is emphasized, highlighting the crucial role of nephrologists.  DOI: 10.52547/ijkd.7595.
Tick infestation causes a significant threat to human and animal health, requiring effective immunological control methods. This study aimed to investigate the potential of recombinant Haemaphysalis longicornis enolase protein for tick vaccine development. The exact mechanism of the recently identified enolase protein from the H. longicornis Jeju strain remains poorly understood. Enolase plays a crucial role in glycolysis, the metabolic process that converts glucose into energy, and is essential for the motility, adhesion, invasion, growth, and differentiation of ticks. In this study, mice were immunized with recombinant enolase, and polyclonal antibodies were generated. Western blot analysis confirmed the specific recognition of enolase by the antiserum. The effects of immunization on tick feeding and attachment were assessed. Adult ticks attached to the recombinant enolase-immunized mice demonstrated longer attachment time, increased blood-sucking abilities, and lower engorgement weight than the controls. The nymphs and larvae had a reduced attachment rate and low engorgement rate compared to the controls. Mice immunized with recombinant enolase expressed in Escherichia coli displayed 90% efficacy in preventing tick infestation. The glycolytic nature of enolase and its involvement in crucial physiological processes makes it an attractive target for disrupting tick survival and disease transmission. Polyclonal antibodies recognize enolase and significantly reduce attachment rates, tick feeding, and engorgement. Our findings indicate that recombinant enolase may be a valuable vaccine candidate for H. longicornis infection in experimental murine model.
Since 2015, countries in the Sahel region have implemented large-scale seasonal malaria chemoprevention (SMC). However, the mass use of sulfadoxine-pyrimethamine (SP) plus amodiaquine impacts the genetic diversity of malaria parasites and their sensitivity to antimalarials. This study aimed to describe and compare the genetic diversity and SP resistance of Plasmodium falciparum strains in Mali and Niger. We collected 400 blood samples in Mali and Niger from children aged 3-59 months suspected of malaria. Of them, 201 tested positive (Niger, 111, 55.2%; Mali, 90, 44.8%). Polymorphism of merozoite surface protein 1 (msp1) genetic marker showed 201 allotypes. The frequency of the RO33 allotype was significantly higher in Niger (63.6%) than in Mali (39.3%). There was no significant difference in the frequency of the K1 and MAD20 allotypes between the 2 countries. The multiplicity of infection was 2 allotypes per patient in Mali and one allotype per patient in Niger. The prevalence of strains with the triple mutants Pfdhfr51I/Pfdhfr59R/Pfdhps436A/F/H and Pfdhfr51I/Pfdhfr59R/Pfdhps437G was 18.1% and 30.2%, respectively, and 7.7% carried the quadruple mutant Pfdhfr51I/Pfdhfr59R/Pfdhps436A/F/H/Pfdhps437G. Despite the significant genetic diversity of parasite populations, the level of SP resistance was comparable between Mali and Niger. The frequency of mutations conferring resistance to SP still allows its effective use in intermittent preventive treatment in pregnant women and in SMC.
Autophagy is a fundamental catabolic process whereby excessive or damaged cytoplasmic components are degraded through lysosomes to maintain cellular homeostasis. Studies of mTOR signaling have revealed that mTOR controls biomass generation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Primary cilia, the assembly of which depends on kinesin molecular motors, serve as sensory organelles and signaling platforms. Given these pathways' central role in maintaining cellular and physiological homeostasis, a connection between mTOR and primary cilia signaling is starting to emerge in a variety of diseases. In this review, we highlight recent advances in our understanding of the complex crosstalk between the mTOR pathway and cilia and discuss its function in the context of related diseases.
Vision is arguably our most important sense, and its loss brings substantial limitations to daily life for affected individuals. Light is perceived in retinal photoreceptors (PRs), which are highly specialized neurons subdivided into several compartments with distinct functions. The outer segments (OSs) of photoreceptors represent highly specialized primary ciliary compartments hosting the phototransduction cascade, which transforms incoming light into a neuronal signal. Retinal disease can result from various pathomechanisms originating in distinct subcompartments of the PR cell, or in the retinal pigment epithelium which supports the PRs. Dysfunction of primary cilia causes human disorders known as "ciliopathies", in which retinal disease is a common feature. This chapter focuses on PR OSs, discussing the mechanisms controlling their complex structure and composition. A sequence of tightly regulated sorting and trafficking events, both upstream of and within this ciliary compartment, ensures the establishment and maintenance of the adequate proteome and lipidome required for signaling in response to light. We discuss in particular our current understanding of the role of ciliopathy proteins involved in multi-protein complexes at the ciliary transition zone (CC2D2A) or BBSome (BBS1) and how their dysfunction causes retinal disease. While the loss of CC2D2A prevents the fusion of vesicles and delivery of the photopigment rhodopsin to the ciliary base, leading to early OS ultrastructural defects, BBS1 deficiency results in precocious accumulation of cholesterol in mutant OSs and decreased visual function preceding morphological changes. These distinct pathomechanisms underscore the central role of ciliary proteins involved in multiple processes controlling OS protein and lipid composition.